miR146a参与HBV感染造成的肝细胞Ⅰ型干扰素应答抑制的分子机制研究
|
摘要
研究目的：
乙型肝炎病毒的感染(CHB)是严重威胁我国人类健康的重大传染病,其迁延不愈的特点不仅给患者和社会带来沉重的负担,也对公共卫生产生巨大隐患。Ⅰ型干扰素是目前治疗CHB和相关疾病最常用的药物,然而仅对约1/3的患者有效,且容易产生耐药性,大大限制了其临床治疗效果,这种现象在临床上称为“干扰素抵抗”。产生这一现象的主要原因是,HBV感染后机体免疫平衡被破坏,造成免疫功能低下或者紊乱。
研究表明,HBV病毒可以在分子、细胞、器官和个体等多个层次上对机体抗病毒天然免疫和获得性免疫产生抑制和耐受,这是造成HBV感染容易慢性化的分子免疫基础。肝脏免疫学认为,抑制进而完全清除HBV离不开机体抗病毒免疫反应的激活,而正常的免疫功能也是药物治疗取得理想疗效不可或缺的因素。然而,目前以针对“干扰素抵抗”为代表的HBV负向免疫调节作用的研究相对滞后,限制了抗HBV免疫治疗药物的开发。
miRNA是近年来发现的一类主要在转录后水平上对基因表达进行调节的短片段非编码RNA。在哺乳动物细胞内,miRNA可通过序列特异性的不完全互补作用抑制靶基因mRNA的翻译。研究发现,miRNA在抗病毒天然免疫反应中,既可以成为典型的基因调控因子,也能够成为直接靶向转录物而发挥干扰作用的免疫效应分子。同时,在HBV的致病过程中,宿主miRNA同样参与了病毒复制、转录、包装等自身生命活动,通过调控宿主基因参与了炎症反应、免疫损伤、促进自身潜伏、诱发继发性疾病乃至肝癌,成为研究HBV与宿主相互作用的重要窗口。
本研究的目的是通过对HBV存在条件下肝细胞内miRNA表达水平的检测和筛选,寻找可能参与HBV诱导胞内天然免疫抑制,特别是“干扰素抵抗”的miRNA。通过干预miRNA的表达水平,分析相关miRNA与HBV感染之间的关系,并研究其是否参与了HBV造成的肝细胞胞内抗病毒免疫应答信号的调节,探究其与“干扰素抵抗”是否相关。利用生物信息学和分子生物学的手段鉴定在这一过程中miRNA发挥作用的关键靶分子,并寻找HBV感染与相关分子之间动态水平的关联。最后,利用HBV复制性小鼠模型，研究靶向于预特定miRNA能否改变HBV的体内感染过程,为开发基于miRNA的新型抗HBV免疫药物提供理论和实验依据。
研究方法：
首先,以整合了HBV全基因组的HepG2.2.15细胞和无HBV存在的对照细胞HepG2为模型,利用miRNA特异性RT和qPCR两种技术,我们定量分析了细胞内与免疫相关的15种niRNA的丰度,观察了HBV感染对肝细胞内免疫相关miRNA的调节作用,发现miR146家族的两个成员,特别足miR146a在HBV整合的肝细胞系内的表达明显高于对照细胞,并通过核糖核酸酶保护实验确认了miR146a在两种细胞系内的绝对水平;在HepG2.2.15细胞和HepG2细胞中,利用特异性引物并通过RT-qPCR定量分析：niR146a初级转录物和次级前体的表达,并采用荧光素酶报告基因实验分析两种细胞中miR146a启动子的活性。
其次,通过基因工程的方法,我们构建了HBV全基因组和四种主要蛋白元件的真核表达载体，并分别将其转染到HepG2细胞中,利用RT-qPCR技术检测了HBV基因组及其蛋白组分的存在对肝细胞内miR146a表达的影响。同时,我们利用HBV全基因组表达载体尾静脉高压注射的方法建立了HBV复制性小鼠模型,通过灌流法分离小鼠肝实质细胞,接着用RT-qPCR法检测了其中miR146a的表达,同时用ELISA法检测了小鼠血清中HBsAg的水平,分析了二者之间的关系,并再次确认了HBV存在对(?)niR146a表达水平的影响。
接下来,我们将化学合成的miR146a抑制物转染入高表达miR146a的HepG2.2.15细胞,利用RT-qPCR和ELISA检测其对HBV转录和抗原表达的影响。与此同时,将miR146a模拟物或其过表达载体与HBV全基因组载体联合转染低表达miR146a的HepG2细胞,检测了其对HBV转求和抗原表达的影响。为进行体内研究,我们构建了靶向miR146a家族成员的niR146a sponge沉默载体与miR146a过表达载体pcDNA3-miR146a,然后分别与HBV全基因组载体配伍,联合高压注射小鼠,ELISA检测小鼠血清HBsAg和HBeAg的含量,并用免疫组织化学分析小鼠肝组织HBsAg和HBcAg的表达,探索了体内靶向干预肝细胞内miR146a的表达水平后对HBV体内感染周期的影响。
在分析miR146a对Ⅰ型干扰素信号转导通路影响研究中,首先对miR146a进行干预,然后再进行Ⅰ型干扰素处理,利用RT-qPCR和Western Blotting技术比较干预前后细胞中MxA、ISG15、OAS-1、IFIT3等主要抗病毒基因的转录和蛋白表达水平；在先期相关研究的基础上,比较了两种细胞miR146a靶基因-转录因子STAT1的表达：构建了包含miR146a识别区域的UTR的荧光素酶报告基因载体,并用于评价靶基因翻译活性。采用荧光素酶报告基因实验,我们分析了STAT1发生差异表达的分子基础；在此基础上,利用miR146a过表达和沉默策略,检测了肝细胞中miR146a调控STAT1表达的机制,并通过改变肝细胞内miR146a的水平,分析了miR146a对STAT1翻译活性的调节作用。通过对HBV阳性和阴性肝细胞癌临床标本的分析,我们进一步确认了STAT1的表达与HBV感染之间存在相关性。
最后,通过生物信息学手段,我们分析了可能参与抗HBV天然免疫应答过程的17种干扰素诱导基因与miR146a之间的关系,其中有三个基因被预测可能成为miR146a的直接靶点。我们构建了针对IFIT3的UTR区荧光素酶报告基因载体,证实了IFIT3是miR146a的一个新的直接靶点。在两种模型细胞中,我们比较了该基因和已知miR146a靶基因TRAF6翻译活性与miR146a表达水平之间的关系。基于已知的IFIT3和TRAF6生物学功能,我们又通过体外转录合成了RLR信号通路的配基5'ppp-RNA(简称3p-RNA)。首先通过预先沉默(?)niR146a的方法,利用RT-qPCR分析了肝细胞对3p-RNA免疫刺激作用的敏感性；接着进行IFIT3过表达实验,利用RT-qPCR和ELISA方法,分析其对肝细胞3p-RNA刺激的正向作用和直接抗HBV活性；通过荧光素酶报告基因实验分析了miR146a对肝细胞内TRAF6翻译的调节作用。
研究结果：
1.肝癌细胞内miR146a的高表达与HBV感染密切相关
HBV感染造成了多种免疫相关miRNA的表达水平发生变化,其中以miR146家族最为明显；在整合有HBV的肝癌细胞系HepG2.2.15和PLC/PRF/5中,miR146a的表达水平明显高于无HBV存在的阴性细胞系；HepG2.2.15中miR146a初级转录本和颈环前体的表达也明显高于对照细胞HepG2; HBV感染引起miR146a上调依赖于NF-κB。
2.HBV感染引起了肝细胞内miR146a表达水平的升高
我们成功建立HBV完整基因组和四个主要蛋白成分X蛋白、核心蛋白、表面抗原小蛋白和HBV的DNA多聚酶的真核表达载体；瞬时转染HBV全基因组载体后,HepG2细胞中(?)niR146a的表达水平明显提高,并且HBV基因组表达载体稳定化的HepG2细胞地也有相同的现象;HBV的四个主要的蛋白产物对上调肝细胞miR146a的表达有共同但不均等的贡献;在HBV复制性小鼠中,小鼠原代肝实质细胞内miR146a的表达与血清HBsAg的表达水平呈一定程度的正相关。
3. miR146a可以促进HBV的复制和转录
流式细胞术结果证实,荧光标记的miR146a模拟物和阻遏物可以有效的导入靶细胞。定量PCR检测结果发现,它们均可以有效地改变肝细胞内源性miRNA的表达水平;我们自行设计结构的miR146a sponge载体也可以有效地沉默HepG2.2.15细胞内miR146a的水平：
在上述实验系统的支撑下,在HepG2细胞模型中,进行模拟物或者质粒载体过表达miR146a,可以促进瞬转HBV的转录,而通过阻遏物或者质粒载体沉默niR146则可以抑制HBV的转录和翻译。通过尾静脉高压注射的的方式向小鼠肝脏输送miR146a过表达载体,可以升高小鼠血清HBsAg和HBeAg抗原的表达水平和肝细胞内HBsAg的表达,而输送靶向miR146a的sponge沉默载体后,小鼠血清HBsAg和HBeAg抗原的含量明显降低,且肝细胞内HBsAg的表达亦显著下降。同时,抑制miR146a的水平不影响(?)AepG2.2.15细胞的增殖;miR146b模拟物和HBV全基因组载体共转染HepG2细胞并不能明显影响HBV的转录,miR146b模拟物转染也未影响HepG2.2.15细胞分泌HBV抗原的能力。
基于RNA二级结构稳定性和保守性的生物信息学预测认为,miR146a不会直接靶向HBV RNA。
4. miR146a可以抑制肝细胞内Ⅰ型干扰素的信号转导
将miR146a模拟物转染入HepG2细胞后,可以抑制细胞对后续IFN-α刺激的应答,表现为ISG15、MxA、OAS-1、IFIT3等干扰素诱导基因转录的减弱和蛋白合成的下降。在miR146a稳定过表达的HepG2细胞上,也可以观察到类似的现象;而将miR146a阻遏物转染入HepG2.2.15细胞后,则会产生相反的作用,表现为ISG15、MxA、OAS-1、IFIT3等干扰素诱导基因转录的增强和蛋白合成的增加。
5.HBV感染诱导的miR146a的高表达抑制了肝细胞内STAT1的水平
HepG2.2.15细胞中STAT1的基础表达水平低于HepG2;与临床HBV阴性肝细胞癌细胞相比,HBV阳性肝细胞癌细胞中STAT1的水平明显降低；将rniR146a阻遏物转染入HepG2.2.15细胞后,可以明显上调STAT1蛋白的表达,而不影响胞内mRNA的水平,而转染miR146a的模拟物则会产生相反的结果。报告基因检测显示,HepG2.2.15细胞中STAT1的基础翻译水平明显低于HepG2细胞,这可能是其蛋白表达低于HepG2勺原因之一；提高HepG2细胞内miR146a勺水平可以序列依赖性地抑制STAT1的翻译,而抑制HepG2.215细胞内miR146a的水平则能够序列依赖性地促进STAT1的翻译。
6.人IFIT3基因是miR146a的一个新的直接靶基因
利用四种生物信息学软件,我们对17种抗HBV宿主基因与miR146a关系进行分析,其中有三个基因可能成为rniR146a的直接靶基因,分别是IFIT3、RIG-I和RNase L:针对IFIT3的荧光素酶报告实验证实,IFIT3是：miR146a的直接靶基因。
HepG2.2.15细胞中IFIT3基因的基础翻译水平低于HepG2细胞,且与STAT1的转录后调节相似,HepG2.2.15细胞中IFIT3基因的翻译水平受到胞内miR146a的抑制,向HepG2.2.15细胞内转染miR146a抑制物能够序列依赖性地促进IFIT3的翻译。
7. miR146a抑制了肝细胞内5'ppp-RNA刺激对I型干扰素的诱导作用
向HepG2.2.15细胞内转染miR146a抑制物后,5'ppp-RNA对HepG2.2.15细胞的免疫刺激作用显著增强,促进内源性Ⅰ型干扰素的诱导表达;在HepG2.2.15细胞内过表达IFIT3后,同样可以促进5'ppp-RNA引起的Ⅰ型干扰素的诱生。
另一种参与天然免疫应答并诱导Ⅰ型干扰素分子TRAF6,也受到miR-146a的转录后抑制,其在HepG2.2.15细胞中的翻译明显低于HepG2细胞。这可能是miR146a通过转录后抑制方式参与HBV感染肝细胞后内源性Ⅰ型干扰素诱导的新的分子机制。
将IFIT3过表达载体转入HepG2.2.15后,胞内HBV mRNA的转求水平显著受到明显抑制,提示IFIT3可能具有直接的抗HBV活性。
结论：
1.HBV基因组织的肝细胞系HepG2.2.15中miR146a的表达水平显著高于(?)AepG2细胞,主要分子机制是转求活性增强。
2. miR146a的存在促进了HBV的生命活动和自身存续,生物信息学预测显示这种左右很可能不是通过直接与病毒RNA相互作用实现的。
3. miR146a通过对靶基因STAT1翻译的抑制,负调了肝细胞对Ⅰ型干扰素诱导的抗病毒反应的应答能力,而抑制miR146a的功能则可以提高Ⅰ型干扰素体内外抗病毒效果。
4.人IFIT3是miR146a的直接的靶基因之一,且可能具有直接的抗HBV作用。
5. miR146a通过对靶基因IFIT3和TRAF6翻译的抑制,负调5'ppp-RNA对肝细胞内源Ⅰ型干扰素的诱导,而抑制miR146a的功能可以增强5'ppp-RNA的刺激作用,促进Ⅰ型干扰索的转录和表达。
意义：
miR146a高表达是HBV感染改变肝细胞表现遗传状态的主要表现之一;它通过调控多种与内源性Ⅰ型干扰素诱导和激活有关的信号分子,对胞内抗病毒天然免疫产生钝化作用,而有利于HBV的存续；靶向沉默miR146a则能够打破HBV感染造成的肝脏免疫抑制状态,促进HBV的清除,而针对miR-146a的阻遏分子则有望开发成为可以有效逆转“干扰索抵抗”,提高干扰素临床抗HBV疗效的免疫增强药物。
Object:
HBV causes acute and chronic hepatitis and results in life-long infected, presenting a significant threat to public health. Interferon-α (IFN-α) has been used in the treatment of chronic HBV-related diseases for many years, but only30%of the patients show positive response to IFN-α, and many patients become resistant to IFN-α. This phenomenon is called "IFN-α resistance", which impairs IFN-α anti-HBV effect severely and therefore contributes to the damage, defect and disorder of host anti-viral immunity during HBV infection
It is believed that HBV can impair anti-viral innate and adaptive immune response at molecular, cellular, organic and holistic levels, which lead to chronic infection to a certain extent. In the view of Liver immunology, activation of host anti-viral immunity is essential for HBV inhibition and clearance, and at the same time, is also required for desirable treatment effectiveness. Until now, research on negative immune regulation of HBV including "IFN resistance" is still delayed, which restricts the progress to develop new anti-HBV drugs.
microRNAs (miRNAs), a big family of small non-coding RNAs, were found to serve the pivotal function of regulating gene expression at posttranscriptional level in recent years, which always inhibit mRNA translation by sequence-dependent "mis-match" in mammalian cells. It is reported that, both virus-and host-encoded miRNAs would be important regulators or executors involved in viral pathogenesis and immunity. During the whole period of HBV infection, miRNAs not only regulate replication, transcription and package itself, but also participate in liver inflammation, immune injury, latency, secondary cases such as hepatocellular carcinoma, which become a new viewpoint to explore human-HBV interaction.
This research is designed to explore the role of miRNA in HBV-induced inhibition of innate immunity, especially "IFN resistance" by screening the expression level of immune-related miRNA between HBV-carried hepatoma cell and negative ones. We used many miRNA interference tools to analyze the interaction between HBV infection and miRNA changes, to determine whether miRNA was involved in the inhibition of intracellular innate immune pathway including "IFN resistance" after HBV infection. Using bioinformatic and molecular tools to look for key factors, we then identify the correlation between HBV and immune moleculars. At last, miRNA-targeting technology was employed to determine whether miRNA changes can favor anti-HBV response in HBV-carried mouse model.
Methods:
15immune-related miRNAs, including miR21, miR26a/b, miR106, miR132, miR146a/b, miR147, miR155, miR181, miR196a, miR296and miR448, were examined by stem-loop special miRNA qRT-PCR assay in HBV-carried HepG2.2.15cells and their counterpart HBV-negative HepG2cells. The miR146a expression levels were further confirmed by ribonuclease protection assay, also miR146a primary transcript (nearly200bp) and its stem-loop precursor (-100bp) were examined using special primers. Luciferase assay was used to test miRNA promoter activity.
LMP-HBV1.2(a new HBV genome expression vector based on pAAV-HBV1.2) and polymerase (HBp), HBV core protein (HBc), HBx and Small HBV surface protein expression vectors based on pEGFP-N1were transfected into HepG2cells. HBV-carried BALB/c mice were established by hydrodynamic tail vein injection with pAAV-HBV1.2. After2weeks, serum was harvested, and HBsAg levels were detected by ELISA. Then primary mouse hepatocytes were isolated by collagenase perfusion. The effect of miR146a on HBV infection was determined by transferring HBV plasmid, and miR146a mimics, inhibitors or expression/silencing vectors in human hepatoma cells (HepG2, and HepG2.2.15) and in mouse by hydrodynamic tail vein injection. The change of miR146a was confirmed by stem-loop special miRNA qRT-PCR assay.
In hepatoma cells, the effect of miR146a on IFN-a response was measured by RT-qPCR and Western Blotting, to test the transcription and translation including MxA、ISG15、OAS-1、IFIT3respectively. To explorer the mechanism of miR146a on STAT1expression, luciferase reporter vector containing STAT13'-UTR and its seed specific mutant control were constructed and cotransfected with miR146a mimics or inhibitors into hepatoma cells. The direct targeting effect of miR146a was validated by Luciferase reporter assay. STAT1protein level was also compared among HBV-positive and negative clinical samples.
Bioinformatic analysis was carried out to look for new miR146a direct targets within17anti-viral factors, and3'-UTR of candidate targets were cloned to luciferase reporter vector for experiment validation. Luciferase assay was used to identify the exact target, IFIT3. Then, IFIT3and TRAF63'-UTR luciferase constructs and its seed specific mutant control were cotransfected into hepatoma cells to evaluate translation efficiency. The impact of miR146a on the translation of these two genes at posttranscriptional level was also estimated by luciferase assay. To determine the promotion of miR146a inhibition to type I interferon induction,5'ppp-RNA was transcripted in vitro, and then introduced into IIepG2.2.15cell after miR146a silencing by miR146a sponge vector. RT-qPCR of type I interferon was utilized to quantitate the increasing fold.
Results:
1. miR146a level was closely correlated to HBV infection in human hepatoma cells
Two members of miR146family, miR146a and miR146b, showed a significant up-regulation in HepG2.2.15cells among15chosen miRNAs, especially miR146a. The expression of miR146a primary transcript and its stem-loop precursor were also higher in MepG2.2.15cells than that in HepG2cells. Promoter activity assay suggested that the transcription of miR146a was enhanced due to higher NF-κB activity.
2. HBV infection induced miR146a expression both in vitro and in vivo
Stem-loop special miRNA qRT-PCR assay showed that miR146a was increased both in HepG2cells transfected with pAAV-HBV1.2transiently and in cells stabilizated by HBV genome vector. Four HBV main proteins all seemed to contribute to up-regulaion of miR146a when they were transfected into HepG2cells individually. The serum HBsAg level revealed a positive relationship with mature miR146a level in hepatocytes from HBV-carried mice.
3. miR146a accelerated HBV replication
Chemical synthesized miR146a mimics and inhibitors could interference endogenous level of miR146a effectively, as well as miR146a expression or silencing vector, pcDNA3-miR146a and psiRNE-PGK-miR146a-sponge.
miR146a over-expression would ameliorate HBx and HBs/p mRNA transcription in HepG2cells. On the other hand, miR146a inhibitors down-regulated HBx and HBs/p mRNA levels in HepG2.2.15cells, as well as the secretion of HBeAg level in HepG2.2.15cell supernatant. And miR146a mimics showed the opposite influence on HBeAg secretion in HepG2.2.15cells. In vivo, mice co-injected with pAAV-HBV1.2and pcDNA3-miR146a plasmids by hydrodynamic tail vein injection showed higher level of serum HBsAg and HBeAg than that in pAAV-HBV1.2+pcDNA3treated mice.
Bioinformatic analysis suggested that miR146a could hardly bind to HBV RNA directly.
4. miR146a inhibited activation of type I interferon signaling
miR146a overexpression by miR146a mimics or expression vector decreased the sensitivity of HepG2cells to IFN-α stimulation, showing lower mRNA and protein level of several ISGs, including MxA, ISG15, OAS-1and IFIT3. On contrary, After HepG2.2.15cells were treated with miR146a inhibitor for24hours, the mRNA and protein levels of these genes were significantly increased in response to IFN-a.
5. miR146a induced by HBV infection suppressed STAT1expression in hepatoma cells
The basic level of STAT1was lower in HepG2.2.15cells than that in HepG2 cells and luciferase assay demonstrated STAT1was regulated at a post transcriptional level. The protein level of STAT1was up-regulated alter miR146a silenced in HepG2.2.15cells by treating with miR146a inhibitors, whereas down-regulated in HepG2cells treated with miR146a mimics with unchanged mRNA level. Reporter gene assay showed that miR146a exerted down-regulatory effect on STAT1via a sequence-dependent manner related to3"-UTR of STAT1mRNA in hepatoma cells. Additionally, STAT1protein level in HBV-positive samples of HCC patients was significantly lower than that in HBV-negative sample, implying the possibility of miR146a in suppressing STAT1expression.
6. Human IFIT3is a potential target of miR146a
IFIT3s R1G-I and RNase L were predicted to be candidate target of miR146a by four algorithms. Luciferase gene assay showed that miR146a also exerted down-regulatory effect on IFIT3translation by binding to3'-UTR of IFff3mRNA via a sequence-dependent manner in hepatoma cells.
The basic level of1F1T3was also lower in HepG2.2.15cells than that in HepG2cells. Luciferase assay demonstrated that IF1T3translation could be partly recovered by miR146a inhibition in HepG2.2.15cells.
7. miR146a repressed the induction of type Ⅰ IFN by5'ppp-RNA stimulation in hepatoma cells
miR146a pre-silencing increased the induction fold of type I IFN after5"ppp-RNA transfection in HepG2.2.15cells, and IFIT3over-expression could achieve similar phenomenon.
Luciferase assay demonstrated that TRAF6. another miR146a target and key adaptor for R1G-I signaling, was also regulated at translational level by miR146a via a "mis-match" paired interaction in hepatoma cells.
Furthermore, IFIT3may inhibit HBV transcription directly either in an unknown pathway.
Conclusion
1. miR146a expression in HBV-carried hepatoma cell line, HepG2.2.15, was higher than that in HepG2cells, which due to enhanced activity of miR146a promoter.
2. miR146a promoted HBV life cycle both in vitro and in vivo, which was independent on the direct interaction between miR146a and HBV RNA.
3. miR146a negatively regulated type I IFN response by targeting STAT1in hepatoma cells, and silencing miR146a would increase anti-HBV effect of type I IFN both in vitro and in vivo.
4. Human IFIT3was a new target of miR146a, and could probably inhibit HBV directly.
5. miR146a also negatively regulated5'ppp-RNA-dependent type I IFN induction in hepatoma cells by targeting IFIT3and TRAF6. Also, inhibition of miR146a would recover impaired intracellular innate immunity and augment type I IFN expression in hepatoma cells.
Our findings demonstrated that miR146a up-regulation was one of the important epigenetic characteristics of hepatocyte during HBV infection; miR146a would attenuate type I IFN production and play roles via targeting essential signaling factors, including STAT1, IFIT3and TRAF6, which facilitated HBV life cycle; silencing miR146a could reverse HBV-induced immune inhibitory status in liver and expedite HBV clearance, which may be a promising strategy to design new drugs that can break "IFN-resistance".
乙型肝炎病毒的感染(CHB)是严重威胁我国人类健康的重大传染病,其迁延不愈的特点不仅给患者和社会带来沉重的负担,也对公共卫生产生巨大隐患。Ⅰ型干扰素是目前治疗CHB和相关疾病最常用的药物,然而仅对约1/3的患者有效,且容易产生耐药性,大大限制了其临床治疗效果,这种现象在临床上称为“干扰素抵抗”。产生这一现象的主要原因是,HBV感染后机体免疫平衡被破坏,造成免疫功能低下或者紊乱。
研究表明,HBV病毒可以在分子、细胞、器官和个体等多个层次上对机体抗病毒天然免疫和获得性免疫产生抑制和耐受,这是造成HBV感染容易慢性化的分子免疫基础。肝脏免疫学认为,抑制进而完全清除HBV离不开机体抗病毒免疫反应的激活,而正常的免疫功能也是药物治疗取得理想疗效不可或缺的因素。然而,目前以针对“干扰素抵抗”为代表的HBV负向免疫调节作用的研究相对滞后,限制了抗HBV免疫治疗药物的开发。
miRNA是近年来发现的一类主要在转录后水平上对基因表达进行调节的短片段非编码RNA。在哺乳动物细胞内,miRNA可通过序列特异性的不完全互补作用抑制靶基因mRNA的翻译。研究发现,miRNA在抗病毒天然免疫反应中,既可以成为典型的基因调控因子,也能够成为直接靶向转录物而发挥干扰作用的免疫效应分子。同时,在HBV的致病过程中,宿主miRNA同样参与了病毒复制、转录、包装等自身生命活动,通过调控宿主基因参与了炎症反应、免疫损伤、促进自身潜伏、诱发继发性疾病乃至肝癌,成为研究HBV与宿主相互作用的重要窗口。
本研究的目的是通过对HBV存在条件下肝细胞内miRNA表达水平的检测和筛选,寻找可能参与HBV诱导胞内天然免疫抑制,特别是“干扰素抵抗”的miRNA。通过干预miRNA的表达水平,分析相关miRNA与HBV感染之间的关系,并研究其是否参与了HBV造成的肝细胞胞内抗病毒免疫应答信号的调节,探究其与“干扰素抵抗”是否相关。利用生物信息学和分子生物学的手段鉴定在这一过程中miRNA发挥作用的关键靶分子,并寻找HBV感染与相关分子之间动态水平的关联。最后,利用HBV复制性小鼠模型，研究靶向于预特定miRNA能否改变HBV的体内感染过程,为开发基于miRNA的新型抗HBV免疫药物提供理论和实验依据。
研究方法：
首先,以整合了HBV全基因组的HepG2.2.15细胞和无HBV存在的对照细胞HepG2为模型,利用miRNA特异性RT和qPCR两种技术,我们定量分析了细胞内与免疫相关的15种niRNA的丰度,观察了HBV感染对肝细胞内免疫相关miRNA的调节作用,发现miR146家族的两个成员,特别足miR146a在HBV整合的肝细胞系内的表达明显高于对照细胞,并通过核糖核酸酶保护实验确认了miR146a在两种细胞系内的绝对水平;在HepG2.2.15细胞和HepG2细胞中,利用特异性引物并通过RT-qPCR定量分析：niR146a初级转录物和次级前体的表达,并采用荧光素酶报告基因实验分析两种细胞中miR146a启动子的活性。
其次,通过基因工程的方法,我们构建了HBV全基因组和四种主要蛋白元件的真核表达载体，并分别将其转染到HepG2细胞中,利用RT-qPCR技术检测了HBV基因组及其蛋白组分的存在对肝细胞内miR146a表达的影响。同时,我们利用HBV全基因组表达载体尾静脉高压注射的方法建立了HBV复制性小鼠模型,通过灌流法分离小鼠肝实质细胞,接着用RT-qPCR法检测了其中miR146a的表达,同时用ELISA法检测了小鼠血清中HBsAg的水平,分析了二者之间的关系,并再次确认了HBV存在对(?)niR146a表达水平的影响。
接下来,我们将化学合成的miR146a抑制物转染入高表达miR146a的HepG2.2.15细胞,利用RT-qPCR和ELISA检测其对HBV转录和抗原表达的影响。与此同时,将miR146a模拟物或其过表达载体与HBV全基因组载体联合转染低表达miR146a的HepG2细胞,检测了其对HBV转求和抗原表达的影响。为进行体内研究,我们构建了靶向miR146a家族成员的niR146a sponge沉默载体与miR146a过表达载体pcDNA3-miR146a,然后分别与HBV全基因组载体配伍,联合高压注射小鼠,ELISA检测小鼠血清HBsAg和HBeAg的含量,并用免疫组织化学分析小鼠肝组织HBsAg和HBcAg的表达,探索了体内靶向干预肝细胞内miR146a的表达水平后对HBV体内感染周期的影响。
在分析miR146a对Ⅰ型干扰素信号转导通路影响研究中,首先对miR146a进行干预,然后再进行Ⅰ型干扰素处理,利用RT-qPCR和Western Blotting技术比较干预前后细胞中MxA、ISG15、OAS-1、IFIT3等主要抗病毒基因的转录和蛋白表达水平；在先期相关研究的基础上,比较了两种细胞miR146a靶基因-转录因子STAT1的表达：构建了包含miR146a识别区域的UTR的荧光素酶报告基因载体,并用于评价靶基因翻译活性。采用荧光素酶报告基因实验,我们分析了STAT1发生差异表达的分子基础；在此基础上,利用miR146a过表达和沉默策略,检测了肝细胞中miR146a调控STAT1表达的机制,并通过改变肝细胞内miR146a的水平,分析了miR146a对STAT1翻译活性的调节作用。通过对HBV阳性和阴性肝细胞癌临床标本的分析,我们进一步确认了STAT1的表达与HBV感染之间存在相关性。
最后,通过生物信息学手段,我们分析了可能参与抗HBV天然免疫应答过程的17种干扰素诱导基因与miR146a之间的关系,其中有三个基因被预测可能成为miR146a的直接靶点。我们构建了针对IFIT3的UTR区荧光素酶报告基因载体,证实了IFIT3是miR146a的一个新的直接靶点。在两种模型细胞中,我们比较了该基因和已知miR146a靶基因TRAF6翻译活性与miR146a表达水平之间的关系。基于已知的IFIT3和TRAF6生物学功能,我们又通过体外转录合成了RLR信号通路的配基5'ppp-RNA(简称3p-RNA)。首先通过预先沉默(?)niR146a的方法,利用RT-qPCR分析了肝细胞对3p-RNA免疫刺激作用的敏感性；接着进行IFIT3过表达实验,利用RT-qPCR和ELISA方法,分析其对肝细胞3p-RNA刺激的正向作用和直接抗HBV活性；通过荧光素酶报告基因实验分析了miR146a对肝细胞内TRAF6翻译的调节作用。
研究结果：
1.肝癌细胞内miR146a的高表达与HBV感染密切相关
HBV感染造成了多种免疫相关miRNA的表达水平发生变化,其中以miR146家族最为明显；在整合有HBV的肝癌细胞系HepG2.2.15和PLC/PRF/5中,miR146a的表达水平明显高于无HBV存在的阴性细胞系；HepG2.2.15中miR146a初级转录本和颈环前体的表达也明显高于对照细胞HepG2; HBV感染引起miR146a上调依赖于NF-κB。
2.HBV感染引起了肝细胞内miR146a表达水平的升高
我们成功建立HBV完整基因组和四个主要蛋白成分X蛋白、核心蛋白、表面抗原小蛋白和HBV的DNA多聚酶的真核表达载体；瞬时转染HBV全基因组载体后,HepG2细胞中(?)niR146a的表达水平明显提高,并且HBV基因组表达载体稳定化的HepG2细胞地也有相同的现象;HBV的四个主要的蛋白产物对上调肝细胞miR146a的表达有共同但不均等的贡献;在HBV复制性小鼠中,小鼠原代肝实质细胞内miR146a的表达与血清HBsAg的表达水平呈一定程度的正相关。
3. miR146a可以促进HBV的复制和转录
流式细胞术结果证实,荧光标记的miR146a模拟物和阻遏物可以有效的导入靶细胞。定量PCR检测结果发现,它们均可以有效地改变肝细胞内源性miRNA的表达水平;我们自行设计结构的miR146a sponge载体也可以有效地沉默HepG2.2.15细胞内miR146a的水平：
在上述实验系统的支撑下,在HepG2细胞模型中,进行模拟物或者质粒载体过表达miR146a,可以促进瞬转HBV的转录,而通过阻遏物或者质粒载体沉默niR146则可以抑制HBV的转录和翻译。通过尾静脉高压注射的的方式向小鼠肝脏输送miR146a过表达载体,可以升高小鼠血清HBsAg和HBeAg抗原的表达水平和肝细胞内HBsAg的表达,而输送靶向miR146a的sponge沉默载体后,小鼠血清HBsAg和HBeAg抗原的含量明显降低,且肝细胞内HBsAg的表达亦显著下降。同时,抑制miR146a的水平不影响(?)AepG2.2.15细胞的增殖;miR146b模拟物和HBV全基因组载体共转染HepG2细胞并不能明显影响HBV的转录,miR146b模拟物转染也未影响HepG2.2.15细胞分泌HBV抗原的能力。
基于RNA二级结构稳定性和保守性的生物信息学预测认为,miR146a不会直接靶向HBV RNA。
4. miR146a可以抑制肝细胞内Ⅰ型干扰素的信号转导
将miR146a模拟物转染入HepG2细胞后,可以抑制细胞对后续IFN-α刺激的应答,表现为ISG15、MxA、OAS-1、IFIT3等干扰素诱导基因转录的减弱和蛋白合成的下降。在miR146a稳定过表达的HepG2细胞上,也可以观察到类似的现象;而将miR146a阻遏物转染入HepG2.2.15细胞后,则会产生相反的作用,表现为ISG15、MxA、OAS-1、IFIT3等干扰素诱导基因转录的增强和蛋白合成的增加。
5.HBV感染诱导的miR146a的高表达抑制了肝细胞内STAT1的水平
HepG2.2.15细胞中STAT1的基础表达水平低于HepG2;与临床HBV阴性肝细胞癌细胞相比,HBV阳性肝细胞癌细胞中STAT1的水平明显降低；将rniR146a阻遏物转染入HepG2.2.15细胞后,可以明显上调STAT1蛋白的表达,而不影响胞内mRNA的水平,而转染miR146a的模拟物则会产生相反的结果。报告基因检测显示,HepG2.2.15细胞中STAT1的基础翻译水平明显低于HepG2细胞,这可能是其蛋白表达低于HepG2勺原因之一；提高HepG2细胞内miR146a勺水平可以序列依赖性地抑制STAT1的翻译,而抑制HepG2.215细胞内miR146a的水平则能够序列依赖性地促进STAT1的翻译。
6.人IFIT3基因是miR146a的一个新的直接靶基因
利用四种生物信息学软件,我们对17种抗HBV宿主基因与miR146a关系进行分析,其中有三个基因可能成为rniR146a的直接靶基因,分别是IFIT3、RIG-I和RNase L:针对IFIT3的荧光素酶报告实验证实,IFIT3是：miR146a的直接靶基因。
HepG2.2.15细胞中IFIT3基因的基础翻译水平低于HepG2细胞,且与STAT1的转录后调节相似,HepG2.2.15细胞中IFIT3基因的翻译水平受到胞内miR146a的抑制,向HepG2.2.15细胞内转染miR146a抑制物能够序列依赖性地促进IFIT3的翻译。
7. miR146a抑制了肝细胞内5'ppp-RNA刺激对I型干扰素的诱导作用
向HepG2.2.15细胞内转染miR146a抑制物后,5'ppp-RNA对HepG2.2.15细胞的免疫刺激作用显著增强,促进内源性Ⅰ型干扰素的诱导表达;在HepG2.2.15细胞内过表达IFIT3后,同样可以促进5'ppp-RNA引起的Ⅰ型干扰素的诱生。
另一种参与天然免疫应答并诱导Ⅰ型干扰素分子TRAF6,也受到miR-146a的转录后抑制,其在HepG2.2.15细胞中的翻译明显低于HepG2细胞。这可能是miR146a通过转录后抑制方式参与HBV感染肝细胞后内源性Ⅰ型干扰素诱导的新的分子机制。
将IFIT3过表达载体转入HepG2.2.15后,胞内HBV mRNA的转求水平显著受到明显抑制,提示IFIT3可能具有直接的抗HBV活性。
结论：
1.HBV基因组织的肝细胞系HepG2.2.15中miR146a的表达水平显著高于(?)AepG2细胞,主要分子机制是转求活性增强。
2. miR146a的存在促进了HBV的生命活动和自身存续,生物信息学预测显示这种左右很可能不是通过直接与病毒RNA相互作用实现的。
3. miR146a通过对靶基因STAT1翻译的抑制,负调了肝细胞对Ⅰ型干扰素诱导的抗病毒反应的应答能力,而抑制miR146a的功能则可以提高Ⅰ型干扰素体内外抗病毒效果。
4.人IFIT3是miR146a的直接的靶基因之一,且可能具有直接的抗HBV作用。
5. miR146a通过对靶基因IFIT3和TRAF6翻译的抑制,负调5'ppp-RNA对肝细胞内源Ⅰ型干扰素的诱导,而抑制miR146a的功能可以增强5'ppp-RNA的刺激作用,促进Ⅰ型干扰索的转录和表达。
意义：
miR146a高表达是HBV感染改变肝细胞表现遗传状态的主要表现之一;它通过调控多种与内源性Ⅰ型干扰素诱导和激活有关的信号分子,对胞内抗病毒天然免疫产生钝化作用,而有利于HBV的存续；靶向沉默miR146a则能够打破HBV感染造成的肝脏免疫抑制状态,促进HBV的清除,而针对miR-146a的阻遏分子则有望开发成为可以有效逆转“干扰索抵抗”,提高干扰素临床抗HBV疗效的免疫增强药物。
Object:
HBV causes acute and chronic hepatitis and results in life-long infected, presenting a significant threat to public health. Interferon-α (IFN-α) has been used in the treatment of chronic HBV-related diseases for many years, but only30%of the patients show positive response to IFN-α, and many patients become resistant to IFN-α. This phenomenon is called "IFN-α resistance", which impairs IFN-α anti-HBV effect severely and therefore contributes to the damage, defect and disorder of host anti-viral immunity during HBV infection
It is believed that HBV can impair anti-viral innate and adaptive immune response at molecular, cellular, organic and holistic levels, which lead to chronic infection to a certain extent. In the view of Liver immunology, activation of host anti-viral immunity is essential for HBV inhibition and clearance, and at the same time, is also required for desirable treatment effectiveness. Until now, research on negative immune regulation of HBV including "IFN resistance" is still delayed, which restricts the progress to develop new anti-HBV drugs.
microRNAs (miRNAs), a big family of small non-coding RNAs, were found to serve the pivotal function of regulating gene expression at posttranscriptional level in recent years, which always inhibit mRNA translation by sequence-dependent "mis-match" in mammalian cells. It is reported that, both virus-and host-encoded miRNAs would be important regulators or executors involved in viral pathogenesis and immunity. During the whole period of HBV infection, miRNAs not only regulate replication, transcription and package itself, but also participate in liver inflammation, immune injury, latency, secondary cases such as hepatocellular carcinoma, which become a new viewpoint to explore human-HBV interaction.
This research is designed to explore the role of miRNA in HBV-induced inhibition of innate immunity, especially "IFN resistance" by screening the expression level of immune-related miRNA between HBV-carried hepatoma cell and negative ones. We used many miRNA interference tools to analyze the interaction between HBV infection and miRNA changes, to determine whether miRNA was involved in the inhibition of intracellular innate immune pathway including "IFN resistance" after HBV infection. Using bioinformatic and molecular tools to look for key factors, we then identify the correlation between HBV and immune moleculars. At last, miRNA-targeting technology was employed to determine whether miRNA changes can favor anti-HBV response in HBV-carried mouse model.
Methods:
15immune-related miRNAs, including miR21, miR26a/b, miR106, miR132, miR146a/b, miR147, miR155, miR181, miR196a, miR296and miR448, were examined by stem-loop special miRNA qRT-PCR assay in HBV-carried HepG2.2.15cells and their counterpart HBV-negative HepG2cells. The miR146a expression levels were further confirmed by ribonuclease protection assay, also miR146a primary transcript (nearly200bp) and its stem-loop precursor (-100bp) were examined using special primers. Luciferase assay was used to test miRNA promoter activity.
LMP-HBV1.2(a new HBV genome expression vector based on pAAV-HBV1.2) and polymerase (HBp), HBV core protein (HBc), HBx and Small HBV surface protein expression vectors based on pEGFP-N1were transfected into HepG2cells. HBV-carried BALB/c mice were established by hydrodynamic tail vein injection with pAAV-HBV1.2. After2weeks, serum was harvested, and HBsAg levels were detected by ELISA. Then primary mouse hepatocytes were isolated by collagenase perfusion. The effect of miR146a on HBV infection was determined by transferring HBV plasmid, and miR146a mimics, inhibitors or expression/silencing vectors in human hepatoma cells (HepG2, and HepG2.2.15) and in mouse by hydrodynamic tail vein injection. The change of miR146a was confirmed by stem-loop special miRNA qRT-PCR assay.
In hepatoma cells, the effect of miR146a on IFN-a response was measured by RT-qPCR and Western Blotting, to test the transcription and translation including MxA、ISG15、OAS-1、IFIT3respectively. To explorer the mechanism of miR146a on STAT1expression, luciferase reporter vector containing STAT13'-UTR and its seed specific mutant control were constructed and cotransfected with miR146a mimics or inhibitors into hepatoma cells. The direct targeting effect of miR146a was validated by Luciferase reporter assay. STAT1protein level was also compared among HBV-positive and negative clinical samples.
Bioinformatic analysis was carried out to look for new miR146a direct targets within17anti-viral factors, and3'-UTR of candidate targets were cloned to luciferase reporter vector for experiment validation. Luciferase assay was used to identify the exact target, IFIT3. Then, IFIT3and TRAF63'-UTR luciferase constructs and its seed specific mutant control were cotransfected into hepatoma cells to evaluate translation efficiency. The impact of miR146a on the translation of these two genes at posttranscriptional level was also estimated by luciferase assay. To determine the promotion of miR146a inhibition to type I interferon induction,5'ppp-RNA was transcripted in vitro, and then introduced into IIepG2.2.15cell after miR146a silencing by miR146a sponge vector. RT-qPCR of type I interferon was utilized to quantitate the increasing fold.
Results:
1. miR146a level was closely correlated to HBV infection in human hepatoma cells
Two members of miR146family, miR146a and miR146b, showed a significant up-regulation in HepG2.2.15cells among15chosen miRNAs, especially miR146a. The expression of miR146a primary transcript and its stem-loop precursor were also higher in MepG2.2.15cells than that in HepG2cells. Promoter activity assay suggested that the transcription of miR146a was enhanced due to higher NF-κB activity.
2. HBV infection induced miR146a expression both in vitro and in vivo
Stem-loop special miRNA qRT-PCR assay showed that miR146a was increased both in HepG2cells transfected with pAAV-HBV1.2transiently and in cells stabilizated by HBV genome vector. Four HBV main proteins all seemed to contribute to up-regulaion of miR146a when they were transfected into HepG2cells individually. The serum HBsAg level revealed a positive relationship with mature miR146a level in hepatocytes from HBV-carried mice.
3. miR146a accelerated HBV replication
Chemical synthesized miR146a mimics and inhibitors could interference endogenous level of miR146a effectively, as well as miR146a expression or silencing vector, pcDNA3-miR146a and psiRNE-PGK-miR146a-sponge.
miR146a over-expression would ameliorate HBx and HBs/p mRNA transcription in HepG2cells. On the other hand, miR146a inhibitors down-regulated HBx and HBs/p mRNA levels in HepG2.2.15cells, as well as the secretion of HBeAg level in HepG2.2.15cell supernatant. And miR146a mimics showed the opposite influence on HBeAg secretion in HepG2.2.15cells. In vivo, mice co-injected with pAAV-HBV1.2and pcDNA3-miR146a plasmids by hydrodynamic tail vein injection showed higher level of serum HBsAg and HBeAg than that in pAAV-HBV1.2+pcDNA3treated mice.
Bioinformatic analysis suggested that miR146a could hardly bind to HBV RNA directly.
4. miR146a inhibited activation of type I interferon signaling
miR146a overexpression by miR146a mimics or expression vector decreased the sensitivity of HepG2cells to IFN-α stimulation, showing lower mRNA and protein level of several ISGs, including MxA, ISG15, OAS-1and IFIT3. On contrary, After HepG2.2.15cells were treated with miR146a inhibitor for24hours, the mRNA and protein levels of these genes were significantly increased in response to IFN-a.
5. miR146a induced by HBV infection suppressed STAT1expression in hepatoma cells
The basic level of STAT1was lower in HepG2.2.15cells than that in HepG2 cells and luciferase assay demonstrated STAT1was regulated at a post transcriptional level. The protein level of STAT1was up-regulated alter miR146a silenced in HepG2.2.15cells by treating with miR146a inhibitors, whereas down-regulated in HepG2cells treated with miR146a mimics with unchanged mRNA level. Reporter gene assay showed that miR146a exerted down-regulatory effect on STAT1via a sequence-dependent manner related to3"-UTR of STAT1mRNA in hepatoma cells. Additionally, STAT1protein level in HBV-positive samples of HCC patients was significantly lower than that in HBV-negative sample, implying the possibility of miR146a in suppressing STAT1expression.
6. Human IFIT3is a potential target of miR146a
IFIT3s R1G-I and RNase L were predicted to be candidate target of miR146a by four algorithms. Luciferase gene assay showed that miR146a also exerted down-regulatory effect on IFIT3translation by binding to3'-UTR of IFff3mRNA via a sequence-dependent manner in hepatoma cells.
The basic level of1F1T3was also lower in HepG2.2.15cells than that in HepG2cells. Luciferase assay demonstrated that IF1T3translation could be partly recovered by miR146a inhibition in HepG2.2.15cells.
7. miR146a repressed the induction of type Ⅰ IFN by5'ppp-RNA stimulation in hepatoma cells
miR146a pre-silencing increased the induction fold of type I IFN after5"ppp-RNA transfection in HepG2.2.15cells, and IFIT3over-expression could achieve similar phenomenon.
Luciferase assay demonstrated that TRAF6. another miR146a target and key adaptor for R1G-I signaling, was also regulated at translational level by miR146a via a "mis-match" paired interaction in hepatoma cells.
Furthermore, IFIT3may inhibit HBV transcription directly either in an unknown pathway.
Conclusion
1. miR146a expression in HBV-carried hepatoma cell line, HepG2.2.15, was higher than that in HepG2cells, which due to enhanced activity of miR146a promoter.
2. miR146a promoted HBV life cycle both in vitro and in vivo, which was independent on the direct interaction between miR146a and HBV RNA.
3. miR146a negatively regulated type I IFN response by targeting STAT1in hepatoma cells, and silencing miR146a would increase anti-HBV effect of type I IFN both in vitro and in vivo.
4. Human IFIT3was a new target of miR146a, and could probably inhibit HBV directly.
5. miR146a also negatively regulated5'ppp-RNA-dependent type I IFN induction in hepatoma cells by targeting IFIT3and TRAF6. Also, inhibition of miR146a would recover impaired intracellular innate immunity and augment type I IFN expression in hepatoma cells.
Our findings demonstrated that miR146a up-regulation was one of the important epigenetic characteristics of hepatocyte during HBV infection; miR146a would attenuate type I IFN production and play roles via targeting essential signaling factors, including STAT1, IFIT3and TRAF6, which facilitated HBV life cycle; silencing miR146a could reverse HBV-induced immune inhibitory status in liver and expedite HBV clearance, which may be a promising strategy to design new drugs that can break "IFN-resistance".
引文
[1]Bruss V. Hepatitis B virus morphogenesis. World J Gastroenterol 2007; 13:65-73.
[2]Jazayeri SM, Alavian SM, Carman WF. Hepatitis B virus:origin and evolution. Journal of Viral Hepatitis 2010; 17:229-235.
[3]Chu CM, Liaw YF. Hepatitis B surface antigen seroclearance during chronic HBV infection. Antivir Ther 2010;15:133-143.
[4]Mizukoshi E, Sidney J, Livingston B, Ghany M, Hoofnagle JH, Sette A, et al. Cellular immune responses to the hepatitis B virus polymerase. J Immunol 2004;173:5863-5871.
[5]Tang H, Oishi N, Kaneko S, Murakami S. Molecular functions and biological roles of hepatitis B virus x protein. Cancer Sci 2006;97:977-983.
[6]Guo H, Mao R, Block TM, Guo JT. Production and function of the cytoplasmic deproteinized relaxed circular DNA of hepadnaviruses. J Virol 2010;84:387-396.
[7]Grimm D, Thimme R, Blum HE. HBV life cycle and novel drug targets. Hepatol Int 2011;5:644-653.
[8]Sommer G, Heise T. Posttranscriptional control of HBV gene expression. Front Biosci 2008;13:5533-5547.
[9]Z B-O. Innate immunity:cells, receptors, and signaling pathways. Arch Immunol Ther Exp (Warsz) 2005;53.
[10]Turvey SE, Broide DH. Innate immunity. Journal of Allergy and Clinical Immunology 2010;125:S24-S32.
[11]Gao B, Jeong WI, Tian Z. Liver:An organ with predominant innate immunity. Hepatology 2008;47:729-736.
[12]Li Z, Diehl AM. Innate immunity in the liver. Curr Opin Gastroenterol 2003; 19:565-571.
[13]Parker GA, Picut CA. Immune Functioning in Non lymphoid Organs:The Liver. Toxicologic Pathology 2011;40:237-247.
[14]Seki S, Habu Y, Kawamura T, Takeda K, Dobashi H, Ohkawa T, et al. The liver as a crucial organ in the first line of host defense:the roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+T cells in T helper 1 immune responses. Immunol Rev 2000:174:35-46.
[15]Kadowaki N, Liu YJ. Natural type I interferon-producing cells as a link between innate and adaptive immunity. Hum Immunol 2002;63:1126-1132.
[16]Chen Y, Wei H, Gao B, Hu Z, Zheng S, Tian Z. Activation and function of hepatic NK cells in hepatitis B infection:an underinvestigated innate immune response. Journal of Viral Hepatitis 2005;12:38-45.
[17]Baron JL, Gardiner L, Nishimura S, Shinkai K, Locksley R, Ganem D. Activation of a nonclassical NKT cell subset in a transgenic mouse model of hepatitis B virus infection. Immunity 2002; 16:583-594.
[18]Boehme KW, Compton T. Innate Sensing of Viruses by Toll-Like Receptors. Journal of Virology 2004;78:7867-7873.
[19]于新蕾.,吕丽萍.,闫舫.,张艳宇.,周锡鹏.,马平.,et al.乙型肝炎病毒攻击肝细胞对β干扰素表达的影响.军事医学2011：35：587-592.
[20]Bonjardim CA, Ferreira PCP, Kroon EG. Interferons:Signaling, antiviral and viral evasion. Immunology Letters 2009; 122:1-11.
[21]Chen J, Wang XM, Wu XJ, Wang Y, Zhao H, Shen B, et al. Intrahepatic levels of PD-1/PD-L correlate with liver inflammation in chronic hepatitis B. Inflamm Res 2011:60:47-53.
[22]石翠翠.,谢青.,赵钢德.,黄谦.,项晓刚.,龚邦东.,et al.肝细胞表面PD-L1的表达及干扰素和HBV对其表达的调节.肝脏2010;15：23-25.
[23]Kakimi K, Lane TE, Wieland S, Asensio VC, Campbell IL, Chisari FV, et al. Blocking chemokine responsive to gamma-2/interferon (IFN)-gamma inducible protein and monokine induced by IFN-gamma activity in vivo reduces the pathogenetic but not the antiviral potential of hepatitis B virus-specific cytotoxic T lymphocytes. J Exp Med 2001; 194:1755-1766.
[24]Kimura K, Kakimi K, Wieland S, Guidotti LG, Chisari FV. Activated intrahepatic antigen-presenting cells inhibit hepatitis B virus replication in the liver of transgenic mice. J Immunol 2002:169:5188-5195.
[25]Kimura K, Kakimi K, Wieland S, Guidotti LG, Chisari FV. Interleukin-18 inhibits hepatitis B virus replication in the livers of transgenic mice. J Virol 2002;76:10702-10707.
[26]Kakimi K, Guidotti LG, Koezuka Y, Chisari FV. Natural killer T cell activation inhibits hepatitis B virus replication in vivo. J Exp Med 2000; 192:921-930.
[27]Smith PL, Lombardi G, Foster GR. Type I interferons and the innate immune response--more than just antiviral cytokines. Mol Immunol 2005:42:869-877.
[28]Touzot M, Soumelis V, Asselah T. A dive into the complexity of type I interferon antiviral functions. Journal of Hepatology 2012;56:726-728.
[29]Peltekian C, Gordien E, Garreau F, Meas-Yedid V, Soussan P, Willams V, et al. Human MxA protein participates to the interferon-related inhibition of hepatitis B virus replication in female transgenic mice.J Hepatol 2005:43:965-972.
[30]Chai Y, Huang H-L, Hu D-J, Luo X, Tao Q-S, Zhang X-L, et al. IL-29 and IFN-α regulate the expression of MxA,2',5'-OAS and PKR genes in association with the activation of Raf-MEK-ERK and PI3K-AKT signal pathways in HepG2.2.15 cells. Molecular Biology Reports 2010;38:139-143.
[31]Fensterl V, Sen GC. The 1SG56/IFIT1 gene family. J Interferon Cytokine Res 2011:31:71-78.
[32]Turelli P, Mangeat B, Jost S, Vianin S, Trono D. Inhibition of hepatitis B virus replication by APOBEC3G. Science 2004:303:1829.
[33]Robek MD, Wieland SF, Chisari FV. Inhibition of hepatitis B virus replication by interferon requires proteasome activity. J Virol 2002:76:3570-3574.
[34]Alsharifi M, Mullbacher A, Regner M. Interferon type Ⅰ responses in primary and secondary infections. Immunol Cell Biol 2008:86:239-245.
[35]梁蔚芳.聚乙二醇化干扰素α-2a与干扰素α-2a 在体内和体外的抗乙型肝炎病毒活性研究[博士学位论文]：第一军医大学;2007.
[36]曾伟强.派罗欣联合阿德福 韦酯治疗乙型肝炎效果观察.中国热带医学 2006,6;822-823.
[37]Gough DJ, Levy DE, Johnstone RW, Clarke CJ. IFNy signaling-Does it mean JAK-STAT? Cytokine & Growth Factor Reviews 2008:19:383-394.
[38]张夏华.,吴广通.,周萍.泛昔洛韦联合干扰素γ 抗HBV的临床疗效观察.中国现代应用药学杂志 2005;22：262-264.
[39]Guidotti LG, Chisari FV. Immunobiology and pathogenesis of viral hepatitis. Annu Rev Pathol 2006:1:23-61.
[40]Chisari FV. Hepatitis B virus transgenic mice:models of viral immunobiology and pathogenesis. CurrTop Microbiol Immunol 1996;206:149-173.
[41]Wieland S, Thimme R, Purcell RH, Chisari FV. Genomic analysis of the host response to hepatitis B virus infection. Proc Natl Acad Sci U S A 2004; 101:6669-6674.
[42]Raimondo G, Pollicino T, Cacciola I, Squadrito G. Occult hepatitis B virus infection. J Hepatol 2007;46:160-170.
[43]Samal J, Kandpal M, Vivekanandan P. Molecular mechanisms underlying occult hepatitis B virus infection. Clin Microbiol Rev 2012;25:142-163.
[44]Liaw YF. Impact of hepatitis B therapy on the long-term outcome of liver disease. Liver Int 2011;31 Suppl 1:117-121.
[45]Ghany MG, Doo EC. Antiviral resistance and hepatitis B therapy. Hepatology 2009;49:S174-184.
[46]Tillmann HL. Antiviral therapy and resistance with hepatitis B virus infection. World J Gastroenterol 2007; 13:125-140.
[47]Zoulim F. Antiviral therapy of chronic hepatitis B:can we clear the virus and prevent drug resistance? Antivir Chem Chemother 2004;15:299-305.
[48]Fournier C, Zoulim F. Antiviral therapy of chronic hepatitis B:prevention of drug resistance. Clin Liver Dis 2007; 11:869-892, ix.
[49]Genin P, Lin R, Hiscott J, Civas A. Differential regulation of human interferon A gene expression by interferon regulatory factors 3 and 7. Mol Cell Biol 2009;29:3435-3450.
[50]Lei CQ, Zhong B, Zhang Y, Zhang J, Wang S, Shu HB. Glycogen synthase kinase 3beta regulates IRF3 transcription factor-mediated antiviral response via activation of the kinase TBK1. Immunity 2010;33:878-889.
[51]Levy DE, Marie I, Smith E, Prakash A. Enhancement and diversification of IFN induction by lRF-7-mediated positive feedback. J Interferon Cytokine Res 2002;22:87-93.
[52]Heydtmann M. Macrophages in hepatitis B and hepatitis C virus infections. J Virol 2009;83:2796-2802.
[53]Cui GY, Diao HY. Recognition of HBV antigens and HBV DNA by dendritic cells. Hepatobiliary Pancreat Dis Int 2010;9:584-592.
[54]钱志平.,郑建铭.,朱梦琪.,王新宇.,陈明泉.,et al.乙型肝炎病毒抑制巨噬细胞TLR3、Mda-5和RIG-I表达.肝脏2010：15：425-428.
[55]安宝燕.,谢青.,下晖.,贾妮娜.,沈怀诚.,蔡伟.,et al.干扰素调节因子3在慢性乙型肝炎患者外周血树突状细胞中的表达及意义.世界华人消化杂志2008;16：1873-1879.
[56]Wieland SF, Vega RG, Muller R, Evans CF, Hiibush B, Guidotti LG, et al. Searching for interferon-induced genes that inhibit hepatitis B virus replication in transgenic mouse hepatocytes. J Virol 2003;77:1227-1236.
[57]胡静.干扰素诱生剂刺激肝细胞产生细胞因子研究[硕士学位论文]：重庆医科大学；2008.
[58]Han Q, Zhang C, Zhang J, Tian Z. Reversal of hepatitis B virus-induced immune tolerance by an immunostimulatory 3p-HBx-siRNAs in a retinoic acid inducible gene I-dependent manner. Hepatology 201];54:1179-1189.
[59]吴瑶瑶 陈HBcAg和TBK1对dsRNA诱导肝细胞表达IFN-β的调节作用[硕十学位论文]：浙江大学;2008.
[60]Wu J, Meng Z, Jiang M, Pei R, Trippler M, Broering R, et al. Hepatitis B virus suppresses toll-like receptor-mediated innate immune responses in murine parenchymal and nonparenchymal liver cells. Hepatology 2009;49:1132-1140.
[61]Yu S, Chen J, Wu M, Chen H, Kato N, Yuan Z. Hepatitis B virus polymerase inhibits RIG-I-and Toll-like receptor 3-mediated beta interferon induction in human hepatocytes through interference with interferon regulatory factor 3 activation and dampening of the interaction between TBK1/IKKepsilon and DDX3. J Gen Virol 2010;91:2080-2090.
[62]Wei C, Ni C, Song T, Liu Y, Yang X, Zheng Z, et al. The hepatitis B virus X protein disrupts innate immunity by downregulating mitochondrial antiviral signaling protein. J Immunol 2010;185:1158-1168.
[63]Kumar M, Jung SY, Hodgson AJ, Madden CR, Qin J, Slagle BL. Hepatitis B virus regulatory HBx protein binds to adaptor protein IPS-1 and inhibits the activation of beta interferon. J Virol 2011;85:987-995.
[64]Wang X, Li Y, Mao A, Li C, Tien P. Hepatitis B virus X protein suppresses virus-triggered IRF3 activation and IFN-beta induction by disrupting the VISA-associated complex. Cell Mol Immunol 2010;7:341-348.
[65]Jiang J, Tang H. Mechanism of inhibiting type I interferon induction by hepatitis B virus X protein. Protein Cell 2010; 1:1106-1117.
[66]Schindler C,Levy DE, Decker T. JAK-STAT signaling:from interferons to cytokines. J Biol Chem 2007;282:20059-20063.
[67]Schoggins JW, Rice CM. Interferon-stimulated genes and their antiviral effector functions. Current Opinion in Virology 2011; 1:519-5.25.
[68]Liu M, Hao Y, Ding H, Yang D, Lu M. Interleukin-10 is expressed in HepG2.2.15 cells and regulated by STAT1 pathway. J Huazhong Univ Sci Technolog Med Sci 2011;31:625-631.
[69]樊和斌.,郭亚兵.,王宝菊.,朱幼芙.,吴爱华.,侯金林.,et al.慢性乙型肝炎患者肝组织及外周血单核细胞内IFN-α/β受体的表达及其临床意义.南方医科大学学报2008;28：979-981.
[70]任苏平.乙型肝炎病毒对I型干扰素受体表达的影响[硕士学位论文]：浙江大学；2000.
[71]Chung Y-H. Hepatitis B virus X protein inhibits extracellular IFN-a-mediated signal transduction by downregulation of type I IFN receptor. International Journal of Molecular Medicine 2012.
[72]Guan SH, Lu M, Grunewald P, Roggendorf M, Gerken G, Schlaak JF. Interferon-alpha response in chronic hepatitis B-transfected HepG2.2.15 cells is partially restored by lamivudine treatment. World J Gastroenterol 2007; 13:228-235.
[73]熊炜.干扰素诱导的MyD88蛋白抑制乙型肝炎病毒复制的研究[博士学位论文]：复旦大学；2004.
[74]王迅.α干扰素诱导的细胞蛋白与乙型肝炎病毒相互作用关系的研究[博士学位论文]：复旦大学博士学位论文;2004.
[75]邬敏.乙型肝炎病毒抑制洳干扰素诱导的MyD88表达的机制研究[硕士学位论文]：复旦大学;2007.
[76]林珊珊.MyD88蛋白抑制乙型肝炎病毒基因复制的分子机制研究[博士学位论文]：复旦大学：2007.
[77]Wu M, Xu Y, Lin S, Zhang X, Xiang L, Yuan Z. Hepatitis B virus polymerase inhibits the interferon-inducible MyD88 promoter by blocking nuclear translocation of Statl. J Gen Virol 2007:88:3260-3269.
[78]于士颜.乙型肝炎病毒(HBV)拮抗IFN-a信号通路的初步研究[硕士学位论文]：复旦大学;2005.
[79]王林.乙型肝炎病毒Tp相关蛋白抗干扰素作用研究[博士学位论文]：福建医科大学;2009.
[80]管世鹤.,杨凯.,潘颖.乙型肝炎病毒抑制α干扰素诱导抗病毒蛋白表达的研究.安徽医科大学学报2011；46：324-327.
[81]管世鹤.,杨凯.,陆蒙吉.,卢银平.,杨东亮.乙型肝炎病毒及其抗原成分对干扰素信号传导途径分子和抗病毒蛋白表达的影响.中华肝脏病杂志2011;19：440-444.
[82]Siebler J, Protzer U, Wirtz S, Schuchmann M, Hohler T, Galle PR, et al. Overexpression of STAT-1 by adenoviral gene transfer does not inhibit hepatitis B virus replication. Eur J Gastroenterol Hepatol 2006;18:167-174.
[83]Zhang Q, Wang Y, Wei L, Jiang D, Wang JH, Rao HY, et al. Role of ISGF3 in modulating the anti-hepatitis B virus activity of interferon-alpha in vitro. J Gastroenterol Hepatol 2008;23:1747-1761.
[84]Christen V, Treves S, Duong FH, Heim MH. Activation of endoplasmic reticulum stress response by hepatitis viruses up-regulates protein phosphatase 2A. Hepatology 2007;46:558-565.
[85]Li J, Chen F, Zheng M, Zhu H, Zhao D, Liu W, et al. Inhibition of STAT1 methylation is involved in the resistance of hepatitis B virus to Interferon alpha. Antiviral Research 2010;85:463-469.
[86]程中乐.,管世鹤.,杨凯.,潘颖SOCS1对a干扰素调节乙肝病毒复制作用的影响.安徽医科大学学报2011：46：228-230.
[87]Su C, Hou Z, Zhang C, Tian Z, Zhang J. Ectopic expression of microRNA-155 enhances innate antiviral immunity against HBV infection in human hepatoma cells. Virol J 2011;8:354.
[88]Fish EN, Uddin S, Korkmaz M, Majchrzak B, Druker BJ, Platanias LC. Activation of a CrkL-stat5 signaling complex by type I interferons. J Biol Chem 1999;274:571-573.
[89]Uddin S, Lekmine F, Sharma N, Majchrzak B, Mayer I, Young PR, et al. The Racl/p38 mitogen-activated protein kinase pathway is required for interferon alpha-dependent transcriptional activation but not serine phosphorylation of Stat proteins. J Biol Chem 2000;275:27634-27640.
[90]Uddin S, Majchrzak B, Woodson J, Arunkumar P, Alsayed Y, Pine R, et al. Activation of the p38 mitogen-activated protein kinase by type I interferons. J Biol Chem 1999;274:30127-30131.
[91]Uddin S, Sassano A, Deb DK, Verma A, Majchrzak B, Rahman A, et al. Protein kinase C-delta (PKC-delta) is activated by type I interferons and mediates phosphorylation of Statl on serine 727. J Biol Chem 2002;277:14408-14416.
[92]Haller O, Kochs G. Human MxA protein:an interferon-induced dynamin-like GTPase with broad antiviral activity. J Interferon Cytokine Res 2011;31:79-87.
[93]Li N, Zhang L, Chen L, Feng W, Xu Y, Chen F, et al. MxA inhibits hepatitis B virus replication by interaction with core protein HBcAg. Hepatology 2012.
[94]Gordien E, Rosmorduc O, Peltekian C, Garreau F, Brechot C, Kremsdorf D. Inhibition of hepatitis B virus replication by the interferon-inducible MxA protein. J Virol 2001;75:2684-2691.
[95]杨凯.HBV及其抗原成分拮抗α干扰素抗病毒活性的初步研究[硕士学位论文]：安徽医科大学;2010.
[96]Rosmorduc O, Petit MA, Pol S, Capel F, Bortolotti F, Berthelot P, et al. In vivo and in vitro expression of defective hepatitis B virus particles generated by spliced hepatitis B virus RNA. Hepatology 1995;22:10-19.
[97]Fernandez M, Quiroga JA, Carreno V. Hepatitis B virus downregulates the human interferon-inducible MxA promoter through direct interaction of precore/core proteins. J Gen Virol 2003;84:2073-2082.
[98]Rosmorduc O, Sirma H, Soussan P, Gordien E, Lebon P, Horisberger M, et al. Inhibition of interferon-inducible MxA protein expression by hepatitis B virus capsid protein. J Gen Virol 1999;80 (Pt5):1253-1262.
[99]Yang YL, Reis LF, Pavlovic J, Aguzzi A, Schafer R, Kumar A, et al. Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase. EMBOJ 1995; 14:6095-6106.
[100]Park I-H, Baek K-W, Cho E-Y, Ahn B-Y. PKR-dependent mechanisms of interferon-a for inhibiting hepatitis B virus replication. Molecules and Cells 2011;32:167-172.
[101]Chen GG, Lai PB, Ho RL, Chan PK, Xu H, Wong J, et al. Reduction of double-stranded RNA-activated protein kinase in hepatocellular carcinoma associated with hepatitis B virus. J Med Virol 2004;73:187-194.
[102]Han Q, Zhang C, Zhang J, Tian Z. Involvement of activation of PKR in HBx-siRNA-mediated innate immune effects on HBV inhibition. PLoS One 2011;6:e27931.
[103]Takaori-Kondo A. APOBEC family proteins:novel antiviral innate immunity. Int J Hematol 2006;83:213-216.
[104]Cullen BR. Role and mechanism of action of the APOBEC3 family of antiretroviral resistance factors. J Virol 2006;80:1067-1076.
[105]Suspene R. Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo. Proceedings of the National Academy of Sciences 2005;102:8321-8326.
[106]Bonvin M, Greeve J. Hepatitis B:modern concepts in pathogenesis--APOBEC3 cytidine deaminases as effectors in innate immunity against the hepatitis B virus. Curr Opin Infect Dis 2008;21:298-303.
[107]Bishop KN, Holmes RK, Sheehy AM, Davidson NO, Cho SJ, Malim MM. Cytidine deamination of retroviral DNA by diverse APOBEC proteins. Curr Biol 2004; 14:1392-1396.
[108]Kim BK, Revill PA, Ahn SH. HBV genotypes:relevance to natural history, pathogenesis and treatment of chronic hepatitis B. Antivir Ther 2011;16:1169-1186.
[109]Erhardt A, Blondin D, Hauck K, Sagir A, Kohnle T, Heintges T, et al. Response to interferon alfa is hepatitis B virus genotype dependent:genotype A is more sensitive to interferon than genotype D. Gut 2005;54:1009-1013.
[110]Ma JC, Wang LW, Li XJ, Liao YF, Hu XY, Gong ZJ. Relationship between HBV genotypes and anti-viral therapeutic efficacy of interferon-alpha. Hepatobiliary Pancreat Dis Int 2007;6:166-171.
[111]中华医学会肝病学分会.慢性乙型肝炎防治指南(2010年版).中华肝病杂志2011;19：13-24,
[112]邵光.干扰素作用下乙肝病毒C基因区变异机制的体外研究及HepG2.2.15细胞在研究中的应用[硕士学位论文]：中山大学;2006.
[113]Liu CJ, Kao JH. Genetic variability of hepatitis B virus and response to antiviral therapy. Antivir Ther 2008; 13:613-624.
[114]王卫峰.,周晓东.干扰素作用下乙型肝炎病毒变异的体外实验研究.中华肝脏病杂志2005;13：793-794.
[115]刘彦华,倪旭.HBV 前 C区基因变异与乙肝病毒复制的相关性.河北医药2007;29：1056-1058.
[116]Chotiyaputta W, Lok AS. Hepatitis B virus variants. Nat Rev Gastroenterol Hepatol 2009;6:453-462.
[117]Mason A, Wick M, White H, Perrillo R. Hepatitis B virus replication in diverse cell types during chronic hepatitis B virus infection. Hepatology 1993;18:781-789.
[118]Schildgen O. Host factors may influence response to antiviral therapy in chronic hepatitis B virus infections. Medical Hypotheses 2011;76:417-420.
[119]卢年芳.,郑瑞强.,林华.,黄爱龙.,杨德刚.不同亚型IFN-α对STAT1表达的影响.江苏医药2006；32：901-903.
[120]He XX, Chang Y, Jiang HJ, Tang F, Meng FY, Xie OH, et al. Persistent effect of IFNAR-I genetic polymorphism on the long-term pathogenesis of chronic HBV infection. Viral Immunol 2010:23:251-257.
[121]Hsieh YY, Chang CC, Hsu CM, Wan L, Chen SY, Lin WH, et al. JAK-1 rs2780895 C-related genotype and allele but not JAK-1 rs10789166, rs4916008, rs2780885, rs17127114, and rs3806277 are associated with higher susceptibility to asthma. Genet Test Mol Biomarkers 2011;15:841-847.
[122]Zhu ZZ, Di JZ, Gu WY, Cong WM, Gawron A, Wang Y, et al. Association of genetic polymorphisms in STAT1 gene with increased risk of hepatocellular carcinoma. Oncology 2010;78:382-388.
[123]Ren S, Yu H, Zhang H, Liu Y, Huang Y, Ma L, et al. Polymorphisms of interferon-inducible genes OAS associated with interferon-alpha treatment response in chronic HBV infection. Antiviral Res 2011;89:232-237.
[124]Abe H, Hayes CN, Ochi H, Tsuge M, Miki D, Hiraga N, et al. Inverse association of IL28B genotype and liver mRNA expression of genes promoting or suppressing antiviral state. J Med Virol 2011;83:1597-1607.
[125]孔晓飞.干扰素信号传导系统与乙型肝炎相关性研究[硕十学位论文]：上海第二医科大学;2004.
[126]Durbin JE, Hackenmiller R, Simon MC, Levy DE. Targeted disruption of the mouse Statl gene results in compromised innate immunity to viral disease. Cell 1996;84:443-450.
[127]陈禄彪.,彭晓谋.,曹红.,张宇峰.,徐启桓.,高志良.OAS-1基因SNP rs 10774671(?)慢性HBV感染者自发性HBeAg血清转换的关系.中华实验和临床病毒学杂志2009;23：35-37.
1. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RN As with antisense complementarity to lin-14. Cell.1993 Dec 3;75(5):843-54.
2. Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell.1993 Dec 3;75(5):855-62.
3. Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Mailer B, et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature.2000 Nov 2;408(6808):86-9.
4. Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP. MicroRNAs in plants. Genes Dev. 2002 Jul 1;16(13):1616-26.
5. Hamilton AJ, Baulcombe DC. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science.1999 Oct 29;286(5441):950-2.
6. Ambros V, Bartel B, Bartel DP, Burge CB, Carrington JC, Chen X, et al. A uniform system for microRNA annotation. RNA.2003 Mar;9(3):277-9.
7. Griffiths-Jones S. The microRNA Registry. Nucleic Acids Res.2004 Jan 1;32(Database issue):D109-11.
8. Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ. miRBase:microRNA sequences, targets and gene nomenclature. Nucleic Acids Res.2006 Jan 1;34(Database issue):D140-4.
9. Kozomara A, Griffiths-Jones S. miRBase:integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res.2011 Jan;39(Database issue):D 152-7.
10. Winter J, Jung S, Keller S, Gregory RI, Diederichs S. Many roads to maturity:microRNA biogenesis pathways and their regulation. Nat Cell Biol.2009 Mar;11(3):228-34.
11. Ying SY, Lin SL. Intronic microRNAs. Biochem Biophys Res Commun.2005 Jan 21;326(3):515-20.
12. Sibley CR, Seow Y, Saayman S, Dijkstra KK, El Andaloussi S, Weinberg MS, et al. The biogenesis and characterization of mammalian microRNAs of mirtron origin. Nucleic Acids Res.2012 Jan;40(1):438-48.
13. Kim VN. MicroRNA biogenesis:coordinated cropping and dicing. Nat Rev Mol Cell Biol.2005 May;6(5):376-85.
14. Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH, et al. MicroRNA genes are transcribed by RNA polymerase 11. EMBO J.2004 Oct 13;23(20):4051-60.
15. Cullen BR. Transcription and processing of human microRNA precursors. Mol Cell.2004 Dec 22;16(6):861-5.
16. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. The nuclear RNase Ⅲ Drosha initiates microRNA processing. Nature.2003 Sep 25;425(6956):415-9.
17. Landthaler M, Yalcin A, Tuschl T. The human DiGeorge syndrome critical region gene 8 and Its D. melanogaster homolog are required for miRNA biogenesis. Curr Biol.2004 Dec 14; 14(23):2162-7.
18. Yi R, Qin Y, Macara IG, Cullen BR. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev.2003 Dec 15;17(24):3011-6.
19. Zeng Y, Cullen BR. Structural requirements for pre-microRNA binding and nuclear export by Exportin 5. Nucleic Acids Res.2004;32(16):4776-85.
20. Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K, et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature.2005 Aug 4;436(7051):740-4.
21. Lee Y, Hur I, Park SY, Kim YK, Suh MR, Kim VN. The role of PACT in the RNA silencing pathway. EMBO J.2006 Feb 8;25(3):522-32.
22. Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell. 2003 Oct 17;115(2):209-16.
23. Song JJ, Smith SK, Hannon GJ, Joshua-Tor L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science.2004 Sep 3;305(5689):1434-7.
24. Meister G, Tuschl T. Mechanisms of gene silencing by double-stranded RNA. Nature.2004 Sep 16;431(7006):343-9.
25. Krol J, Loedige I, Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet.2010 Sep; 11 (9):597-610.
26. Wu L, Fan J, Belasco JG. MicroRNAs direct rapid deadenylation of mRNA. Proc Natl Acad Sci U SA.2006 Mar14;103(11):4034-9.
27. Okamura K, Ishizuka A, Siomi H, Siomi MC. Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev.2004 Jul 15; 18(14):1655-66.
28. Bartel DP. MicroRNAs:genomics, biogenesis, mechanism, and function. Cell.2004 Jan 23;116(2):281-97.
29. Lewis BP, Burge CB, Bartel DP. Conserved Seed Pairing, Often Flanked by Adenosines, Indicates that Thousands of Human Genes are MicroRNA Targets. Cell.2005; 120(1):15-20.
30. Duursma AM, Kedde M, Schrier M, le Sage C, Agami R. miR-148 targets human DNMT3b protein coding region. RNA.2008 May;14(5):872-7.
31. Lee I, Ajay SS, Yook JI, Kim HS, Hong SH, Kim NH, et al. New class of microRNA targets containing simultaneous 5'-UTR and 3'-UTR interaction sites. Genome Res.2009 Jul; 19(7):1175-83.
32. Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation:microRNAs can up-regulate translation. Science.2007 Dec 21;318(5858):1931-4.
33. Djuranovic S, Nahvi A, Green R. miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay. Science.2012 Apr 13;336(6078):237-40.
34. Smith SM, Murray DW. An overview of microRNA methods:expression profiling and target identification. Methods Mol Biol.2012,823:119-38.
35. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of mammalian microRNA targets. Cell.2003 Dec 26; 115(7):787-98.
36. Rehmsmeier M, Steffen P, Hochsmann M, Giegerich R. Fast and effective prediction of microRNA/target duplexes. RNA.2004 Oct; 10(10):1507-17.
37. Takada S, Asahara H. Current strategies for microRNA research. Mod Rheumatol.2012 Jan 12.
38. Farh KK, Grimson A, Jan C, Lewis BP, Johnston WK, Lim LP, et al. The widespread impact of mammalian MicroRNAs on mRNA repression and evolution. Science.2005 Dec 16;310(5755):1817-21.
39. Chang J, Nicolas E, Marks D, Sander C, Lerro A, Buendia MA, et al. miR-122, a mammalian liver-specific microRNA, is processed from her mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1. RNA Biol.2004 Jul; 1(2):106-13.
40. de Planell-Saguer M, Rodicio MC. Analytical aspects of microRNA in diagnostics:a review. Anal Chim Acta.2011 Aug 12;699(2):134-52.
41. Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elcgans. Science.2001 Oct 26;294(5543):862-4.
42. Bernardo BC, Charchar FJ, Lin RC, McMullen JR. A microRNA guide for clinicians and basic scientists:background and experimental techniques. Heart LungCirc.2012 Mar;21(3):131-42.
43. Lai EC. Predicting and validating microRNA targets. Genome Biol.2004;5(9):115.
44. Lim LP, Lau NC, Weinstein EG, Abdelhakim A, Yekta S. Rhoades MW, et al. The microRNAs of Caenorhabditis elegans. Genes Dev.2003 Apr 15; 17(8):991-1008.
45. Doran J, Strauss WM. Bio-informatic trends for the determination of miRNA-target interactions in mammals. DNA Cell Biol.2007 May;26(5):353-60.
46. Liu H, Yue D, Zhang L, Chen Y, Gao SJ, Huang Y. A Bayesian approach for identifying miRNA targets by combining sequence prediction and gene expression profiling. BMC Genomics.2010; 11 Suppl3:S12.
47. Zhou X, Ruan J, Wang G, Zhang W. Characterization and identification of microRNA core promoters in four model species. PLoS Comput Biol.2007 Mar 9;3(3):e37.
48. Stern-Ginossar N, Elefant N, Zimmermann A, Wolf DG, Saleh N, Biton M, et al. Host immune system gene targeting by a viral miRNA. Science.2007 Jul 20;317(5836):376-81.
49. Zhang Y. miRU:an automated plant miRNA target prediction server. Nucleic Acids Res.2005 Jul 1;33(Web Server issue):W701-4.
50. Rodriguez A, Griffiths-Jones S, Ashurst JL, Bradley A. Identification of mammalian microRNA host genes and transcription units. Genome Res.2004 Oct;14(10A):1902-10.
51. Mu W, Zhang W. Bioinformatic Resources of microRNA Sequences, Gene Targets, and Genetic Variation. Front Genet.2012;3:31.
52. Tora L, Dong H, Paquette M, Williams A, Zoeller RT, Wade M, et al. Thyroid Hormone May Regulate mRNA Abundance in Liver by Acting on MicroRNAs. PLoS One.2010;5(8):el2136.
53. Vergoulis T, Vlachos IS, Alexiou P, Georgakilas G, Maragkakis M, Reczko M, et al. TarBase 6.0: capturing the exponential growth of miRNA targets with experimental support. Nucleic Acids Res. 2012 Jan;40(Database issue):D222-9.
54. Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP. MicroRNA targeting specificity in mammals:determinants beyond seed pairing. Mol Cell.2007 Jul 6;27(1):91-105.
55. Krek A, Grun D, Poy MN, Wolf R, Rosenberg L, Epstein EJ, et al. Combinatorial microRNA target predictions. Nat Genet.2005 May;37(5):495-500.
56. Gamazon ER, Im HK, Duan S, Lussier YA, Cox NJ, Dolan ME, et al. Exprtarget:an integrative approach to predicting human microRNA targets. PLoS One.2010;5(10):e13534.
57. Wang X. miRDB:a microRNA target prediction and functional annotation database with a wiki interface. RNA.2008 Jun;14(6):1012-7.
58. Walter AE, Turner DH, Kim J, Lyttle MH, Muller P, Mathews DH, et al. Coaxial stacking of helixes enhances binding of oligoribonucleotides and improves predictions of RNA folding. Proc Natl Acad Sci U S A.1994 Sep 27;91(20):9218-22.
59. Lindgreen S, Gardner PP, Krogh A. MASTR:multiple alignment and structure prediction of non-coding RNAs using simulated annealing. Bioinformatics.2007 Dec 15;23(24):3304-11.
60. Will S, Reiche K, Hofacker IL, Stadler PF, Backofen R. Inferring noncoding RNA families and classes by means of genome-scale structure-based clustering. PLoS Comput Biol.2007 Apr 13;3(4):e65.
61. Hofacker IL. Vienna RNA secondary structure server. Nucleic Acids Res.2003 Jul 1;31(13):3429-31.
62. Li SC, Pan CY, Lin WC. Bioinformatic discovery of microRNA precursors from human ESTs and introns. BMC Genomics.2006;7:164.
63. Kiezun A, Artzi S, Modai S, Volk N, Isakov O, Shomron N. miRviewer:a multispecies microRNA homologous viewer. BMC Res Notes.2012;5:92.
64. Zhang Y, Yang Y, Zhang H, Jiang X, Xu B, Xue Y, et al. Prediction of novel pre-microRNAs with high accuracy through boosting and SVM. Bioinformatics.2011 May 15;27(10):1436-7.
65. Wu Y, Wei B, Liu H, Li T, Rayner S. MiRPara:a SVM-based software tool for prediction of most probable microRNA coding regions in genome scale sequences. BMC Bioinformatics.2011; 12:107.
66. Thieme CJ, Gramzow L, Lobbes D, Theissen G. SplamiR--prediction of spliced miRN As in plants. Bioinformatics.2011 May 1;27(9):1215-23.
67. Szczesniak MW, Deorowicz S, Gapski J, Kaczynski L, Makalowska I. miRNEST database:an integrative approach in microRNA search and annotation. Nucleic Acids Res.2012 Jan;40(Database issue):D 198-204.
68. Gkirtzou K, Tsamardinos I, Tsakalides P, Poirazi P. MatureBayes:a probabilistic algorithm for identifying the mature miRNA within novel precursors. PLoS One.2010;5(8):e11843.
69. Hackenberg M, Sturm M, Langenberger D, Falcon-Perez JM, Aransay AM. miRanalyzer:a microRNA detection and analysis tool for next-generation sequencing experiments. Nucleic Acids Res. 2009 Jul;37(Web Server issue):W68-76.
70. Mitra R, Bandyopadhyay S. MultiMiTar:a novel multi objective optimization based miRNA-target prediction method. PLoS One.2011;6(9):e24583.
71. Reczko M, Maragkakis M, Alexiou P, Grosse I, Hatzigeorgiou AG. Functional microRNA targets in protein coding sequences. Bioinformatics.2012 Mar 15;28(6):771-6.
72. Elefant N, Berger A, Shein H, Hofree M, Margalit H, Altuvia Y. RepTar:a database of predicted cellular targets of host and viral miRNAs. Nucleic Acids Res.2011 Jan;39(Database issue):D188-94.
73. Le Brigand K, Robbe-Sermesant K, Mari B, Barbry P. MiRonTop:mining microRNAs targets across large scale gene expression studies. Bioinformatics.2010 Dec 15;26(24):3131-2.
74. Xie F, Zhang B. Target-align:a tool for plant microRNA target identification. Bioinformatics. 2010 Dec 1;26(23):3002-3.
75. Reyes-Herrera PH, Ficarra E, Acquaviva A, Macii E. miREE:miRNA recognition elements ensemble. BMC Bioinformatics.2011; 12:454.
76. Dai X, Zhao PX. psRNATarget:a plant small RNA target analysis server. Nucleic Acids Res.2011 Jul;39(Web Server issue):W155-9.
77. Sualp M, Can T. Using network context as a filter for miRNA target prediction. Biosystems. 2011;105(3):201-9.
78. Huang GT, Athanassiou C, Benos PV. mirConnX:condition-specific mRNA-microRNA network integrator. Nucleic Acids Res.2011 Jul;39(Web Server issue):W416-23.
79. Xiao F, Zuo Z, Cai G, Kang S, Gao X, Li T. mi Records:an integrated resource for microRNA-target interactions. Nucleic Acids Res.2009 Jan;37(Database issue):D105-10.
80. Qiu C, Wang D, Wang E, Cui Q. An upstream interacting context based framework for the computational inference of microRNA functions. Mol Biosyst.2012 Apr 3;8(5):1492-8.
81. Laczny C, Leidinger P, Haas J, Ludwig N, Backes C, Gerasch A, et al. miRTrail-a comprehensive webserver for analyzing gene and miRNA patterns to enhance the understanding of regulatory mechanisms in diseases. BMC Bioinformatics.2012; 13:36.
82. Bruno AE, Li L, Kalabus JL, Pan Y, Yu A, Hu Z. miRdSNP:a database of disease-associated SNPs and microRNA target sites on 3'UTRs of human genes. BMC Genomics.2012;13:44.
83. Xu X, Zhao Y, Simon R. Gene Set Expression Comparison kit for BRB-ArrayTools. Bioinformatics.2008 Jan 1;24(I):137-9.
84. Hua Y, Duan S,Murmann AE, Larsen N, Kjems J, Lund AH, et al. miRConnect:identifying effector genes of miRNAs and miRNA families in cancer cells. PLoS One.2011;6(10):e26521.
85. Mathelier A, Carbone A. MIReNA:finding microRNAs with high accuracy and no learning at genome scale and from deep sequencing data. Bioinformatics.2010 Sep 15;26(18):2226-34.
86. Hendrix D, Levine M, Shi W. miRTRAP, a computational method for the systematic identification of miRNAs from high throughput sequencing data. Genome Biol.2010; 11(4):R39.
87. Rodriguez A, Vigorito E, Clare S, Warren MV, Couttet P, Soond DR, et al. Requirement of bic/microRNA-155 for normal immune function. Science.2007 Apr 27;316(5824):608-11.
88. van Rooij E. The art of microRNA research. Circ Res.2011 Jan 21;108(2):219-34.
89. Willenbrock H, Salomon J, Sokilde R, Barken KB, Hansen TN, Nielsen FC, et al. Quantitative miRNA expression analysis:comparing microarrays with next-generation sequencing. RNA.2009 Nov;15(11):2028-34.
90. Git A, Dvinge H, Salmon-Divon M, Osborne M, Kutter C, Hadfield J, et al. Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRN A expression. RNA.2010 May; 16(5):991-1006.
91. Fehniger TA, Wylie T, Germino E, Leong JW, Magrini VJ, Koul S, et al. Next-generation sequencing identifies the natural killer cell microRN A transcriptome. Genome Res.2010 Nov;20(11):1590-604.
92. Hafner M, Landthaler M, Burger L, Khorshid M, Hausser J, Berninger P, et al. PAR-CliP--a method to identify transcriptome-wide the binding sites of RNA binding proteins. J Vis Exp.2010(41).
93. Keene JD, Komisarow JM, Friedersdorf MB. RIP-Chip:the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nat Protoc.2006; 1(1):302-7.
94. Thomson DW, Bracken CP, Goodall GJ. Experimental strategies for microRNA target identification. Nucleic Acids Res.2011 Sep 1;39(16):6845-53.
95. Wang WX, Wilfred BR, Hu Y, Stromberg AJ, Nelson PT. Anti-Argonaute RIP-Chip shows that miRNA transfections alter global patterns of mRNA recruitment to microribonucleoprotein complexes. RNA.2009;16(2):394-404.
96. Tan LP, Seinen E, Duns G, de Jong D, Sibon OC, Poppema S, et al. A high throughput experimental approach to identify miRNA targets in human cells. Nucleic Acids Res.2009 Nov;37(20):e137.
97. Huang Y, Zou Q, Wang SP, Tang SM, Zhang GZ, Shen XJ. The discovery approaches and detection methods of microRNAs. Mol Biol Rep.2011 Aug;38(6):4125-35.
1. Seeger C, Mason WS. Hepatitis B virus biology. Microbiol Mol Biol Rev.2000 Mar;64(1):51-68.
2. Chang JJ, Lewin SR. Immunopathogenesis of hepatitis B virus infection. Immunol Cell Biol. 2007 Jan;85(1):16-23.
3. Tsai HT, Tsai TH, Lu TM, Yang CC. Immunopathology of hepatitis B virus infection. Int Rev Immunol.2008;27(6):427-46.
4. Hoofnagle JH, Doo E, Liang TJ, Fleischer R, Lok AS. Management of hepatitis B:summary of a clinical research workshop. Hepatology.2007 Apr;45(4):1056-75.
5. Duong A, Mousa SA. Current status of nucleoside antivirals in chronic hepatitis B. Drugs Today (Barc).2009 Oct;45(10):751-61.
6. Buster EH, Janssen HL. Antiviral treatment for chronic hepatitis B virus infection--immune modulation or viral suppression? Neth J Med.2006 Jun;64(6):175-85.
7. Lin CL, Kao JH. Recent advances in the treatment of chronic hepatitis B. Expert Opin Pharmacother.2011 Sep;12(13):2025-40.
8. Niederhauser C. Reducing the risk of hepatitis B virus transfusion-transmitted infection. J Blood Med.2011;2:91-102.
9. Sayed D, Abdellatif M. MicroRNAs in development and disease. Physiol Rev.2011 Jul;91(3):827-87.
10. Taganov KD, Boldin MP, Baltimore D. MicroRNAs and immunity:tiny players in a big field. Immunity.2007 Feb;26(2):133-7.
11. Thai TH, Calado DP, Casola S, Ansel KM, Xiao C, Xue Y, et al. Regulation of the germinal center response by microRNA-155. Science.2007 Apr 27;316(5824):604-8.
12. Liu G, Min H, Yue S, Chen CZ. Pre-miRNA loop nucleotides control the distinct activities of mir-181 a-1 and mir-181 c in early T cell development. PLoS One.2008;3(10):e3592.
13. Li QJ, Chau J, Ebert PJ, Sylvester G, Min H, Liu G, et al. miR-181a is an intrinsic modulator of T cell sensitivity and selection. Cell.2007 Apr 6; 129(1):147-61.
14. Fazi F, Rosa A, Fatica A, Gelmetti V, De Marchis ML, Nervi C, et al. A Minicircuitry Comprised of MicroRNA-223 and Transcription Factors NFI-A and C/EBPa Regulates Human Granulopoiesis. Cell.2005;123(5):819-31.
15. Rodriguez A, Vigorito E, Clare S, Warren MV, Couttet P, Soond DR, et al. Requirement of bic/microRNA-155 for normal immune function. Science.2007 Apr 27;316(5824):608-11.
16. Vigorito E, Perks KL, Abreu-Goodger C, Bunting S, Xiang Z, Kohlhaas S, et al. microRNA-155 regulates the generation of immunoglobulin class-switched plasma cells. Immunity.2007 Dec;27(6):847-59.
17. Xiao C, Calado DP, Galler G, Thai T-H, Patterson HC, Wang J, et al. MiR-150 Controls B Cell Differentiation by Targeting the Transcription Factor c-Myb. Cell.2007; 131(1):146-59.
18. de Yebenes VG, Belver L, Pisano DG, Gonzalez S, Villasante A, Croce C, et al. miR-181b negatively regulates activation-induced cytidine deaminase in B cells. Journal of Experimental Medicine.2008;205(10):2199-206.
19. Ma F, Xu S, Liu X, Zhang Q, Xu X, Liu M, et al. The microRNA miR-29 controls innate and adaptive immune responses to intracellular bacterial infection by targeting interferon-y. Nature Immunology.2011; 12(9):861-9.
20. Chen XM, Splinter PL, O'Hara SP, LaRusso NF. A cellular micro-RNA, let-7i, regulates Toll-like receptor 4 expression and contributes to cholangiocyte immune responses against Cryptosporidium parvum infection. J Biol Chem.2007 Sep 28;282(39):28929-38.
21. Hou J, Wang P, Lin L, Liu X, Ma F, An H, et al. MicroRNA-146a Feedback Inhibits RIG-I-Dependent Type I IFN Production in Macrophages by Targeting TRAF6, IRAKI, and IRAK2. The Journal of Immunology.2009;183(3):2150-8.
22. Murphy E, Vanicek J, Robins H, Shenk T, Levine AJ. Suppression of immediate-early viral gene expression by herpesvirus-coded microRNAs:implications for latency. Proc Natl Acad Sci U S A.2008 Apr 8;105(14):5453-8.
23. Choy EY, Siu KL, Kok KH, Lung RW, Tsang CM, To KF, et al. An Epstein-Barr virus-encoded microRNA targets PUMA to promote host cell survival. J Exp Med.2008 Oct 27;205(11):2551-60.
24. Umbach JL, Kramer MF, Jurak I, Karnowski HW, Coen DM, Cullen BR. MicroRNAs expressed by herpes simplex virus 1 during latent infection regulate viral mRNAs. Nature.2008 Aug 7;454(7205):780-3.
25. Lecellier CH, Dunoyer P, Arar K, Lehmann-Che J, Eyquem S, Himber C, et al. A cellular microRNA mediates antiviral defense in human cells. Science.2005 Apr 22;308(5721):557-60.
26. Gao P, Wong CC-L, Tung EK-K, Lee JM-F, Wong C-M, Ng IO-L. Deregulation of microRNA expression occurs early and accumulates in early stages of HBV-associated multistep hepatocarcinogenesis. Journal of Hepatology.2011;54(6):1177-84.
27. Ladeiro Y, Couchy G, Balabaud C, Bioulac-Sage P, Pelletier L, Rebouissou S, et al. MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations. Hepatology.2008;47(6):1955-63.
28. Guan X-y, Mizuguchi Y, Mishima T, Yokomuro S, Arima Y, Kawahigashi Y, et al. Sequencing and Bioinformatics-Based Analyses of the microRNA Transcriptome in Hepatitis B-Related Hepatocellular Carcinoma. PLoS One.2011;6(1):e 15304.
29. Zhou J, Yu L, Gao X, Hu J, Wang J, Dai Z, et al. Plasma microRNA panel to diagnose hepatitis B virus-related hepatocellular carcinoma. J Clin Oncol.2011 Dec 20;29(36):4781-8.
30. Li LM, Hu ZB, Zhou ZX, Chen X, Liu FY. Zhang JF. et al. Serum microRNA Profiles Serve as Novel Biomarkers for HBV Infection and Diagnosis of HBV-Positive Hepatocarcinoma. Cancer Research.2010;70(23):9798-807.
31. Wang S, Qiu L, Yan X, Jin W, Wang Y, Chen L, et al. Loss of microRNA 122 expression in patients with hepatitis B enhances hepatitis B virus replication through cyclin G1-modulated P53 activity. Hepatology.2012;55(3):730-41.
32. Li R-F. miR-122 inhibits viral replication and cell proliferation in hepatitis B virus-related hepatocellular carcinoma and targets NDRG3. Oncology Reports.2011.
33. Qiu L, Fan H, Jin W, Zhao B, Wang Y, Ju Y, et al. miR-122-induced down-regulation of HO-1 negatively affects miR-122-mediated suppression of HBV. Biochemical and Biophysical Research Communications.2010;398(4):771-7.
34. Chen Y, Shen A, Rider PJ, Yu Y, Wu K, Mu Y, et al. A liver-specific microRNA binds to a highly conserved RNA sequence of hepatitis B virus and negatively regulates viral gene expression and replication. The FASEB Journal.2011;25(12):4511-21.
35. Potenza N, Papa U, Mosca N, Zerbini F, Nobile V, Russo A. Human microRNA hsa-miR-125a-5p interferes with expression of hepatitis B virus surface antigen. Nucleic Acids Research. 2011;39(12):5157-63.
36. Zhang G-1, Li Y-x, Zheng S-q, Liu M, Li X, Tang H. Suppression of hepatitis B virus replication by microRNA-199a-3p and microRNA-210. Antiviral Research.2010;88(2):169-75.
37. Zhang X, Zhang E, Ma Z, Pei R, Jiang M, Schlaak JF, et al. Modulation of hepatitis 13 virus replication and hepatocyte differentiation by MicroRNA-1. Hepatology.2011;53(5):1476-85.
38. Guo H, Liu H, Mitchelson K, Rao H, Luo M, Xie L, et al. MicroRNAs-372/373 promote the expression of hepatitis B virus through the targeting of nuclear factor I/B. Hepatology. 2011;54(3):808-19.
39. Hu W, Wang X, Ding X, Li Y, Zhang X, Xie P, et al. MicroRNA-141 Represses HBV Replication by Targeting PPARA. PLoS One.2012;7(3):e34165.
40.杜锐.microRNA在乙肝病毒复制中的作用及机制研究.第四军医大学博士学位论文.2009.
41. Liu WH, Yeh SH, Chen PJ. Role of microRNAs in hepatitis B virus replication and pathogenesis. Biochim Biophys Acta.2011 Nov-Dec; 1809(11-12):678-85.
42. Su C, Hou Z, Zhang C, Tian Z, Zhang J. Ectopic expression of microRNA-155 enhances innate antiviral immunity against HBV infection in human hepatoma cells. Virology Journal.2011;8(1):354.
43. Huang LR, Wu HL, Chen PJ, Chen DS. An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection. Proceedings of the National Academy of Sciences. 2006;103(47):17862-7.
44. Dickins RA, McJunkin K, Hernando E, Premsrirut PK, Krizhanovsky V, Burgess DJ, et al. Tissue-specific and reversible RNA interference in transgenic mice. Nat Genet.2007 Jul;39(7):914-21.
45. Schmeisser H, Mejido J, Balinsky CA, Morrow AN, Clark CR, Zhao T, et al. Identification of Alpha Interferon-Induced Genes Associated with Antiviral Activity in Daudi Cells and Characterization of IFIT3 as a Novel Antiviral Gene. Journal of Virology.2010:84(20):10671-80.
46. Taganov K.D. NF-B-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proceedings of the National Academy of Sciences. 2006; 103(33):12481-6.
47. Han Q, Zhang C, Zhang J, Tian Z. Reversal of hepatitis B virus-induced immune tolerance by an immunostimulatory 3p-HBx-siRNAs in a retinoic acid inducible gene 1-dependent manner. Hepatology. 2011;54(4):1179-89.
48. Tang Y, Luo X, Cui H, Ni X, Yuan M, Guo Y, et al. MicroRNA-146a contributes to abnormal activation of the type 1 interferon pathway in human lupus by targeting the key signaling proteins. Arthritis & Rheumatism.2009;60(4):1065-75.
49. Ramkissoon SH, Mainwaring LA, Sloand EM, Young NS, Kajigaya S. Nonisotopic detection of microRNA using digoxigenin labeled RNA probes. Molecular and Cellular Probes.2006;20(1):1-4.
50. Cameron JE, Yin Q, Fewell C, Lacey M, McBride J, Wang X, et al. Epstein-Barr virus latent membrane protein 1 induces cellular MicroRNA miR-146a, a modulator of lymphocyte signaling pathways. J Virol.2008 Feb;82(4):1946-58.
51. Labbaye C, Spinello I, Quaranta MT, Pelosi E, Pasquini L, Petrucci E, et al. A three-step pathway comprising PLZF/miR-146a/CXCR4 controls megakaryopoiesis. Nat Cell Biol.2008 JuI;10(7):788-801.
52. Tomankova T, Petrek M, Gallo J, Kriegova E. MicroRNAs:emerging regulators of immune-mediated diseases. Scand J Immunol.2011 Oct 11.
53. Paik JH, Jang JY, Jeon YK, Kim WY, Kim TM, Heo DS, et al. MicroRNA-146a Downregulates NF B Activity via Targeting TRAF6 and Functions as a Tumor Suppressor Having Strong Prognostic Implications in NK/T Cell Lymphoma. Clinical Cancer Research.2011; 17(14):4761-71.
54. Li L, Chen XP, Li YJ. MicroRNA-146a and Human Disease. Scandinavian Journal of Immunology.2010;71 (4):227-31.
55. Lu L-F, Boldin MP, Chaudhry A, Lin L-L, Taganov KD, Hanada T, et al. Function of miR-146a in Controlling Treg Cell-Mediated Regulation of Thl Responses. Cell.2010;142(6):914-29.
56. Friedman RC, Farh KKH, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Research.2008;19(1):92-105.
57. Rehmsmeier M.Fast and effective prediction of microRNA/target duplexes. Rna. 2004;10(10):1507-17.
58. John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS. Human MicroRNA Targets. PLoS Biology.2004;2(11):e363.
59. Krek A, Grun D, Poy MN, Wolf R, Rosenberg L, Epstein EJ, et al. Combinatorial microRNA target predictions. Nat Genet.2005 May;37(5):495-500.
60. Hornung V, Ellegast J, Kim S, Brzozka K, Jung A, Kato H, et al.5'-Triphosphate RNA is the ligand for RIG-I. Science.2006 Nov 10;314(5801):994-7.
61. Baum A, Garcia-Sastre A. Differential recognition of viral RNA by RIG-I. Virulence.2011 Mar-Apr;2(2):166-9.
62. Guo H, Jiang D, Ma D, Chang J, Dougherty AM, Cuconati A, et al. Activation of pattern recognition receptor-mediated innate immunity inhibits the replication of hepatitis B virus in human hepatocyte-derived cells. J Virol.2009 Jan;83(2):847-58.
63. Pichlmair A, Lassnig C, Eberle C-A, Gorna MW, Baumann CL, Burkard TR, et al. IFIT1 is an antiviral protein that recognizes 5'-triphosphate RNA. Nature Immunology.2011;12(7):624-30.
64. Liu XY, Chen W, Wei B, Shan YF, Wang C. IFN-Induced TPR Protein IFIT3 Potentiates Antiviral Signaling by Bridging MAVS and TBK1. The Journal of Immunology.2011;187(5):2559-68.
65. Pedersen IM, Cheng G, Wieland S, Volinia S, Croce CM, Chisari FV, et al. Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature.2007;449(7164):919-22.
66. Yang CH, Yue J, Fan M, Pfeffer LM. IFN Induces miR-21 through a Signal Transducer and Activator of Transcription 3-Dependent Pathway as a Suppressive Negative Feedback on IFN-Onduced Apoptosis. Cancer Research.2010;70(20):8108-16.
67. Witwer KW, Sisk JM, Gama L, Clements JE. MicroRNA Regulation of IFN- Protein Expression: Rapid and Sensitive Modulation of the Innate Immune Response. The Journal of Immunology. 2010;184(5):2369-76.
68. Sharma A, Kumar M, Aich J, Hariharan M, Brahmachari SK, Agrawal A, et al. Posttranscriptional regulation of interleukin-10 expression by hsa-miR-106a. Proc Natl Acad Sci U S A.2009 Apr 7;106(14):5761-6.
69. Lagos D, Pollara G, Henderson S, Gratrix F, Fabani M, Milne RSB, et al. miR-132 regulates antiviral innate immunity through suppression of the p300 transcriptional co-activator. Nature Cell Biology.2010;12(5):513-9.
70. Liu G, Friggeri A, Yang Y, Park YJ, Tsuruta Y, Abraham E. miR-147, a microRNA that is induced upon Toll-like receptor stimulation, regulates murine macrophage inflammatory responses. Proceedings of the National Academy of Sciences.2009; 106(37):15819-24.
71. Tili E, Michaille JJ, Cimino A, Costinean S, Dumitru CD, Adair B, et al. Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible roles in regulating the response to endotoxin shock. J Immunol.2007 Oct 15;179(8):5082-9.
72. Qi P, Dou TH, Geng L, Zhou FG, Gu X, Wang H, et al. Association of a variant in MIR 196A2 with susceptibility to hepatocellular carcinoma in male Chinese patients with chronic hepatitis B virus infection. Hum Immunol.2010 Jun;71(6):621-6.
73. Griffiths-Jones S. The microRNA Registry. Nucleic Acids Res.2004 Jan I;32(Database issue):D109-11.
74.Willenbrock H, Salomon J, Sokilde R, Barken KB, Hansen TN, Nielsen FC, et al. Quantitative miRNA expression analysis:comparing microarrays with next-generation sequencing. Rna.2009 Nov; 15(11):2028-34.
75. Pradervand S, Weber J, Lemoine F, Consales F, Paillusson A, Dupasquier M, et al. Concordance among digital gene expression, microarrays, and qPCR when measuring differential expression of microRNAs. BioTechniques.2010;48(3):219-22.
76. Meyer SU, Pfaffl MW, Ulbrich SE. Normalization strategies for microRNA profiling experiments: a'normal' way to a hidden layer of complexity? Biotechnology Letters.2010;32(12):1777-88.
77. Liang Z, Zhou H, Zheng H, Wu J. Expression levels of microRNAs are not associated with their regulatory activities. Biology Direct.2011;6(1):43.
78. Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. Widespread changes in protein synthesis induced by microRNAs. Nature.2008 Sep 4;455(7209):58-63.
79. Si ML, Zhu S, Wu H, Lu Z, Wu F, Mo YY. miR-21-mediated tumor growth. Oncogene. 2006;26(19):2799-803.
80. Xie SY, Li YJ, Wang PY, Jiao F, Zhang S, Zhang WJ. miRNA-regulated expression of oncogencs and tumor suppressor genes in the cisplatin-inhibited growth of K562 cells. Oncol Rep.2010 Jun;23(6):1693-700.
81. Tam W, Dahlberg JE. miR-155/BIC as an oncogenic microRNA. Genes, Chromosomes and Cancer.2006;45(2):211-2.
82. Jopling C. Liver-specific microRNA-122:Biogenesis and function. RNA Biol.2012 Feb 1;9(2).
83.张小勇.倪明.陈姗姗.吕婷婷.余冰.陆蒙吉.肝脏内源性microRNA调控乙型肝炎病毒基因的表达与复制.医学分子生物学杂志.2008;5(3)：189-93.
84. Ji F, Yang B, Peng X, Ding H, You H, Tien P. Circulating microRNAs in hepatitis B virus-infected patients. J Viral Hepat.2011 Jul; 18(7):e242-51.
85.郝美君.郑素军.丁惠国.黄爱龙MicroRNA-122作用于HBx影响乙肝病毒复制.生物医学工程学杂志.2011;28(4)：784-9.
86.金文松.肝特异性miR-122负向调控IFN-alpha抗乙肝病毒活力.福建农林大学硕十学位论文.2010.
87. Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T. Identification of tissue-specific microRNAs from mouse. Curr Biol.2002 Apr 30;12(9):735-9.
88. Hsu SD, Chu CH, Tsou AP, Chen SJ, Chen HC, Hsu PW, et al. miRNAMap 2.0:genomic maps of microRNAs in metazoan genomes. Nucleic Acids Res.2008 Jan;36(Database issue):D165-9.
89. Rusca N, Monticelli S. MiR-146a in Immunity and Disease. Mol Biol Int.2011;2011:437301.
90. Zhang Z-Z. Hepatitis B virus and hepatocellular carcinoma at the miRNA level. World Journal of Gastroenterology.2011;17(28):3353.
91. Liu Y, Zhao JJ, Wang CM, Li MY, Han P, Wang L, et al. Altered expression profiles of microRNAs in a stable hepatitis B virus-expressing cell line. Chin Med J (Engl).2009 Jan 5;122(1):10-4.
92. Sun B, Karin M. NF-κB signaling, liver disease and hepatoprotective agents. Oncogene. 2008;27(48):6228-44.
93. Hildt E, Saher G, Bruss V, Hofschneider PH. The hepatitis B virus large surface protein (LHBs) is a transcriptional activator. Virology.1996 Nov 1;225(1):235-9.
94. Liu Y, Lou G, Wu W, Zheng M, Shi Y, Zhao D, et al. Involvement of the NF-κB pathway in multidrug resistance induced by HBx in a hepatoma cell line. Journal of Viral Hepatitis. 2011;18(10):e439-e46.
95. Tang H, Oishi N, Kaneko S, Murakami S. Molecular functions and biological roles of hepatitis B virus x protein. Cancer Sci.2006 Oct;97(10):977-83.
96. Wu J, Meng Z, Jiang M, Pei R, Trippler M, Broering R, et al. Hepatitis B virus suppresses toll-like receptor-mediated innate immune responses in murine parenchymal and nonparenchymal liver cells. Hepatology.2009 Apr;49(4):1132-40.
97. Murakami S. Hepatitis B virus X protein:a multifunctional viral regulator. J Gastroenterol.2001 Oct;36(10):651-60.
98. Nahid MA, Pauley KM, Satoh M, Chan EKL. miR-146a Is Critical for Endotoxin-induced Tolerance:IMPLICATION IN INNATE IMMUNITY. Journal of Biological Chemistry. 2009;284(50):34590-9.
99. Punj V, Matta H, Schamus S, Tamewitz A, Anyang B, Chaudhary PM. Kaposi's sarcoma-associated herpesvirus-encoded viral FLICE inhibitory protein (vFLIP) K13 suppresses CXCR4 expression by upregulating miR-146a. Oncogene.2009:29(12):1835-44.
100. Cameron JE, Yin Q, Fewell C, Lacey M, McBride J, Wang X, et al. Epstein-Barr Virus Latent Membrane Protein 1 Induces Cellular MicroRNA miR-146a, a Modulator of Lymphocyte Signaling Pathways. Journal of Virology.2007;82(4):1946-58.
101. Wu G, Yu F, Xiao Z, Xu K, Xu J, Tang W, et al. Hepatitis B virus X protein downregulates expression of the miR-16 family in malignant hepatocytes in vitro. Br J Cancer.2011 Jun 28; 105(1):146-53.
102. Bui-Nguyen TM, Pakala SB, Sirigiri DR, Martin E, Murad F, Kumar R. Stimulation of inducible nitric oxide by hepatitis B virus transactivator protein HBx requires MTA1 coregulator. J Biol Chem. 2010 Mar 5;285(10):6980-6.
103. Wang Y, Lu Y, Toh ST. Sung WK, Tan P, Chow P, et al. Lethal-7 is down-regulated by the hepatitis B virus x protein and targets signal transducer and activator of transcription 3. J Hepatol.2010 Jul;53(1):57-66.
104. Shan C, Zhang S, Cui W, You X, Kong G, Du Y, et al. Hepatitis B virus X protein activates CD59 involving DNA binding and let-7i in protection of hepatoma and hepatic cells from complement attack. Carcinogenesis.2011 Aug;32(8):1190-7.
105. Kong G, Zhang J, Zhang S. Shan C, Ye L, Zhang X. Upregulated microRNA-29a by hepatitis B virus X protein enhances hepatoma cell migration by targeting PTEN in cell culture model. PLoS One. 2011;6(5):e19518.
106. Yip WK, Cheng AS, Zhu R, Lung RW, Tsang DP, Lau SS, et al. Carboxyl-terminal truncated HBx regulates a distinct microRNA transcription program in hepatocellular carcinoma development. PLoS One.2011;6(8):e22888.
107. Jurkin J, Schichl YM, Koeffel R, Bauer T, Richter S. Konradi S, et al. miR-146a Is Differentially Expressed by Myeloid Dendritic Cell Subsets and Desensitizes Cells to TLR2-Dependent Activation. The Journal of Immunology.2010;184(9):4955-65.
108. Lukiw WJ, Zhao Y, Cui JG. An NF-B-sensitive Micro RNA-146a-mediated Inflammatory Circuit in Alzheimer Disease and in Stressed Human Brain Cells. Journal of Biological Chemistry. 2008;283(46):31315-22.
109. Tomokuni A, Eguchi H, Tomimaru Y, Wada H, Kawamoto K, Kobayashi S, et al. miR-146a suppresses the sensitivity to interferon-a in hepatocellular carcinoma cells. Biochemical and Biophysical Research Communications.2011;414(4):675-80.
110. Motsch N, Pfuhl T, Mrazek J, Barth S, Grasser FA. Hpstein-Barr virus-encoded latent membrane protein I (LMP1) induces the expression of the cellular microRNA miR-146a. RNA Biol.2007 Nov;4(3):131-7.
111. Meraz MA, White JM, Sheehan KC, Bach EA, Rodig SJ, Dighe AS, et al. Targeted disruption of the Slatl gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell.1996 Feb9;84(3):431-42.
112. Park C, Li S, Cha E, Schindler C. Immune response in Stat2 knockout mice. Immunity.2000 Dec;13(6):795-804.
113. Boldin MP, Taganov KD, Rao DS, Yang L, Zhao JL, Kalwani M, et al. miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice. J Exp Med.2011 Jun 6;208(6):1189-201.
114. Hurst DR, Edmonds MD, Scott GK, Benz CC, Vaidya KS, Welch DR. Breast cancer metastasis suppressor 1 up-regulates miR-146, which suppresses breast cancer metastasis. Cancer Res.2009 Feb 15;69(4):1279-83.
115. Kogo R, Mimori K, Tanaka F, Komune S, Mori M. Clinical significance of miR-146a in gastric cancer cases. Clin Cancer Res.2011 Ju11;17(13):4277-84.
116. Perske C, Lahat N, Sheffy Levin S, Bitterman H, Hemmerlein B, Rahat MA. Loss of inducible nitric oxide synthase expression in the mouse renal cell carcinoma cell line RENCA is mediated by microRNA miR-146a. Am J Pathol.2010 Oct;177(4):2046-54.
117. Wang X, Tang S, Le SY, Lu R, Rader JS, Meyers C, et al. Aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth. PLoS One. 2008;3(7):e2557.
118. Mei J, Bachoo R, Zhang CL. MicroRNA-146a inhibits glioma development by targeting Notch 1. Mol Cell Biol.2011 Sep;31 (17)3584-92.
119. Liu DZ, Ander BP, Tian Y, Stamova B, Jickling GC, Davis RR, et al. Integrated analysis of mRNA and microRNA expression in mature neurons, neural progenitor cells and neuroblastoma cells. Gene. 2012 Mar 10;495(2):120-7.
120. Lin SL, Chiang A, Chang D, Ying SY. Loss of mir-146a function in hormone-refractory prostate cancer. Rna.2008 Mar;14(3):417-24.
121. Xu B, Wang N, Wang X, Tong N, Shao N, Tao J, et al. MiR-146a suppresses tumor growth and progression by targeting EGFR pathway and in a p-ERK-dependent manner in castration-resistant prostate cancer. Prostate.2011 Dec 7.
122. Li Y, VandenBoom TG, Wang Z, Kong D, Ali S, Philip PA, et al. miR-146a Suppresses Invasion of Pancreatic Cancer Cells. Cancer Research.2010;70(4):1486-95.
123. Karakatsanis A, Papaconstantinou I, Gazouli M, Lyberopoulou A, Polymeneas G, Voros D. Expression of microRNAs, miR-21, miR-31, miR-122, miR-145, miR-146a, miR-200c, miR-221, miR-222, and miR-223 in patients with hepatocellular carcinoma or intrahepatic cholangiocarcinoma and its prognostic significance. Mol Carcinog.2011 Dec 27.
124. Labbaye C, Spinello I, Quaranta MT, Pelosi E, Pasquini L, Petrucci E, et al. A three-step pathway comprising PLZF/miR-146a/CXCR4 controls megakaryopoiesis. Nature Cell Biology. 2008;10(7):788-801.
125. Qiu L-X, He J, Wang M-Y, Zhang R-X, Shi T-Y, Zhu M-L, et al. The association between common genetic variant of microRNA-146a and cancer susceptibility. Cytokine.2011;56(3):695-8.
126.扶琼.SLE相关基因IFIT3的功能及分子机制研究.上海交通大学博十学位论文.2008.
127. Hacker H, Redecke V, Blagoev B, Kratchmarova I, Hsu LC, Wang GG, et al. Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6. Nature.2006 Jan 12;439(7073):204-7.
128. Yoboua F, Martel A, Duval A, Mukawera E, Grandvaux N. Respiratory Syncytial Virus-Mediated NF-B p65 Phosphorylation at Serine 536 Is Dependent on RIG-I, TRAF6, and IKK Journal of Virology.2010;84(14):7267-77.
129. He JQ, Zarnegar B, Oganesyan G, Saha SK, Yamazaki S, Doyle SE, et al. Rescue of TRAF3-null mice by pi00 NF-kappa B deficiency. J Exp Med.2006 Oct 30;203(11):2413-8.
130. Yoneyama M, Fujita T. RNA recognition and signal transduction by RIG-I-like receptors. Immunol Rev.2009 Jan;227(1):54-65.
131. Yoshida R, Takaesu G, Yoshida H, Okamoto F, Yoshioka T, Choi Y, et al. TRAF6 and MEKK1 play a pivotal role in the RIG-I-like helicase antiviral pathway. J Biol Chem.2008 Dec 26;283(52):36211-20.
132. Bernardo BC, Charchar FJ, Lin RC, McMullen JR. A microRNA guide for clinicians and basic scientists:background and experimental techniques. Heart Lung Circ.2012 Mar;21(3):131-42.
[2]Jazayeri SM, Alavian SM, Carman WF. Hepatitis B virus:origin and evolution. Journal of Viral Hepatitis 2010; 17:229-235.
[3]Chu CM, Liaw YF. Hepatitis B surface antigen seroclearance during chronic HBV infection. Antivir Ther 2010;15:133-143.
[4]Mizukoshi E, Sidney J, Livingston B, Ghany M, Hoofnagle JH, Sette A, et al. Cellular immune responses to the hepatitis B virus polymerase. J Immunol 2004;173:5863-5871.
[5]Tang H, Oishi N, Kaneko S, Murakami S. Molecular functions and biological roles of hepatitis B virus x protein. Cancer Sci 2006;97:977-983.
[6]Guo H, Mao R, Block TM, Guo JT. Production and function of the cytoplasmic deproteinized relaxed circular DNA of hepadnaviruses. J Virol 2010;84:387-396.
[7]Grimm D, Thimme R, Blum HE. HBV life cycle and novel drug targets. Hepatol Int 2011;5:644-653.
[8]Sommer G, Heise T. Posttranscriptional control of HBV gene expression. Front Biosci 2008;13:5533-5547.
[9]Z B-O. Innate immunity:cells, receptors, and signaling pathways. Arch Immunol Ther Exp (Warsz) 2005;53.
[10]Turvey SE, Broide DH. Innate immunity. Journal of Allergy and Clinical Immunology 2010;125:S24-S32.
[11]Gao B, Jeong WI, Tian Z. Liver:An organ with predominant innate immunity. Hepatology 2008;47:729-736.
[12]Li Z, Diehl AM. Innate immunity in the liver. Curr Opin Gastroenterol 2003; 19:565-571.
[13]Parker GA, Picut CA. Immune Functioning in Non lymphoid Organs:The Liver. Toxicologic Pathology 2011;40:237-247.
[14]Seki S, Habu Y, Kawamura T, Takeda K, Dobashi H, Ohkawa T, et al. The liver as a crucial organ in the first line of host defense:the roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+T cells in T helper 1 immune responses. Immunol Rev 2000:174:35-46.
[15]Kadowaki N, Liu YJ. Natural type I interferon-producing cells as a link between innate and adaptive immunity. Hum Immunol 2002;63:1126-1132.
[16]Chen Y, Wei H, Gao B, Hu Z, Zheng S, Tian Z. Activation and function of hepatic NK cells in hepatitis B infection:an underinvestigated innate immune response. Journal of Viral Hepatitis 2005;12:38-45.
[17]Baron JL, Gardiner L, Nishimura S, Shinkai K, Locksley R, Ganem D. Activation of a nonclassical NKT cell subset in a transgenic mouse model of hepatitis B virus infection. Immunity 2002; 16:583-594.
[18]Boehme KW, Compton T. Innate Sensing of Viruses by Toll-Like Receptors. Journal of Virology 2004;78:7867-7873.
[19]于新蕾.,吕丽萍.,闫舫.,张艳宇.,周锡鹏.,马平.,et al.乙型肝炎病毒攻击肝细胞对β干扰素表达的影响.军事医学2011：35：587-592.
[20]Bonjardim CA, Ferreira PCP, Kroon EG. Interferons:Signaling, antiviral and viral evasion. Immunology Letters 2009; 122:1-11.
[21]Chen J, Wang XM, Wu XJ, Wang Y, Zhao H, Shen B, et al. Intrahepatic levels of PD-1/PD-L correlate with liver inflammation in chronic hepatitis B. Inflamm Res 2011:60:47-53.
[22]石翠翠.,谢青.,赵钢德.,黄谦.,项晓刚.,龚邦东.,et al.肝细胞表面PD-L1的表达及干扰素和HBV对其表达的调节.肝脏2010;15：23-25.
[23]Kakimi K, Lane TE, Wieland S, Asensio VC, Campbell IL, Chisari FV, et al. Blocking chemokine responsive to gamma-2/interferon (IFN)-gamma inducible protein and monokine induced by IFN-gamma activity in vivo reduces the pathogenetic but not the antiviral potential of hepatitis B virus-specific cytotoxic T lymphocytes. J Exp Med 2001; 194:1755-1766.
[24]Kimura K, Kakimi K, Wieland S, Guidotti LG, Chisari FV. Activated intrahepatic antigen-presenting cells inhibit hepatitis B virus replication in the liver of transgenic mice. J Immunol 2002:169:5188-5195.
[25]Kimura K, Kakimi K, Wieland S, Guidotti LG, Chisari FV. Interleukin-18 inhibits hepatitis B virus replication in the livers of transgenic mice. J Virol 2002;76:10702-10707.
[26]Kakimi K, Guidotti LG, Koezuka Y, Chisari FV. Natural killer T cell activation inhibits hepatitis B virus replication in vivo. J Exp Med 2000; 192:921-930.
[27]Smith PL, Lombardi G, Foster GR. Type I interferons and the innate immune response--more than just antiviral cytokines. Mol Immunol 2005:42:869-877.
[28]Touzot M, Soumelis V, Asselah T. A dive into the complexity of type I interferon antiviral functions. Journal of Hepatology 2012;56:726-728.
[29]Peltekian C, Gordien E, Garreau F, Meas-Yedid V, Soussan P, Willams V, et al. Human MxA protein participates to the interferon-related inhibition of hepatitis B virus replication in female transgenic mice.J Hepatol 2005:43:965-972.
[30]Chai Y, Huang H-L, Hu D-J, Luo X, Tao Q-S, Zhang X-L, et al. IL-29 and IFN-α regulate the expression of MxA,2',5'-OAS and PKR genes in association with the activation of Raf-MEK-ERK and PI3K-AKT signal pathways in HepG2.2.15 cells. Molecular Biology Reports 2010;38:139-143.
[31]Fensterl V, Sen GC. The 1SG56/IFIT1 gene family. J Interferon Cytokine Res 2011:31:71-78.
[32]Turelli P, Mangeat B, Jost S, Vianin S, Trono D. Inhibition of hepatitis B virus replication by APOBEC3G. Science 2004:303:1829.
[33]Robek MD, Wieland SF, Chisari FV. Inhibition of hepatitis B virus replication by interferon requires proteasome activity. J Virol 2002:76:3570-3574.
[34]Alsharifi M, Mullbacher A, Regner M. Interferon type Ⅰ responses in primary and secondary infections. Immunol Cell Biol 2008:86:239-245.
[35]梁蔚芳.聚乙二醇化干扰素α-2a与干扰素α-2a 在体内和体外的抗乙型肝炎病毒活性研究[博士学位论文]：第一军医大学;2007.
[36]曾伟强.派罗欣联合阿德福 韦酯治疗乙型肝炎效果观察.中国热带医学 2006,6;822-823.
[37]Gough DJ, Levy DE, Johnstone RW, Clarke CJ. IFNy signaling-Does it mean JAK-STAT? Cytokine & Growth Factor Reviews 2008:19:383-394.
[38]张夏华.,吴广通.,周萍.泛昔洛韦联合干扰素γ 抗HBV的临床疗效观察.中国现代应用药学杂志 2005;22：262-264.
[39]Guidotti LG, Chisari FV. Immunobiology and pathogenesis of viral hepatitis. Annu Rev Pathol 2006:1:23-61.
[40]Chisari FV. Hepatitis B virus transgenic mice:models of viral immunobiology and pathogenesis. CurrTop Microbiol Immunol 1996;206:149-173.
[41]Wieland S, Thimme R, Purcell RH, Chisari FV. Genomic analysis of the host response to hepatitis B virus infection. Proc Natl Acad Sci U S A 2004; 101:6669-6674.
[42]Raimondo G, Pollicino T, Cacciola I, Squadrito G. Occult hepatitis B virus infection. J Hepatol 2007;46:160-170.
[43]Samal J, Kandpal M, Vivekanandan P. Molecular mechanisms underlying occult hepatitis B virus infection. Clin Microbiol Rev 2012;25:142-163.
[44]Liaw YF. Impact of hepatitis B therapy on the long-term outcome of liver disease. Liver Int 2011;31 Suppl 1:117-121.
[45]Ghany MG, Doo EC. Antiviral resistance and hepatitis B therapy. Hepatology 2009;49:S174-184.
[46]Tillmann HL. Antiviral therapy and resistance with hepatitis B virus infection. World J Gastroenterol 2007; 13:125-140.
[47]Zoulim F. Antiviral therapy of chronic hepatitis B:can we clear the virus and prevent drug resistance? Antivir Chem Chemother 2004;15:299-305.
[48]Fournier C, Zoulim F. Antiviral therapy of chronic hepatitis B:prevention of drug resistance. Clin Liver Dis 2007; 11:869-892, ix.
[49]Genin P, Lin R, Hiscott J, Civas A. Differential regulation of human interferon A gene expression by interferon regulatory factors 3 and 7. Mol Cell Biol 2009;29:3435-3450.
[50]Lei CQ, Zhong B, Zhang Y, Zhang J, Wang S, Shu HB. Glycogen synthase kinase 3beta regulates IRF3 transcription factor-mediated antiviral response via activation of the kinase TBK1. Immunity 2010;33:878-889.
[51]Levy DE, Marie I, Smith E, Prakash A. Enhancement and diversification of IFN induction by lRF-7-mediated positive feedback. J Interferon Cytokine Res 2002;22:87-93.
[52]Heydtmann M. Macrophages in hepatitis B and hepatitis C virus infections. J Virol 2009;83:2796-2802.
[53]Cui GY, Diao HY. Recognition of HBV antigens and HBV DNA by dendritic cells. Hepatobiliary Pancreat Dis Int 2010;9:584-592.
[54]钱志平.,郑建铭.,朱梦琪.,王新宇.,陈明泉.,et al.乙型肝炎病毒抑制巨噬细胞TLR3、Mda-5和RIG-I表达.肝脏2010：15：425-428.
[55]安宝燕.,谢青.,下晖.,贾妮娜.,沈怀诚.,蔡伟.,et al.干扰素调节因子3在慢性乙型肝炎患者外周血树突状细胞中的表达及意义.世界华人消化杂志2008;16：1873-1879.
[56]Wieland SF, Vega RG, Muller R, Evans CF, Hiibush B, Guidotti LG, et al. Searching for interferon-induced genes that inhibit hepatitis B virus replication in transgenic mouse hepatocytes. J Virol 2003;77:1227-1236.
[57]胡静.干扰素诱生剂刺激肝细胞产生细胞因子研究[硕士学位论文]：重庆医科大学；2008.
[58]Han Q, Zhang C, Zhang J, Tian Z. Reversal of hepatitis B virus-induced immune tolerance by an immunostimulatory 3p-HBx-siRNAs in a retinoic acid inducible gene I-dependent manner. Hepatology 201];54:1179-1189.
[59]吴瑶瑶 陈HBcAg和TBK1对dsRNA诱导肝细胞表达IFN-β的调节作用[硕十学位论文]：浙江大学;2008.
[60]Wu J, Meng Z, Jiang M, Pei R, Trippler M, Broering R, et al. Hepatitis B virus suppresses toll-like receptor-mediated innate immune responses in murine parenchymal and nonparenchymal liver cells. Hepatology 2009;49:1132-1140.
[61]Yu S, Chen J, Wu M, Chen H, Kato N, Yuan Z. Hepatitis B virus polymerase inhibits RIG-I-and Toll-like receptor 3-mediated beta interferon induction in human hepatocytes through interference with interferon regulatory factor 3 activation and dampening of the interaction between TBK1/IKKepsilon and DDX3. J Gen Virol 2010;91:2080-2090.
[62]Wei C, Ni C, Song T, Liu Y, Yang X, Zheng Z, et al. The hepatitis B virus X protein disrupts innate immunity by downregulating mitochondrial antiviral signaling protein. J Immunol 2010;185:1158-1168.
[63]Kumar M, Jung SY, Hodgson AJ, Madden CR, Qin J, Slagle BL. Hepatitis B virus regulatory HBx protein binds to adaptor protein IPS-1 and inhibits the activation of beta interferon. J Virol 2011;85:987-995.
[64]Wang X, Li Y, Mao A, Li C, Tien P. Hepatitis B virus X protein suppresses virus-triggered IRF3 activation and IFN-beta induction by disrupting the VISA-associated complex. Cell Mol Immunol 2010;7:341-348.
[65]Jiang J, Tang H. Mechanism of inhibiting type I interferon induction by hepatitis B virus X protein. Protein Cell 2010; 1:1106-1117.
[66]Schindler C,Levy DE, Decker T. JAK-STAT signaling:from interferons to cytokines. J Biol Chem 2007;282:20059-20063.
[67]Schoggins JW, Rice CM. Interferon-stimulated genes and their antiviral effector functions. Current Opinion in Virology 2011; 1:519-5.25.
[68]Liu M, Hao Y, Ding H, Yang D, Lu M. Interleukin-10 is expressed in HepG2.2.15 cells and regulated by STAT1 pathway. J Huazhong Univ Sci Technolog Med Sci 2011;31:625-631.
[69]樊和斌.,郭亚兵.,王宝菊.,朱幼芙.,吴爱华.,侯金林.,et al.慢性乙型肝炎患者肝组织及外周血单核细胞内IFN-α/β受体的表达及其临床意义.南方医科大学学报2008;28：979-981.
[70]任苏平.乙型肝炎病毒对I型干扰素受体表达的影响[硕士学位论文]：浙江大学；2000.
[71]Chung Y-H. Hepatitis B virus X protein inhibits extracellular IFN-a-mediated signal transduction by downregulation of type I IFN receptor. International Journal of Molecular Medicine 2012.
[72]Guan SH, Lu M, Grunewald P, Roggendorf M, Gerken G, Schlaak JF. Interferon-alpha response in chronic hepatitis B-transfected HepG2.2.15 cells is partially restored by lamivudine treatment. World J Gastroenterol 2007; 13:228-235.
[73]熊炜.干扰素诱导的MyD88蛋白抑制乙型肝炎病毒复制的研究[博士学位论文]：复旦大学；2004.
[74]王迅.α干扰素诱导的细胞蛋白与乙型肝炎病毒相互作用关系的研究[博士学位论文]：复旦大学博士学位论文;2004.
[75]邬敏.乙型肝炎病毒抑制洳干扰素诱导的MyD88表达的机制研究[硕士学位论文]：复旦大学;2007.
[76]林珊珊.MyD88蛋白抑制乙型肝炎病毒基因复制的分子机制研究[博士学位论文]：复旦大学：2007.
[77]Wu M, Xu Y, Lin S, Zhang X, Xiang L, Yuan Z. Hepatitis B virus polymerase inhibits the interferon-inducible MyD88 promoter by blocking nuclear translocation of Statl. J Gen Virol 2007:88:3260-3269.
[78]于士颜.乙型肝炎病毒(HBV)拮抗IFN-a信号通路的初步研究[硕士学位论文]：复旦大学;2005.
[79]王林.乙型肝炎病毒Tp相关蛋白抗干扰素作用研究[博士学位论文]：福建医科大学;2009.
[80]管世鹤.,杨凯.,潘颖.乙型肝炎病毒抑制α干扰素诱导抗病毒蛋白表达的研究.安徽医科大学学报2011；46：324-327.
[81]管世鹤.,杨凯.,陆蒙吉.,卢银平.,杨东亮.乙型肝炎病毒及其抗原成分对干扰素信号传导途径分子和抗病毒蛋白表达的影响.中华肝脏病杂志2011;19：440-444.
[82]Siebler J, Protzer U, Wirtz S, Schuchmann M, Hohler T, Galle PR, et al. Overexpression of STAT-1 by adenoviral gene transfer does not inhibit hepatitis B virus replication. Eur J Gastroenterol Hepatol 2006;18:167-174.
[83]Zhang Q, Wang Y, Wei L, Jiang D, Wang JH, Rao HY, et al. Role of ISGF3 in modulating the anti-hepatitis B virus activity of interferon-alpha in vitro. J Gastroenterol Hepatol 2008;23:1747-1761.
[84]Christen V, Treves S, Duong FH, Heim MH. Activation of endoplasmic reticulum stress response by hepatitis viruses up-regulates protein phosphatase 2A. Hepatology 2007;46:558-565.
[85]Li J, Chen F, Zheng M, Zhu H, Zhao D, Liu W, et al. Inhibition of STAT1 methylation is involved in the resistance of hepatitis B virus to Interferon alpha. Antiviral Research 2010;85:463-469.
[86]程中乐.,管世鹤.,杨凯.,潘颖SOCS1对a干扰素调节乙肝病毒复制作用的影响.安徽医科大学学报2011：46：228-230.
[87]Su C, Hou Z, Zhang C, Tian Z, Zhang J. Ectopic expression of microRNA-155 enhances innate antiviral immunity against HBV infection in human hepatoma cells. Virol J 2011;8:354.
[88]Fish EN, Uddin S, Korkmaz M, Majchrzak B, Druker BJ, Platanias LC. Activation of a CrkL-stat5 signaling complex by type I interferons. J Biol Chem 1999;274:571-573.
[89]Uddin S, Lekmine F, Sharma N, Majchrzak B, Mayer I, Young PR, et al. The Racl/p38 mitogen-activated protein kinase pathway is required for interferon alpha-dependent transcriptional activation but not serine phosphorylation of Stat proteins. J Biol Chem 2000;275:27634-27640.
[90]Uddin S, Majchrzak B, Woodson J, Arunkumar P, Alsayed Y, Pine R, et al. Activation of the p38 mitogen-activated protein kinase by type I interferons. J Biol Chem 1999;274:30127-30131.
[91]Uddin S, Sassano A, Deb DK, Verma A, Majchrzak B, Rahman A, et al. Protein kinase C-delta (PKC-delta) is activated by type I interferons and mediates phosphorylation of Statl on serine 727. J Biol Chem 2002;277:14408-14416.
[92]Haller O, Kochs G. Human MxA protein:an interferon-induced dynamin-like GTPase with broad antiviral activity. J Interferon Cytokine Res 2011;31:79-87.
[93]Li N, Zhang L, Chen L, Feng W, Xu Y, Chen F, et al. MxA inhibits hepatitis B virus replication by interaction with core protein HBcAg. Hepatology 2012.
[94]Gordien E, Rosmorduc O, Peltekian C, Garreau F, Brechot C, Kremsdorf D. Inhibition of hepatitis B virus replication by the interferon-inducible MxA protein. J Virol 2001;75:2684-2691.
[95]杨凯.HBV及其抗原成分拮抗α干扰素抗病毒活性的初步研究[硕士学位论文]：安徽医科大学;2010.
[96]Rosmorduc O, Petit MA, Pol S, Capel F, Bortolotti F, Berthelot P, et al. In vivo and in vitro expression of defective hepatitis B virus particles generated by spliced hepatitis B virus RNA. Hepatology 1995;22:10-19.
[97]Fernandez M, Quiroga JA, Carreno V. Hepatitis B virus downregulates the human interferon-inducible MxA promoter through direct interaction of precore/core proteins. J Gen Virol 2003;84:2073-2082.
[98]Rosmorduc O, Sirma H, Soussan P, Gordien E, Lebon P, Horisberger M, et al. Inhibition of interferon-inducible MxA protein expression by hepatitis B virus capsid protein. J Gen Virol 1999;80 (Pt5):1253-1262.
[99]Yang YL, Reis LF, Pavlovic J, Aguzzi A, Schafer R, Kumar A, et al. Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase. EMBOJ 1995; 14:6095-6106.
[100]Park I-H, Baek K-W, Cho E-Y, Ahn B-Y. PKR-dependent mechanisms of interferon-a for inhibiting hepatitis B virus replication. Molecules and Cells 2011;32:167-172.
[101]Chen GG, Lai PB, Ho RL, Chan PK, Xu H, Wong J, et al. Reduction of double-stranded RNA-activated protein kinase in hepatocellular carcinoma associated with hepatitis B virus. J Med Virol 2004;73:187-194.
[102]Han Q, Zhang C, Zhang J, Tian Z. Involvement of activation of PKR in HBx-siRNA-mediated innate immune effects on HBV inhibition. PLoS One 2011;6:e27931.
[103]Takaori-Kondo A. APOBEC family proteins:novel antiviral innate immunity. Int J Hematol 2006;83:213-216.
[104]Cullen BR. Role and mechanism of action of the APOBEC3 family of antiretroviral resistance factors. J Virol 2006;80:1067-1076.
[105]Suspene R. Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo. Proceedings of the National Academy of Sciences 2005;102:8321-8326.
[106]Bonvin M, Greeve J. Hepatitis B:modern concepts in pathogenesis--APOBEC3 cytidine deaminases as effectors in innate immunity against the hepatitis B virus. Curr Opin Infect Dis 2008;21:298-303.
[107]Bishop KN, Holmes RK, Sheehy AM, Davidson NO, Cho SJ, Malim MM. Cytidine deamination of retroviral DNA by diverse APOBEC proteins. Curr Biol 2004; 14:1392-1396.
[108]Kim BK, Revill PA, Ahn SH. HBV genotypes:relevance to natural history, pathogenesis and treatment of chronic hepatitis B. Antivir Ther 2011;16:1169-1186.
[109]Erhardt A, Blondin D, Hauck K, Sagir A, Kohnle T, Heintges T, et al. Response to interferon alfa is hepatitis B virus genotype dependent:genotype A is more sensitive to interferon than genotype D. Gut 2005;54:1009-1013.
[110]Ma JC, Wang LW, Li XJ, Liao YF, Hu XY, Gong ZJ. Relationship between HBV genotypes and anti-viral therapeutic efficacy of interferon-alpha. Hepatobiliary Pancreat Dis Int 2007;6:166-171.
[111]中华医学会肝病学分会.慢性乙型肝炎防治指南(2010年版).中华肝病杂志2011;19：13-24,
[112]邵光.干扰素作用下乙肝病毒C基因区变异机制的体外研究及HepG2.2.15细胞在研究中的应用[硕士学位论文]：中山大学;2006.
[113]Liu CJ, Kao JH. Genetic variability of hepatitis B virus and response to antiviral therapy. Antivir Ther 2008; 13:613-624.
[114]王卫峰.,周晓东.干扰素作用下乙型肝炎病毒变异的体外实验研究.中华肝脏病杂志2005;13：793-794.
[115]刘彦华,倪旭.HBV 前 C区基因变异与乙肝病毒复制的相关性.河北医药2007;29：1056-1058.
[116]Chotiyaputta W, Lok AS. Hepatitis B virus variants. Nat Rev Gastroenterol Hepatol 2009;6:453-462.
[117]Mason A, Wick M, White H, Perrillo R. Hepatitis B virus replication in diverse cell types during chronic hepatitis B virus infection. Hepatology 1993;18:781-789.
[118]Schildgen O. Host factors may influence response to antiviral therapy in chronic hepatitis B virus infections. Medical Hypotheses 2011;76:417-420.
[119]卢年芳.,郑瑞强.,林华.,黄爱龙.,杨德刚.不同亚型IFN-α对STAT1表达的影响.江苏医药2006；32：901-903.
[120]He XX, Chang Y, Jiang HJ, Tang F, Meng FY, Xie OH, et al. Persistent effect of IFNAR-I genetic polymorphism on the long-term pathogenesis of chronic HBV infection. Viral Immunol 2010:23:251-257.
[121]Hsieh YY, Chang CC, Hsu CM, Wan L, Chen SY, Lin WH, et al. JAK-1 rs2780895 C-related genotype and allele but not JAK-1 rs10789166, rs4916008, rs2780885, rs17127114, and rs3806277 are associated with higher susceptibility to asthma. Genet Test Mol Biomarkers 2011;15:841-847.
[122]Zhu ZZ, Di JZ, Gu WY, Cong WM, Gawron A, Wang Y, et al. Association of genetic polymorphisms in STAT1 gene with increased risk of hepatocellular carcinoma. Oncology 2010;78:382-388.
[123]Ren S, Yu H, Zhang H, Liu Y, Huang Y, Ma L, et al. Polymorphisms of interferon-inducible genes OAS associated with interferon-alpha treatment response in chronic HBV infection. Antiviral Res 2011;89:232-237.
[124]Abe H, Hayes CN, Ochi H, Tsuge M, Miki D, Hiraga N, et al. Inverse association of IL28B genotype and liver mRNA expression of genes promoting or suppressing antiviral state. J Med Virol 2011;83:1597-1607.
[125]孔晓飞.干扰素信号传导系统与乙型肝炎相关性研究[硕十学位论文]：上海第二医科大学;2004.
[126]Durbin JE, Hackenmiller R, Simon MC, Levy DE. Targeted disruption of the mouse Statl gene results in compromised innate immunity to viral disease. Cell 1996;84:443-450.
[127]陈禄彪.,彭晓谋.,曹红.,张宇峰.,徐启桓.,高志良.OAS-1基因SNP rs 10774671(?)慢性HBV感染者自发性HBeAg血清转换的关系.中华实验和临床病毒学杂志2009;23：35-37.
1. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RN As with antisense complementarity to lin-14. Cell.1993 Dec 3;75(5):843-54.
2. Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell.1993 Dec 3;75(5):855-62.
3. Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Mailer B, et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature.2000 Nov 2;408(6808):86-9.
4. Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP. MicroRNAs in plants. Genes Dev. 2002 Jul 1;16(13):1616-26.
5. Hamilton AJ, Baulcombe DC. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science.1999 Oct 29;286(5441):950-2.
6. Ambros V, Bartel B, Bartel DP, Burge CB, Carrington JC, Chen X, et al. A uniform system for microRNA annotation. RNA.2003 Mar;9(3):277-9.
7. Griffiths-Jones S. The microRNA Registry. Nucleic Acids Res.2004 Jan 1;32(Database issue):D109-11.
8. Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ. miRBase:microRNA sequences, targets and gene nomenclature. Nucleic Acids Res.2006 Jan 1;34(Database issue):D140-4.
9. Kozomara A, Griffiths-Jones S. miRBase:integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res.2011 Jan;39(Database issue):D 152-7.
10. Winter J, Jung S, Keller S, Gregory RI, Diederichs S. Many roads to maturity:microRNA biogenesis pathways and their regulation. Nat Cell Biol.2009 Mar;11(3):228-34.
11. Ying SY, Lin SL. Intronic microRNAs. Biochem Biophys Res Commun.2005 Jan 21;326(3):515-20.
12. Sibley CR, Seow Y, Saayman S, Dijkstra KK, El Andaloussi S, Weinberg MS, et al. The biogenesis and characterization of mammalian microRNAs of mirtron origin. Nucleic Acids Res.2012 Jan;40(1):438-48.
13. Kim VN. MicroRNA biogenesis:coordinated cropping and dicing. Nat Rev Mol Cell Biol.2005 May;6(5):376-85.
14. Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH, et al. MicroRNA genes are transcribed by RNA polymerase 11. EMBO J.2004 Oct 13;23(20):4051-60.
15. Cullen BR. Transcription and processing of human microRNA precursors. Mol Cell.2004 Dec 22;16(6):861-5.
16. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. The nuclear RNase Ⅲ Drosha initiates microRNA processing. Nature.2003 Sep 25;425(6956):415-9.
17. Landthaler M, Yalcin A, Tuschl T. The human DiGeorge syndrome critical region gene 8 and Its D. melanogaster homolog are required for miRNA biogenesis. Curr Biol.2004 Dec 14; 14(23):2162-7.
18. Yi R, Qin Y, Macara IG, Cullen BR. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev.2003 Dec 15;17(24):3011-6.
19. Zeng Y, Cullen BR. Structural requirements for pre-microRNA binding and nuclear export by Exportin 5. Nucleic Acids Res.2004;32(16):4776-85.
20. Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K, et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature.2005 Aug 4;436(7051):740-4.
21. Lee Y, Hur I, Park SY, Kim YK, Suh MR, Kim VN. The role of PACT in the RNA silencing pathway. EMBO J.2006 Feb 8;25(3):522-32.
22. Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell. 2003 Oct 17;115(2):209-16.
23. Song JJ, Smith SK, Hannon GJ, Joshua-Tor L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science.2004 Sep 3;305(5689):1434-7.
24. Meister G, Tuschl T. Mechanisms of gene silencing by double-stranded RNA. Nature.2004 Sep 16;431(7006):343-9.
25. Krol J, Loedige I, Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet.2010 Sep; 11 (9):597-610.
26. Wu L, Fan J, Belasco JG. MicroRNAs direct rapid deadenylation of mRNA. Proc Natl Acad Sci U SA.2006 Mar14;103(11):4034-9.
27. Okamura K, Ishizuka A, Siomi H, Siomi MC. Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev.2004 Jul 15; 18(14):1655-66.
28. Bartel DP. MicroRNAs:genomics, biogenesis, mechanism, and function. Cell.2004 Jan 23;116(2):281-97.
29. Lewis BP, Burge CB, Bartel DP. Conserved Seed Pairing, Often Flanked by Adenosines, Indicates that Thousands of Human Genes are MicroRNA Targets. Cell.2005; 120(1):15-20.
30. Duursma AM, Kedde M, Schrier M, le Sage C, Agami R. miR-148 targets human DNMT3b protein coding region. RNA.2008 May;14(5):872-7.
31. Lee I, Ajay SS, Yook JI, Kim HS, Hong SH, Kim NH, et al. New class of microRNA targets containing simultaneous 5'-UTR and 3'-UTR interaction sites. Genome Res.2009 Jul; 19(7):1175-83.
32. Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation:microRNAs can up-regulate translation. Science.2007 Dec 21;318(5858):1931-4.
33. Djuranovic S, Nahvi A, Green R. miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay. Science.2012 Apr 13;336(6078):237-40.
34. Smith SM, Murray DW. An overview of microRNA methods:expression profiling and target identification. Methods Mol Biol.2012,823:119-38.
35. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of mammalian microRNA targets. Cell.2003 Dec 26; 115(7):787-98.
36. Rehmsmeier M, Steffen P, Hochsmann M, Giegerich R. Fast and effective prediction of microRNA/target duplexes. RNA.2004 Oct; 10(10):1507-17.
37. Takada S, Asahara H. Current strategies for microRNA research. Mod Rheumatol.2012 Jan 12.
38. Farh KK, Grimson A, Jan C, Lewis BP, Johnston WK, Lim LP, et al. The widespread impact of mammalian MicroRNAs on mRNA repression and evolution. Science.2005 Dec 16;310(5755):1817-21.
39. Chang J, Nicolas E, Marks D, Sander C, Lerro A, Buendia MA, et al. miR-122, a mammalian liver-specific microRNA, is processed from her mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1. RNA Biol.2004 Jul; 1(2):106-13.
40. de Planell-Saguer M, Rodicio MC. Analytical aspects of microRNA in diagnostics:a review. Anal Chim Acta.2011 Aug 12;699(2):134-52.
41. Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elcgans. Science.2001 Oct 26;294(5543):862-4.
42. Bernardo BC, Charchar FJ, Lin RC, McMullen JR. A microRNA guide for clinicians and basic scientists:background and experimental techniques. Heart LungCirc.2012 Mar;21(3):131-42.
43. Lai EC. Predicting and validating microRNA targets. Genome Biol.2004;5(9):115.
44. Lim LP, Lau NC, Weinstein EG, Abdelhakim A, Yekta S. Rhoades MW, et al. The microRNAs of Caenorhabditis elegans. Genes Dev.2003 Apr 15; 17(8):991-1008.
45. Doran J, Strauss WM. Bio-informatic trends for the determination of miRNA-target interactions in mammals. DNA Cell Biol.2007 May;26(5):353-60.
46. Liu H, Yue D, Zhang L, Chen Y, Gao SJ, Huang Y. A Bayesian approach for identifying miRNA targets by combining sequence prediction and gene expression profiling. BMC Genomics.2010; 11 Suppl3:S12.
47. Zhou X, Ruan J, Wang G, Zhang W. Characterization and identification of microRNA core promoters in four model species. PLoS Comput Biol.2007 Mar 9;3(3):e37.
48. Stern-Ginossar N, Elefant N, Zimmermann A, Wolf DG, Saleh N, Biton M, et al. Host immune system gene targeting by a viral miRNA. Science.2007 Jul 20;317(5836):376-81.
49. Zhang Y. miRU:an automated plant miRNA target prediction server. Nucleic Acids Res.2005 Jul 1;33(Web Server issue):W701-4.
50. Rodriguez A, Griffiths-Jones S, Ashurst JL, Bradley A. Identification of mammalian microRNA host genes and transcription units. Genome Res.2004 Oct;14(10A):1902-10.
51. Mu W, Zhang W. Bioinformatic Resources of microRNA Sequences, Gene Targets, and Genetic Variation. Front Genet.2012;3:31.
52. Tora L, Dong H, Paquette M, Williams A, Zoeller RT, Wade M, et al. Thyroid Hormone May Regulate mRNA Abundance in Liver by Acting on MicroRNAs. PLoS One.2010;5(8):el2136.
53. Vergoulis T, Vlachos IS, Alexiou P, Georgakilas G, Maragkakis M, Reczko M, et al. TarBase 6.0: capturing the exponential growth of miRNA targets with experimental support. Nucleic Acids Res. 2012 Jan;40(Database issue):D222-9.
54. Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP. MicroRNA targeting specificity in mammals:determinants beyond seed pairing. Mol Cell.2007 Jul 6;27(1):91-105.
55. Krek A, Grun D, Poy MN, Wolf R, Rosenberg L, Epstein EJ, et al. Combinatorial microRNA target predictions. Nat Genet.2005 May;37(5):495-500.
56. Gamazon ER, Im HK, Duan S, Lussier YA, Cox NJ, Dolan ME, et al. Exprtarget:an integrative approach to predicting human microRNA targets. PLoS One.2010;5(10):e13534.
57. Wang X. miRDB:a microRNA target prediction and functional annotation database with a wiki interface. RNA.2008 Jun;14(6):1012-7.
58. Walter AE, Turner DH, Kim J, Lyttle MH, Muller P, Mathews DH, et al. Coaxial stacking of helixes enhances binding of oligoribonucleotides and improves predictions of RNA folding. Proc Natl Acad Sci U S A.1994 Sep 27;91(20):9218-22.
59. Lindgreen S, Gardner PP, Krogh A. MASTR:multiple alignment and structure prediction of non-coding RNAs using simulated annealing. Bioinformatics.2007 Dec 15;23(24):3304-11.
60. Will S, Reiche K, Hofacker IL, Stadler PF, Backofen R. Inferring noncoding RNA families and classes by means of genome-scale structure-based clustering. PLoS Comput Biol.2007 Apr 13;3(4):e65.
61. Hofacker IL. Vienna RNA secondary structure server. Nucleic Acids Res.2003 Jul 1;31(13):3429-31.
62. Li SC, Pan CY, Lin WC. Bioinformatic discovery of microRNA precursors from human ESTs and introns. BMC Genomics.2006;7:164.
63. Kiezun A, Artzi S, Modai S, Volk N, Isakov O, Shomron N. miRviewer:a multispecies microRNA homologous viewer. BMC Res Notes.2012;5:92.
64. Zhang Y, Yang Y, Zhang H, Jiang X, Xu B, Xue Y, et al. Prediction of novel pre-microRNAs with high accuracy through boosting and SVM. Bioinformatics.2011 May 15;27(10):1436-7.
65. Wu Y, Wei B, Liu H, Li T, Rayner S. MiRPara:a SVM-based software tool for prediction of most probable microRNA coding regions in genome scale sequences. BMC Bioinformatics.2011; 12:107.
66. Thieme CJ, Gramzow L, Lobbes D, Theissen G. SplamiR--prediction of spliced miRN As in plants. Bioinformatics.2011 May 1;27(9):1215-23.
67. Szczesniak MW, Deorowicz S, Gapski J, Kaczynski L, Makalowska I. miRNEST database:an integrative approach in microRNA search and annotation. Nucleic Acids Res.2012 Jan;40(Database issue):D 198-204.
68. Gkirtzou K, Tsamardinos I, Tsakalides P, Poirazi P. MatureBayes:a probabilistic algorithm for identifying the mature miRNA within novel precursors. PLoS One.2010;5(8):e11843.
69. Hackenberg M, Sturm M, Langenberger D, Falcon-Perez JM, Aransay AM. miRanalyzer:a microRNA detection and analysis tool for next-generation sequencing experiments. Nucleic Acids Res. 2009 Jul;37(Web Server issue):W68-76.
70. Mitra R, Bandyopadhyay S. MultiMiTar:a novel multi objective optimization based miRNA-target prediction method. PLoS One.2011;6(9):e24583.
71. Reczko M, Maragkakis M, Alexiou P, Grosse I, Hatzigeorgiou AG. Functional microRNA targets in protein coding sequences. Bioinformatics.2012 Mar 15;28(6):771-6.
72. Elefant N, Berger A, Shein H, Hofree M, Margalit H, Altuvia Y. RepTar:a database of predicted cellular targets of host and viral miRNAs. Nucleic Acids Res.2011 Jan;39(Database issue):D188-94.
73. Le Brigand K, Robbe-Sermesant K, Mari B, Barbry P. MiRonTop:mining microRNAs targets across large scale gene expression studies. Bioinformatics.2010 Dec 15;26(24):3131-2.
74. Xie F, Zhang B. Target-align:a tool for plant microRNA target identification. Bioinformatics. 2010 Dec 1;26(23):3002-3.
75. Reyes-Herrera PH, Ficarra E, Acquaviva A, Macii E. miREE:miRNA recognition elements ensemble. BMC Bioinformatics.2011; 12:454.
76. Dai X, Zhao PX. psRNATarget:a plant small RNA target analysis server. Nucleic Acids Res.2011 Jul;39(Web Server issue):W155-9.
77. Sualp M, Can T. Using network context as a filter for miRNA target prediction. Biosystems. 2011;105(3):201-9.
78. Huang GT, Athanassiou C, Benos PV. mirConnX:condition-specific mRNA-microRNA network integrator. Nucleic Acids Res.2011 Jul;39(Web Server issue):W416-23.
79. Xiao F, Zuo Z, Cai G, Kang S, Gao X, Li T. mi Records:an integrated resource for microRNA-target interactions. Nucleic Acids Res.2009 Jan;37(Database issue):D105-10.
80. Qiu C, Wang D, Wang E, Cui Q. An upstream interacting context based framework for the computational inference of microRNA functions. Mol Biosyst.2012 Apr 3;8(5):1492-8.
81. Laczny C, Leidinger P, Haas J, Ludwig N, Backes C, Gerasch A, et al. miRTrail-a comprehensive webserver for analyzing gene and miRNA patterns to enhance the understanding of regulatory mechanisms in diseases. BMC Bioinformatics.2012; 13:36.
82. Bruno AE, Li L, Kalabus JL, Pan Y, Yu A, Hu Z. miRdSNP:a database of disease-associated SNPs and microRNA target sites on 3'UTRs of human genes. BMC Genomics.2012;13:44.
83. Xu X, Zhao Y, Simon R. Gene Set Expression Comparison kit for BRB-ArrayTools. Bioinformatics.2008 Jan 1;24(I):137-9.
84. Hua Y, Duan S,Murmann AE, Larsen N, Kjems J, Lund AH, et al. miRConnect:identifying effector genes of miRNAs and miRNA families in cancer cells. PLoS One.2011;6(10):e26521.
85. Mathelier A, Carbone A. MIReNA:finding microRNAs with high accuracy and no learning at genome scale and from deep sequencing data. Bioinformatics.2010 Sep 15;26(18):2226-34.
86. Hendrix D, Levine M, Shi W. miRTRAP, a computational method for the systematic identification of miRNAs from high throughput sequencing data. Genome Biol.2010; 11(4):R39.
87. Rodriguez A, Vigorito E, Clare S, Warren MV, Couttet P, Soond DR, et al. Requirement of bic/microRNA-155 for normal immune function. Science.2007 Apr 27;316(5824):608-11.
88. van Rooij E. The art of microRNA research. Circ Res.2011 Jan 21;108(2):219-34.
89. Willenbrock H, Salomon J, Sokilde R, Barken KB, Hansen TN, Nielsen FC, et al. Quantitative miRNA expression analysis:comparing microarrays with next-generation sequencing. RNA.2009 Nov;15(11):2028-34.
90. Git A, Dvinge H, Salmon-Divon M, Osborne M, Kutter C, Hadfield J, et al. Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRN A expression. RNA.2010 May; 16(5):991-1006.
91. Fehniger TA, Wylie T, Germino E, Leong JW, Magrini VJ, Koul S, et al. Next-generation sequencing identifies the natural killer cell microRN A transcriptome. Genome Res.2010 Nov;20(11):1590-604.
92. Hafner M, Landthaler M, Burger L, Khorshid M, Hausser J, Berninger P, et al. PAR-CliP--a method to identify transcriptome-wide the binding sites of RNA binding proteins. J Vis Exp.2010(41).
93. Keene JD, Komisarow JM, Friedersdorf MB. RIP-Chip:the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nat Protoc.2006; 1(1):302-7.
94. Thomson DW, Bracken CP, Goodall GJ. Experimental strategies for microRNA target identification. Nucleic Acids Res.2011 Sep 1;39(16):6845-53.
95. Wang WX, Wilfred BR, Hu Y, Stromberg AJ, Nelson PT. Anti-Argonaute RIP-Chip shows that miRNA transfections alter global patterns of mRNA recruitment to microribonucleoprotein complexes. RNA.2009;16(2):394-404.
96. Tan LP, Seinen E, Duns G, de Jong D, Sibon OC, Poppema S, et al. A high throughput experimental approach to identify miRNA targets in human cells. Nucleic Acids Res.2009 Nov;37(20):e137.
97. Huang Y, Zou Q, Wang SP, Tang SM, Zhang GZ, Shen XJ. The discovery approaches and detection methods of microRNAs. Mol Biol Rep.2011 Aug;38(6):4125-35.
1. Seeger C, Mason WS. Hepatitis B virus biology. Microbiol Mol Biol Rev.2000 Mar;64(1):51-68.
2. Chang JJ, Lewin SR. Immunopathogenesis of hepatitis B virus infection. Immunol Cell Biol. 2007 Jan;85(1):16-23.
3. Tsai HT, Tsai TH, Lu TM, Yang CC. Immunopathology of hepatitis B virus infection. Int Rev Immunol.2008;27(6):427-46.
4. Hoofnagle JH, Doo E, Liang TJ, Fleischer R, Lok AS. Management of hepatitis B:summary of a clinical research workshop. Hepatology.2007 Apr;45(4):1056-75.
5. Duong A, Mousa SA. Current status of nucleoside antivirals in chronic hepatitis B. Drugs Today (Barc).2009 Oct;45(10):751-61.
6. Buster EH, Janssen HL. Antiviral treatment for chronic hepatitis B virus infection--immune modulation or viral suppression? Neth J Med.2006 Jun;64(6):175-85.
7. Lin CL, Kao JH. Recent advances in the treatment of chronic hepatitis B. Expert Opin Pharmacother.2011 Sep;12(13):2025-40.
8. Niederhauser C. Reducing the risk of hepatitis B virus transfusion-transmitted infection. J Blood Med.2011;2:91-102.
9. Sayed D, Abdellatif M. MicroRNAs in development and disease. Physiol Rev.2011 Jul;91(3):827-87.
10. Taganov KD, Boldin MP, Baltimore D. MicroRNAs and immunity:tiny players in a big field. Immunity.2007 Feb;26(2):133-7.
11. Thai TH, Calado DP, Casola S, Ansel KM, Xiao C, Xue Y, et al. Regulation of the germinal center response by microRNA-155. Science.2007 Apr 27;316(5824):604-8.
12. Liu G, Min H, Yue S, Chen CZ. Pre-miRNA loop nucleotides control the distinct activities of mir-181 a-1 and mir-181 c in early T cell development. PLoS One.2008;3(10):e3592.
13. Li QJ, Chau J, Ebert PJ, Sylvester G, Min H, Liu G, et al. miR-181a is an intrinsic modulator of T cell sensitivity and selection. Cell.2007 Apr 6; 129(1):147-61.
14. Fazi F, Rosa A, Fatica A, Gelmetti V, De Marchis ML, Nervi C, et al. A Minicircuitry Comprised of MicroRNA-223 and Transcription Factors NFI-A and C/EBPa Regulates Human Granulopoiesis. Cell.2005;123(5):819-31.
15. Rodriguez A, Vigorito E, Clare S, Warren MV, Couttet P, Soond DR, et al. Requirement of bic/microRNA-155 for normal immune function. Science.2007 Apr 27;316(5824):608-11.
16. Vigorito E, Perks KL, Abreu-Goodger C, Bunting S, Xiang Z, Kohlhaas S, et al. microRNA-155 regulates the generation of immunoglobulin class-switched plasma cells. Immunity.2007 Dec;27(6):847-59.
17. Xiao C, Calado DP, Galler G, Thai T-H, Patterson HC, Wang J, et al. MiR-150 Controls B Cell Differentiation by Targeting the Transcription Factor c-Myb. Cell.2007; 131(1):146-59.
18. de Yebenes VG, Belver L, Pisano DG, Gonzalez S, Villasante A, Croce C, et al. miR-181b negatively regulates activation-induced cytidine deaminase in B cells. Journal of Experimental Medicine.2008;205(10):2199-206.
19. Ma F, Xu S, Liu X, Zhang Q, Xu X, Liu M, et al. The microRNA miR-29 controls innate and adaptive immune responses to intracellular bacterial infection by targeting interferon-y. Nature Immunology.2011; 12(9):861-9.
20. Chen XM, Splinter PL, O'Hara SP, LaRusso NF. A cellular micro-RNA, let-7i, regulates Toll-like receptor 4 expression and contributes to cholangiocyte immune responses against Cryptosporidium parvum infection. J Biol Chem.2007 Sep 28;282(39):28929-38.
21. Hou J, Wang P, Lin L, Liu X, Ma F, An H, et al. MicroRNA-146a Feedback Inhibits RIG-I-Dependent Type I IFN Production in Macrophages by Targeting TRAF6, IRAKI, and IRAK2. The Journal of Immunology.2009;183(3):2150-8.
22. Murphy E, Vanicek J, Robins H, Shenk T, Levine AJ. Suppression of immediate-early viral gene expression by herpesvirus-coded microRNAs:implications for latency. Proc Natl Acad Sci U S A.2008 Apr 8;105(14):5453-8.
23. Choy EY, Siu KL, Kok KH, Lung RW, Tsang CM, To KF, et al. An Epstein-Barr virus-encoded microRNA targets PUMA to promote host cell survival. J Exp Med.2008 Oct 27;205(11):2551-60.
24. Umbach JL, Kramer MF, Jurak I, Karnowski HW, Coen DM, Cullen BR. MicroRNAs expressed by herpes simplex virus 1 during latent infection regulate viral mRNAs. Nature.2008 Aug 7;454(7205):780-3.
25. Lecellier CH, Dunoyer P, Arar K, Lehmann-Che J, Eyquem S, Himber C, et al. A cellular microRNA mediates antiviral defense in human cells. Science.2005 Apr 22;308(5721):557-60.
26. Gao P, Wong CC-L, Tung EK-K, Lee JM-F, Wong C-M, Ng IO-L. Deregulation of microRNA expression occurs early and accumulates in early stages of HBV-associated multistep hepatocarcinogenesis. Journal of Hepatology.2011;54(6):1177-84.
27. Ladeiro Y, Couchy G, Balabaud C, Bioulac-Sage P, Pelletier L, Rebouissou S, et al. MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations. Hepatology.2008;47(6):1955-63.
28. Guan X-y, Mizuguchi Y, Mishima T, Yokomuro S, Arima Y, Kawahigashi Y, et al. Sequencing and Bioinformatics-Based Analyses of the microRNA Transcriptome in Hepatitis B-Related Hepatocellular Carcinoma. PLoS One.2011;6(1):e 15304.
29. Zhou J, Yu L, Gao X, Hu J, Wang J, Dai Z, et al. Plasma microRNA panel to diagnose hepatitis B virus-related hepatocellular carcinoma. J Clin Oncol.2011 Dec 20;29(36):4781-8.
30. Li LM, Hu ZB, Zhou ZX, Chen X, Liu FY. Zhang JF. et al. Serum microRNA Profiles Serve as Novel Biomarkers for HBV Infection and Diagnosis of HBV-Positive Hepatocarcinoma. Cancer Research.2010;70(23):9798-807.
31. Wang S, Qiu L, Yan X, Jin W, Wang Y, Chen L, et al. Loss of microRNA 122 expression in patients with hepatitis B enhances hepatitis B virus replication through cyclin G1-modulated P53 activity. Hepatology.2012;55(3):730-41.
32. Li R-F. miR-122 inhibits viral replication and cell proliferation in hepatitis B virus-related hepatocellular carcinoma and targets NDRG3. Oncology Reports.2011.
33. Qiu L, Fan H, Jin W, Zhao B, Wang Y, Ju Y, et al. miR-122-induced down-regulation of HO-1 negatively affects miR-122-mediated suppression of HBV. Biochemical and Biophysical Research Communications.2010;398(4):771-7.
34. Chen Y, Shen A, Rider PJ, Yu Y, Wu K, Mu Y, et al. A liver-specific microRNA binds to a highly conserved RNA sequence of hepatitis B virus and negatively regulates viral gene expression and replication. The FASEB Journal.2011;25(12):4511-21.
35. Potenza N, Papa U, Mosca N, Zerbini F, Nobile V, Russo A. Human microRNA hsa-miR-125a-5p interferes with expression of hepatitis B virus surface antigen. Nucleic Acids Research. 2011;39(12):5157-63.
36. Zhang G-1, Li Y-x, Zheng S-q, Liu M, Li X, Tang H. Suppression of hepatitis B virus replication by microRNA-199a-3p and microRNA-210. Antiviral Research.2010;88(2):169-75.
37. Zhang X, Zhang E, Ma Z, Pei R, Jiang M, Schlaak JF, et al. Modulation of hepatitis 13 virus replication and hepatocyte differentiation by MicroRNA-1. Hepatology.2011;53(5):1476-85.
38. Guo H, Liu H, Mitchelson K, Rao H, Luo M, Xie L, et al. MicroRNAs-372/373 promote the expression of hepatitis B virus through the targeting of nuclear factor I/B. Hepatology. 2011;54(3):808-19.
39. Hu W, Wang X, Ding X, Li Y, Zhang X, Xie P, et al. MicroRNA-141 Represses HBV Replication by Targeting PPARA. PLoS One.2012;7(3):e34165.
40.杜锐.microRNA在乙肝病毒复制中的作用及机制研究.第四军医大学博士学位论文.2009.
41. Liu WH, Yeh SH, Chen PJ. Role of microRNAs in hepatitis B virus replication and pathogenesis. Biochim Biophys Acta.2011 Nov-Dec; 1809(11-12):678-85.
42. Su C, Hou Z, Zhang C, Tian Z, Zhang J. Ectopic expression of microRNA-155 enhances innate antiviral immunity against HBV infection in human hepatoma cells. Virology Journal.2011;8(1):354.
43. Huang LR, Wu HL, Chen PJ, Chen DS. An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection. Proceedings of the National Academy of Sciences. 2006;103(47):17862-7.
44. Dickins RA, McJunkin K, Hernando E, Premsrirut PK, Krizhanovsky V, Burgess DJ, et al. Tissue-specific and reversible RNA interference in transgenic mice. Nat Genet.2007 Jul;39(7):914-21.
45. Schmeisser H, Mejido J, Balinsky CA, Morrow AN, Clark CR, Zhao T, et al. Identification of Alpha Interferon-Induced Genes Associated with Antiviral Activity in Daudi Cells and Characterization of IFIT3 as a Novel Antiviral Gene. Journal of Virology.2010:84(20):10671-80.
46. Taganov K.D. NF-B-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proceedings of the National Academy of Sciences. 2006; 103(33):12481-6.
47. Han Q, Zhang C, Zhang J, Tian Z. Reversal of hepatitis B virus-induced immune tolerance by an immunostimulatory 3p-HBx-siRNAs in a retinoic acid inducible gene 1-dependent manner. Hepatology. 2011;54(4):1179-89.
48. Tang Y, Luo X, Cui H, Ni X, Yuan M, Guo Y, et al. MicroRNA-146a contributes to abnormal activation of the type 1 interferon pathway in human lupus by targeting the key signaling proteins. Arthritis & Rheumatism.2009;60(4):1065-75.
49. Ramkissoon SH, Mainwaring LA, Sloand EM, Young NS, Kajigaya S. Nonisotopic detection of microRNA using digoxigenin labeled RNA probes. Molecular and Cellular Probes.2006;20(1):1-4.
50. Cameron JE, Yin Q, Fewell C, Lacey M, McBride J, Wang X, et al. Epstein-Barr virus latent membrane protein 1 induces cellular MicroRNA miR-146a, a modulator of lymphocyte signaling pathways. J Virol.2008 Feb;82(4):1946-58.
51. Labbaye C, Spinello I, Quaranta MT, Pelosi E, Pasquini L, Petrucci E, et al. A three-step pathway comprising PLZF/miR-146a/CXCR4 controls megakaryopoiesis. Nat Cell Biol.2008 JuI;10(7):788-801.
52. Tomankova T, Petrek M, Gallo J, Kriegova E. MicroRNAs:emerging regulators of immune-mediated diseases. Scand J Immunol.2011 Oct 11.
53. Paik JH, Jang JY, Jeon YK, Kim WY, Kim TM, Heo DS, et al. MicroRNA-146a Downregulates NF B Activity via Targeting TRAF6 and Functions as a Tumor Suppressor Having Strong Prognostic Implications in NK/T Cell Lymphoma. Clinical Cancer Research.2011; 17(14):4761-71.
54. Li L, Chen XP, Li YJ. MicroRNA-146a and Human Disease. Scandinavian Journal of Immunology.2010;71 (4):227-31.
55. Lu L-F, Boldin MP, Chaudhry A, Lin L-L, Taganov KD, Hanada T, et al. Function of miR-146a in Controlling Treg Cell-Mediated Regulation of Thl Responses. Cell.2010;142(6):914-29.
56. Friedman RC, Farh KKH, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Research.2008;19(1):92-105.
57. Rehmsmeier M.Fast and effective prediction of microRNA/target duplexes. Rna. 2004;10(10):1507-17.
58. John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS. Human MicroRNA Targets. PLoS Biology.2004;2(11):e363.
59. Krek A, Grun D, Poy MN, Wolf R, Rosenberg L, Epstein EJ, et al. Combinatorial microRNA target predictions. Nat Genet.2005 May;37(5):495-500.
60. Hornung V, Ellegast J, Kim S, Brzozka K, Jung A, Kato H, et al.5'-Triphosphate RNA is the ligand for RIG-I. Science.2006 Nov 10;314(5801):994-7.
61. Baum A, Garcia-Sastre A. Differential recognition of viral RNA by RIG-I. Virulence.2011 Mar-Apr;2(2):166-9.
62. Guo H, Jiang D, Ma D, Chang J, Dougherty AM, Cuconati A, et al. Activation of pattern recognition receptor-mediated innate immunity inhibits the replication of hepatitis B virus in human hepatocyte-derived cells. J Virol.2009 Jan;83(2):847-58.
63. Pichlmair A, Lassnig C, Eberle C-A, Gorna MW, Baumann CL, Burkard TR, et al. IFIT1 is an antiviral protein that recognizes 5'-triphosphate RNA. Nature Immunology.2011;12(7):624-30.
64. Liu XY, Chen W, Wei B, Shan YF, Wang C. IFN-Induced TPR Protein IFIT3 Potentiates Antiviral Signaling by Bridging MAVS and TBK1. The Journal of Immunology.2011;187(5):2559-68.
65. Pedersen IM, Cheng G, Wieland S, Volinia S, Croce CM, Chisari FV, et al. Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature.2007;449(7164):919-22.
66. Yang CH, Yue J, Fan M, Pfeffer LM. IFN Induces miR-21 through a Signal Transducer and Activator of Transcription 3-Dependent Pathway as a Suppressive Negative Feedback on IFN-Onduced Apoptosis. Cancer Research.2010;70(20):8108-16.
67. Witwer KW, Sisk JM, Gama L, Clements JE. MicroRNA Regulation of IFN- Protein Expression: Rapid and Sensitive Modulation of the Innate Immune Response. The Journal of Immunology. 2010;184(5):2369-76.
68. Sharma A, Kumar M, Aich J, Hariharan M, Brahmachari SK, Agrawal A, et al. Posttranscriptional regulation of interleukin-10 expression by hsa-miR-106a. Proc Natl Acad Sci U S A.2009 Apr 7;106(14):5761-6.
69. Lagos D, Pollara G, Henderson S, Gratrix F, Fabani M, Milne RSB, et al. miR-132 regulates antiviral innate immunity through suppression of the p300 transcriptional co-activator. Nature Cell Biology.2010;12(5):513-9.
70. Liu G, Friggeri A, Yang Y, Park YJ, Tsuruta Y, Abraham E. miR-147, a microRNA that is induced upon Toll-like receptor stimulation, regulates murine macrophage inflammatory responses. Proceedings of the National Academy of Sciences.2009; 106(37):15819-24.
71. Tili E, Michaille JJ, Cimino A, Costinean S, Dumitru CD, Adair B, et al. Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible roles in regulating the response to endotoxin shock. J Immunol.2007 Oct 15;179(8):5082-9.
72. Qi P, Dou TH, Geng L, Zhou FG, Gu X, Wang H, et al. Association of a variant in MIR 196A2 with susceptibility to hepatocellular carcinoma in male Chinese patients with chronic hepatitis B virus infection. Hum Immunol.2010 Jun;71(6):621-6.
73. Griffiths-Jones S. The microRNA Registry. Nucleic Acids Res.2004 Jan I;32(Database issue):D109-11.
74.Willenbrock H, Salomon J, Sokilde R, Barken KB, Hansen TN, Nielsen FC, et al. Quantitative miRNA expression analysis:comparing microarrays with next-generation sequencing. Rna.2009 Nov; 15(11):2028-34.
75. Pradervand S, Weber J, Lemoine F, Consales F, Paillusson A, Dupasquier M, et al. Concordance among digital gene expression, microarrays, and qPCR when measuring differential expression of microRNAs. BioTechniques.2010;48(3):219-22.
76. Meyer SU, Pfaffl MW, Ulbrich SE. Normalization strategies for microRNA profiling experiments: a'normal' way to a hidden layer of complexity? Biotechnology Letters.2010;32(12):1777-88.
77. Liang Z, Zhou H, Zheng H, Wu J. Expression levels of microRNAs are not associated with their regulatory activities. Biology Direct.2011;6(1):43.
78. Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. Widespread changes in protein synthesis induced by microRNAs. Nature.2008 Sep 4;455(7209):58-63.
79. Si ML, Zhu S, Wu H, Lu Z, Wu F, Mo YY. miR-21-mediated tumor growth. Oncogene. 2006;26(19):2799-803.
80. Xie SY, Li YJ, Wang PY, Jiao F, Zhang S, Zhang WJ. miRNA-regulated expression of oncogencs and tumor suppressor genes in the cisplatin-inhibited growth of K562 cells. Oncol Rep.2010 Jun;23(6):1693-700.
81. Tam W, Dahlberg JE. miR-155/BIC as an oncogenic microRNA. Genes, Chromosomes and Cancer.2006;45(2):211-2.
82. Jopling C. Liver-specific microRNA-122:Biogenesis and function. RNA Biol.2012 Feb 1;9(2).
83.张小勇.倪明.陈姗姗.吕婷婷.余冰.陆蒙吉.肝脏内源性microRNA调控乙型肝炎病毒基因的表达与复制.医学分子生物学杂志.2008;5(3)：189-93.
84. Ji F, Yang B, Peng X, Ding H, You H, Tien P. Circulating microRNAs in hepatitis B virus-infected patients. J Viral Hepat.2011 Jul; 18(7):e242-51.
85.郝美君.郑素军.丁惠国.黄爱龙MicroRNA-122作用于HBx影响乙肝病毒复制.生物医学工程学杂志.2011;28(4)：784-9.
86.金文松.肝特异性miR-122负向调控IFN-alpha抗乙肝病毒活力.福建农林大学硕十学位论文.2010.
87. Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T. Identification of tissue-specific microRNAs from mouse. Curr Biol.2002 Apr 30;12(9):735-9.
88. Hsu SD, Chu CH, Tsou AP, Chen SJ, Chen HC, Hsu PW, et al. miRNAMap 2.0:genomic maps of microRNAs in metazoan genomes. Nucleic Acids Res.2008 Jan;36(Database issue):D165-9.
89. Rusca N, Monticelli S. MiR-146a in Immunity and Disease. Mol Biol Int.2011;2011:437301.
90. Zhang Z-Z. Hepatitis B virus and hepatocellular carcinoma at the miRNA level. World Journal of Gastroenterology.2011;17(28):3353.
91. Liu Y, Zhao JJ, Wang CM, Li MY, Han P, Wang L, et al. Altered expression profiles of microRNAs in a stable hepatitis B virus-expressing cell line. Chin Med J (Engl).2009 Jan 5;122(1):10-4.
92. Sun B, Karin M. NF-κB signaling, liver disease and hepatoprotective agents. Oncogene. 2008;27(48):6228-44.
93. Hildt E, Saher G, Bruss V, Hofschneider PH. The hepatitis B virus large surface protein (LHBs) is a transcriptional activator. Virology.1996 Nov 1;225(1):235-9.
94. Liu Y, Lou G, Wu W, Zheng M, Shi Y, Zhao D, et al. Involvement of the NF-κB pathway in multidrug resistance induced by HBx in a hepatoma cell line. Journal of Viral Hepatitis. 2011;18(10):e439-e46.
95. Tang H, Oishi N, Kaneko S, Murakami S. Molecular functions and biological roles of hepatitis B virus x protein. Cancer Sci.2006 Oct;97(10):977-83.
96. Wu J, Meng Z, Jiang M, Pei R, Trippler M, Broering R, et al. Hepatitis B virus suppresses toll-like receptor-mediated innate immune responses in murine parenchymal and nonparenchymal liver cells. Hepatology.2009 Apr;49(4):1132-40.
97. Murakami S. Hepatitis B virus X protein:a multifunctional viral regulator. J Gastroenterol.2001 Oct;36(10):651-60.
98. Nahid MA, Pauley KM, Satoh M, Chan EKL. miR-146a Is Critical for Endotoxin-induced Tolerance:IMPLICATION IN INNATE IMMUNITY. Journal of Biological Chemistry. 2009;284(50):34590-9.
99. Punj V, Matta H, Schamus S, Tamewitz A, Anyang B, Chaudhary PM. Kaposi's sarcoma-associated herpesvirus-encoded viral FLICE inhibitory protein (vFLIP) K13 suppresses CXCR4 expression by upregulating miR-146a. Oncogene.2009:29(12):1835-44.
100. Cameron JE, Yin Q, Fewell C, Lacey M, McBride J, Wang X, et al. Epstein-Barr Virus Latent Membrane Protein 1 Induces Cellular MicroRNA miR-146a, a Modulator of Lymphocyte Signaling Pathways. Journal of Virology.2007;82(4):1946-58.
101. Wu G, Yu F, Xiao Z, Xu K, Xu J, Tang W, et al. Hepatitis B virus X protein downregulates expression of the miR-16 family in malignant hepatocytes in vitro. Br J Cancer.2011 Jun 28; 105(1):146-53.
102. Bui-Nguyen TM, Pakala SB, Sirigiri DR, Martin E, Murad F, Kumar R. Stimulation of inducible nitric oxide by hepatitis B virus transactivator protein HBx requires MTA1 coregulator. J Biol Chem. 2010 Mar 5;285(10):6980-6.
103. Wang Y, Lu Y, Toh ST. Sung WK, Tan P, Chow P, et al. Lethal-7 is down-regulated by the hepatitis B virus x protein and targets signal transducer and activator of transcription 3. J Hepatol.2010 Jul;53(1):57-66.
104. Shan C, Zhang S, Cui W, You X, Kong G, Du Y, et al. Hepatitis B virus X protein activates CD59 involving DNA binding and let-7i in protection of hepatoma and hepatic cells from complement attack. Carcinogenesis.2011 Aug;32(8):1190-7.
105. Kong G, Zhang J, Zhang S. Shan C, Ye L, Zhang X. Upregulated microRNA-29a by hepatitis B virus X protein enhances hepatoma cell migration by targeting PTEN in cell culture model. PLoS One. 2011;6(5):e19518.
106. Yip WK, Cheng AS, Zhu R, Lung RW, Tsang DP, Lau SS, et al. Carboxyl-terminal truncated HBx regulates a distinct microRNA transcription program in hepatocellular carcinoma development. PLoS One.2011;6(8):e22888.
107. Jurkin J, Schichl YM, Koeffel R, Bauer T, Richter S. Konradi S, et al. miR-146a Is Differentially Expressed by Myeloid Dendritic Cell Subsets and Desensitizes Cells to TLR2-Dependent Activation. The Journal of Immunology.2010;184(9):4955-65.
108. Lukiw WJ, Zhao Y, Cui JG. An NF-B-sensitive Micro RNA-146a-mediated Inflammatory Circuit in Alzheimer Disease and in Stressed Human Brain Cells. Journal of Biological Chemistry. 2008;283(46):31315-22.
109. Tomokuni A, Eguchi H, Tomimaru Y, Wada H, Kawamoto K, Kobayashi S, et al. miR-146a suppresses the sensitivity to interferon-a in hepatocellular carcinoma cells. Biochemical and Biophysical Research Communications.2011;414(4):675-80.
110. Motsch N, Pfuhl T, Mrazek J, Barth S, Grasser FA. Hpstein-Barr virus-encoded latent membrane protein I (LMP1) induces the expression of the cellular microRNA miR-146a. RNA Biol.2007 Nov;4(3):131-7.
111. Meraz MA, White JM, Sheehan KC, Bach EA, Rodig SJ, Dighe AS, et al. Targeted disruption of the Slatl gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell.1996 Feb9;84(3):431-42.
112. Park C, Li S, Cha E, Schindler C. Immune response in Stat2 knockout mice. Immunity.2000 Dec;13(6):795-804.
113. Boldin MP, Taganov KD, Rao DS, Yang L, Zhao JL, Kalwani M, et al. miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice. J Exp Med.2011 Jun 6;208(6):1189-201.
114. Hurst DR, Edmonds MD, Scott GK, Benz CC, Vaidya KS, Welch DR. Breast cancer metastasis suppressor 1 up-regulates miR-146, which suppresses breast cancer metastasis. Cancer Res.2009 Feb 15;69(4):1279-83.
115. Kogo R, Mimori K, Tanaka F, Komune S, Mori M. Clinical significance of miR-146a in gastric cancer cases. Clin Cancer Res.2011 Ju11;17(13):4277-84.
116. Perske C, Lahat N, Sheffy Levin S, Bitterman H, Hemmerlein B, Rahat MA. Loss of inducible nitric oxide synthase expression in the mouse renal cell carcinoma cell line RENCA is mediated by microRNA miR-146a. Am J Pathol.2010 Oct;177(4):2046-54.
117. Wang X, Tang S, Le SY, Lu R, Rader JS, Meyers C, et al. Aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth. PLoS One. 2008;3(7):e2557.
118. Mei J, Bachoo R, Zhang CL. MicroRNA-146a inhibits glioma development by targeting Notch 1. Mol Cell Biol.2011 Sep;31 (17)3584-92.
119. Liu DZ, Ander BP, Tian Y, Stamova B, Jickling GC, Davis RR, et al. Integrated analysis of mRNA and microRNA expression in mature neurons, neural progenitor cells and neuroblastoma cells. Gene. 2012 Mar 10;495(2):120-7.
120. Lin SL, Chiang A, Chang D, Ying SY. Loss of mir-146a function in hormone-refractory prostate cancer. Rna.2008 Mar;14(3):417-24.
121. Xu B, Wang N, Wang X, Tong N, Shao N, Tao J, et al. MiR-146a suppresses tumor growth and progression by targeting EGFR pathway and in a p-ERK-dependent manner in castration-resistant prostate cancer. Prostate.2011 Dec 7.
122. Li Y, VandenBoom TG, Wang Z, Kong D, Ali S, Philip PA, et al. miR-146a Suppresses Invasion of Pancreatic Cancer Cells. Cancer Research.2010;70(4):1486-95.
123. Karakatsanis A, Papaconstantinou I, Gazouli M, Lyberopoulou A, Polymeneas G, Voros D. Expression of microRNAs, miR-21, miR-31, miR-122, miR-145, miR-146a, miR-200c, miR-221, miR-222, and miR-223 in patients with hepatocellular carcinoma or intrahepatic cholangiocarcinoma and its prognostic significance. Mol Carcinog.2011 Dec 27.
124. Labbaye C, Spinello I, Quaranta MT, Pelosi E, Pasquini L, Petrucci E, et al. A three-step pathway comprising PLZF/miR-146a/CXCR4 controls megakaryopoiesis. Nature Cell Biology. 2008;10(7):788-801.
125. Qiu L-X, He J, Wang M-Y, Zhang R-X, Shi T-Y, Zhu M-L, et al. The association between common genetic variant of microRNA-146a and cancer susceptibility. Cytokine.2011;56(3):695-8.
126.扶琼.SLE相关基因IFIT3的功能及分子机制研究.上海交通大学博十学位论文.2008.
127. Hacker H, Redecke V, Blagoev B, Kratchmarova I, Hsu LC, Wang GG, et al. Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6. Nature.2006 Jan 12;439(7073):204-7.
128. Yoboua F, Martel A, Duval A, Mukawera E, Grandvaux N. Respiratory Syncytial Virus-Mediated NF-B p65 Phosphorylation at Serine 536 Is Dependent on RIG-I, TRAF6, and IKK Journal of Virology.2010;84(14):7267-77.
129. He JQ, Zarnegar B, Oganesyan G, Saha SK, Yamazaki S, Doyle SE, et al. Rescue of TRAF3-null mice by pi00 NF-kappa B deficiency. J Exp Med.2006 Oct 30;203(11):2413-8.
130. Yoneyama M, Fujita T. RNA recognition and signal transduction by RIG-I-like receptors. Immunol Rev.2009 Jan;227(1):54-65.
131. Yoshida R, Takaesu G, Yoshida H, Okamoto F, Yoshioka T, Choi Y, et al. TRAF6 and MEKK1 play a pivotal role in the RIG-I-like helicase antiviral pathway. J Biol Chem.2008 Dec 26;283(52):36211-20.
132. Bernardo BC, Charchar FJ, Lin RC, McMullen JR. A microRNA guide for clinicians and basic scientists:background and experimental techniques. Heart Lung Circ.2012 Mar;21(3):131-42.