幽门螺杆菌非编码小RNA的筛选、鉴定及miRNAs在幽门螺杆菌感染中负向调控炎症反应的作用研究
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摘要
非编码RNA(non-coding RNA,ncRNA)是指不编码蛋白质,以RNA形式发挥生物学作用的一类RNA分子。近年来,非编码RNA作为一类新的调控因子,在原核和真核生物的表达调控中发挥重要的作用。
细菌中的非编码RNA称为小RNA(small non-coding RNAs,sRNAs),它们通过碱基配对识别靶标mRNA,调控mRNA的翻译或稳定性,在转录后水平调节基因的表达。sRNAs是细菌适应环境压力、代谢和细菌毒性的重要调节因子。目前,在大肠杆菌中已经发现70多个sRNAs,此外sRNAs还存在于铜绿假单胞菌、霍乱弧菌、金黄色葡萄球菌、沙门氏菌、鼠疫耶尔森菌、志贺氏菌等细菌中。
微小RNA (microRNAs, miRNAs)是一类长度约为20~25 nt的内源性非编码RNA分子,普遍存在于真核生物中。成熟miRNAs可以与靶mRNAs 3′非翻译区互补结合,诱导靶mRNA降解或抑制靶mRNA的翻译,从而实现转录后基因调控作用。miRNAs在细胞增殖、发育、分化以及肿瘤发生中发挥重要的调节作用。
幽门螺杆菌( Helicobacter pylori, Hp)是慢性胃炎、消化性溃疡的主要致病因子,且与胃癌发病密切相关。Hp的一个显著特性是定植在低pH值的胃酸环境中,这需要Hp能够精细调控细菌的基因表达,以适应其生存环境,然而Hp的转录调节能力非常有限,这提示可能存在新的基因调控因素。同时,Hp感染的另一个特性是复杂的免疫反应,虽然机体对于Hp感染能够引发强烈的细胞与体液免疫,但是并不能够有效地清除细菌,感染状态常常持续存在。因此,Hp在与人类长期共存的过程中,相互构成了一个复杂的网络,以精细调控Hp感染的免疫反应,但确切免疫调控机制仍不清楚。
目前,关于Hp的非编码小RNA的研究很少,而且miRNAs在Hp感染与免疫中的作用也未见报道。因此,本研究首次通过生物信息学方法和一种基于RNase I保护实验的cDNA文库构建方法,筛选和鉴定出Hp中的非编码小RNA;同时,利用miRNAs芯片、实时定量PCR技术和Northern杂交,筛选出Hp感染引起胃上皮细胞差异表达的miRNAs,并以miR-155为深入研究对象,探讨Hp感染诱导miR-155高表达的分子机制,预测和鉴定miR-155的靶基因,研究miR-155在Hp感染中负向调控炎症反应的作用。本研究以非编码小RNA为切入点,将有助于进一步阐明Hp的基因表达调控网络,同时miRNAs也为深入探讨Hp感染中的免疫调控机制提供了新思路。
方法:
1. Hp中sRNAs的生物信息学预测与鉴定。
首先从Hp 26695基因组序列中,提取大于120 nt的基因间隔区(intergenic region,IGR)序列,然后结合sRNAs的保守性、启动子和终止子分析、二级结构预测,从全基因组水平预测Hp中的sRNAs基因。通过Northern杂交和RT-PCR技术,鉴定侯选的sRNAs基因。
2.基于RNase I保护实验原理,构建Hp的自然反义转录子(natural antisense transcripts, NATs)文库。
利用双链RNA(dsRNA)能够耐受RNase I的酶切作用的特性。首先提取Hp 26695的总RNA,RNase If酶切去除单链RNA;通过随机引物和逆转录酶的作用生成cDNA;然后在cDNA分子的5′端加上接头(adapter),在3′端加上poly(A)尾,PCR构建NATs的cDNA克隆文库。
3. Hp中NATs文库的序列分析及鉴定。
对提取的文库质粒进行SmaⅠ/XhoⅠ双酶切鉴定后,将所有阳性克隆进行测序。测序结果与Hp 26695基因组进行BLAST分析,剔除无关序列和冗长序列后,分析候选NATs的基因组定位,并利用Northern Blot和RT-PCR进行鉴定。
4. Hp感染相关miRNAs的筛选与鉴定。
以Hp感染细胞模型和Hp感染患者的胃粘膜组织为研究对象,利用miRNAs芯片、real-time PCR和Northern杂交技术,筛选Hp感染引起胃上皮细胞差异表达的miRNAs。
5. miR-155在Hp感染中负向调控炎症反应的作用研究。
在筛选出的Hp感染相关miRNAs中,我们以miR-155为深入研究的对象,通过启动子分析、荧光素酶实验、信号通路抑制剂实验等方法,探讨Hp感染诱导miR-155高表达的分子机制;结合生物信息学预测、荧光素酶实验、Western blot等方法鉴定miR-155在胃上皮细胞中的靶基因;同时,通过miR-155体外过表达或抑制表达,探讨miR-155负向调控炎症反应的作用及机制。
结果:
1. Hp中sRNAs的生物信息学预测与鉴定。
通过生物信息学预测,在Hp 26695基因组筛选出了6个候选sRNAs,经Northern Blot和RT-PCR鉴定,发现了Hp基因组中的两个新sRNAs,即IG-443和IG-524。基因组定位分析表明,IG-443和IG-524属于自然反义转录子(NATs),分别与相对链上的fliM基因和fumC基因互补,可能以碱基配对的方式调控互补基因的表达。
2.基于RNase I保护实验筛选和鉴定Hp中NATs。
通过基于RNase I保护实验的文库构建策略,我们成功构建了一个含68个克隆的cDNA文库,经Sma I和Xho I双酶切、测序和BLAST分析后,筛选出了6个能在Hp 26695基因组准确定位的候选NATs;经Northern Blot和poly(A)-tailed RT-PCR鉴定,发现了两个新的Hp NATs (NAT-39和NAT-67)。这两个NATs分别与frpB基因和ceuE基因互补,可能与Hp的铁代谢调节有关。
3. Hp感染相关miRNAs的筛选与鉴定。
Hp 26695感染胃上皮细胞GES-1 24h后,miRNAs芯片表明,Hp感染引起了一系列的miRNAs的表达改变,如:miR-155、miR-146a、miR-16等的表达上调,miR-181b、miR-324的表达下调。real-time PCR结果与芯片结果一致,表明miR-155, miR-16和miR-146a分别上调了3.0, 2.1和2.5倍。同时,与Hp阴性的正常胃粘膜组织相比,在Hp感染的慢性胃炎病人的胃粘膜组织中,miR-155的表达量也上调了3.9倍(P<0.05)。
4. Hp感染诱导miR-155高表达的信号通路。
启动子预测表明, miR-155基因的启动子序列中含有NF-κB和AP-1的结合位点。启动子分析、荧光素酶实验、信号通路抑制剂实验表明,NF-κB和AP-1信号通路参与了miR-155的诱导表达,其中AP-1可能在miR-155的诱导表达中起着更为关键的作用。
5. miR-155靶基因的预测与鉴定。
利用miRNAs靶基因预测软件,筛选出IKK-ε、SMAD2和FADD是miR-155的潜在作用靶点;构建靶基因荧光素酶报告载体和GFP报告载体,证实了miR-155能与靶基因的3′UTR结合;Western blot和real-time PCR结果表明,miR-155可以抑制IKK-ε、SMAD2和FADD靶基因的蛋白表达,而且作用机制各不相同,如直接降解mRNA或抑制蛋白的翻译。
6. miR-155抑制Hp感染中炎症因子的表达。
体外过表达miR-155后,能够显著减少Hp感染引起的炎症因子(IL-8、GRO-α)的mRNA和蛋白水平(P<0.05),而且这种抑制作用可能是通过降低NF-κB的活性引起的次级效应,这表明miR-155参与了Hp感染中炎症反应的负反馈调节。
结论:
1.建立了生物信息学预测Hp中sRNAs的方法,且成功筛选出了IG-443和IG-524两个sRNAs,这表明Hp中也存在着sRNAs,它们可能在Hp的基因表达调控中发挥调节作用。
2.通过基于RNase I保护实验的文库构建策略,成功构建Hp的NATs文库,且鉴定出两个新的Hp NATs(NAT-39和NAT-67),它们以碱基配对的方式调控互补基因的表达,可能与Hp的铁代谢调节有关。
3. Hp感染能够引起胃上皮细胞株和胃粘膜组织中miR-155的表达上调,且miR-155作为一类新的负反馈调节因子,参与了Hp感染中炎症反应的调节过程,这为进一步阐明Hp感染的免疫调控机制及Hp的致病性研究提供新方向。
Recently, small non-coding RNAs, which are a group of regulatory RNA molecules normally without a protein-coding function, have attracted great interest as key regulators in both eukaryotic and prokaryotic life.
In bacteria, these regulatory RNAs are also termed as small RNAs or sRNAs. The majority of sRNAs can control gene expression at the post-transcriptional level via base pairing with complementary sequences in target transcripts. Many sRNAs have been identified as crucial regulatory elements in bacterial stress responses, bacterial virulence, quorum sensing and bacterial homeostasis. As a result of recent systematic searches, over 70 sRNAs are now known in Escherichia coli, and more and more sRNAs are found in other pathogens, for example, Salmonella, Yersinia and Vibrio harveyi, etc.
microRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. Mature miRNAs can specifically bind to 3′UTRs of target cellular mRNA in turn triggering mRNA degradation or inhibition of translation. miRNAs can act as key regulators in a wide variety of biological processes and various diseases, including development, cell differentiation, apoptosis, metabolism, and cancer.
Helicobacter pylori (H. pylori) is a major human pathogen which is associated with gastric diseases like chronic active gastritis, peptic ulcer, and gastric carcinoma. A remarkable feature of H. pylori is its ability to survive in the extremely acidic environment of the stomach. It requires regulation of bacterial gene expression to cope with the environmental fluctuations. Despite the limited number of transcriptional regulators, evidence is accumulating for the existence of new and complex circuits regulating gene transcription. Another remarkable feature of the H. pylori infection is its complex immune response. Though strong cellular and humoral immunity is induced during H. pylori infection, the immune and inflammatory response is unable to clear the bacterium, resulting in lifelong bacterial persistence. During its long co-existence with humans, H. pylori, host and environmental factors consist of a complex network to precisely regulate the immune response, however, the regulatory mechanism of this complex system is not clear.
To date, little is known about sRNAs and H. pylori, and the potential role of miRNAs in the immune response to H. pylori infection has not been investigated. Here we first systematically identified sRNAs in H. pylori genome by a computational approach and a novel experimental strategy based on RNase I protection assay. The expression profile of cellular miRNAs during H. pylori infection was analyzed by using microarray, quantitative RT-PCR and Northern blot. Then we choose miR-155 for detailed investigation, defined the underlying mechanism leading to the miR-155 up-regulation by H. pylori, and identified the potential target genes of miR-155, and investigated the possible role of miR-155 as novel negative regulator that help to fine-tune the inflammation response of H. pylori infection. Our results provide insight into the knowledge of regulatory network of H. pylori. Furthermore, the altered miR-155 expression may identify a potential link between miRNAs and immune regulation during H. pylori infection.
Methods
1. Identification of sRNAs in H. pylori by a bioinformatics based approach. A file of all H. pylori Intergenic regions (IGRs) sequences was created by a Perl package based on the gene annotations. Then we systematically identified sRNAs in H. pylori based on gene location, sequence conservation, promoter and terminator search, and secondary structure. The candidate sRNAs were confirmed by Northern blot and RT-PCR.
2. Screening of natural antisense transcripts(NATs) in H. pylori by a novel approach based on RNase I protection assay. Total RNA H. pylori was digested by RNase I to remove single-stranded RNA. Synthesis of the first strand of cDNA was performed with random primer and reverse transcriptase. Then cDNA was tailed with poly(A) at the 3′-end, and a 5′adapter was ligated to 5' phosphorylated cDNA, and a cDNA library of NATs was constructed by PCR.
3. Identification of NATs in H. pylori. After the identification by SmaⅠ/XhoⅠrestriction endonuclease, insert-containing clones were subsequently characterized by DNA sequencing and BLAST analysis. The expression of NATs was confirmed by Northern blot and poly(A)-tailed RT-PCR.
4. The expression profile of miRNAs in gastric epithelial cell lines and gastric mucosal tissues during H. pylori infection was analyzed by using microarray, quantitative RT-PCR and Northern blot.
5. The negative regulatory role of miR-155 in the inflammatory response.
The potential target of miR-155 was identified by luciferase assay and western blot. Promoter analysis and inhibitor experiment were used to investigate the pathway involved in the induction of miR-155. Examination of miR-155 function was performed by overexpression and inhibition of miR-155.
Results:
1. Identification of sRNAs in H. pylori by a bioinformatics based approach.
Among a total of 6 candidate sRNAs initially predicted, two novel sRNAs (IG-443 and IG-524) were confirmed by Northern blot and RT-PCR. Virtually, they were a class of natural antisense transcripts (NATs), which were complementary to partial sequences of the following genes: flagellar motor switch gene (fliM) and fumarase (fumC).
2. Screening and identification of NATs in H. pylori by a novel approach.
We successfully constructed a cDNA library of NATs containing 68 clones. After the identification by SmaⅠ/XhoⅠrestriction endonuclease, a total of 33 insert-containing clones were subsequently characterized by DNA sequencing and BLAST analysis. 6 of all inserts were partially homologous to H. pylori genomic sequences and corresponded to the opposite strands of ORFs. Two novel NATs (NAT-39 and NAT-67) were confirmed by Northern blot and poly(A)-tailed RT-PCR. They were respectively complementary to the following genes: iron-regulated outer membrane protein (frpB) and periplasmic iron-binding protein (ceuE). NAT-39 and NAT-67 may participate in the regulation of iron homeostasis in H. pylori in a sequence complementary manner.
3. Screening and identification of obviously altered miRNAs in response to H. pylori infection.
The expression of miRNAs could be significantly altered during H. pylori infection, including the up-regulation of miR-155, miR-16, and miR-146a, and the down-regulation of miR-181b and miR-324. Consistent with the microarray findings, the results of qRT-PCR showed that miR-155, miR-16, and miR-146a was increased, 3.0, 2.1 and 2.5 fold change, respectively. miR-155 was highly up-regulated in H.pylori-positive patients, with a 3.9-fold increase as compared to the control (P<0.05).
4. The promoter region of miR-155 contained putative NF-κB and AP-1 binding sites. The results of promoter analysis and inhibitor experiment showed that both NF-κB and AP-1 pathways are required for the up-regulation of miR-155 in response to H. pylori, and AP-1 plays a central role in the induction of miR-155.
5. IKK-ε, SMAD2, and FADD are potential targets of miR-155, and miR-155 might down-regulate the target protein through different mechanism, either mRNA degradation or inhibition of translation.
6. miR-155 mimics significantly attenuated the mRNA and protein levels of IL-8 and GRO-α, and the effect of miR-155 in modulating the inflammation may be as a secondary effect through diminishing NF-κB activity. miR-155 may be involved in the negative feedback regulation of inflammation.
Conclusion:
1. We developed a computational approach to identify novel sRNAs in H. pylori, and identified two sRNAs (IG-443 and IG-524). The results indicate that there exist novel sRNAs in H. pylori and these RNAs may play potential role in regulating gene expression.
2. We developed a novel approach based on RNase I protection assay to identify NATs in H. pylori, and identified two NATs (NAT-39 and NAT-67). They may participate in the regulation of iron homeostasis in H. pylori in a sequence complementary manner.
3. H. pylori can stimulate the expression of miR-155 in gastric epithelial cells as well as in gastric mucosal tissues. miR-155 may function as novel negative regulator that help to fine-tune the inflammation response of H. pylori infection. Furthermore, the altered miR-155 expression may identify a potential link between miRNAs and pathogenesis of H. pylori related diseases.
细菌中的非编码RNA称为小RNA(small non-coding RNAs,sRNAs),它们通过碱基配对识别靶标mRNA,调控mRNA的翻译或稳定性,在转录后水平调节基因的表达。sRNAs是细菌适应环境压力、代谢和细菌毒性的重要调节因子。目前,在大肠杆菌中已经发现70多个sRNAs,此外sRNAs还存在于铜绿假单胞菌、霍乱弧菌、金黄色葡萄球菌、沙门氏菌、鼠疫耶尔森菌、志贺氏菌等细菌中。
微小RNA (microRNAs, miRNAs)是一类长度约为20~25 nt的内源性非编码RNA分子,普遍存在于真核生物中。成熟miRNAs可以与靶mRNAs 3′非翻译区互补结合,诱导靶mRNA降解或抑制靶mRNA的翻译,从而实现转录后基因调控作用。miRNAs在细胞增殖、发育、分化以及肿瘤发生中发挥重要的调节作用。
幽门螺杆菌( Helicobacter pylori, Hp)是慢性胃炎、消化性溃疡的主要致病因子,且与胃癌发病密切相关。Hp的一个显著特性是定植在低pH值的胃酸环境中,这需要Hp能够精细调控细菌的基因表达,以适应其生存环境,然而Hp的转录调节能力非常有限,这提示可能存在新的基因调控因素。同时,Hp感染的另一个特性是复杂的免疫反应,虽然机体对于Hp感染能够引发强烈的细胞与体液免疫,但是并不能够有效地清除细菌,感染状态常常持续存在。因此,Hp在与人类长期共存的过程中,相互构成了一个复杂的网络,以精细调控Hp感染的免疫反应,但确切免疫调控机制仍不清楚。
目前,关于Hp的非编码小RNA的研究很少,而且miRNAs在Hp感染与免疫中的作用也未见报道。因此,本研究首次通过生物信息学方法和一种基于RNase I保护实验的cDNA文库构建方法,筛选和鉴定出Hp中的非编码小RNA;同时,利用miRNAs芯片、实时定量PCR技术和Northern杂交,筛选出Hp感染引起胃上皮细胞差异表达的miRNAs,并以miR-155为深入研究对象,探讨Hp感染诱导miR-155高表达的分子机制,预测和鉴定miR-155的靶基因,研究miR-155在Hp感染中负向调控炎症反应的作用。本研究以非编码小RNA为切入点,将有助于进一步阐明Hp的基因表达调控网络,同时miRNAs也为深入探讨Hp感染中的免疫调控机制提供了新思路。
方法:
1. Hp中sRNAs的生物信息学预测与鉴定。
首先从Hp 26695基因组序列中,提取大于120 nt的基因间隔区(intergenic region,IGR)序列,然后结合sRNAs的保守性、启动子和终止子分析、二级结构预测,从全基因组水平预测Hp中的sRNAs基因。通过Northern杂交和RT-PCR技术,鉴定侯选的sRNAs基因。
2.基于RNase I保护实验原理,构建Hp的自然反义转录子(natural antisense transcripts, NATs)文库。
利用双链RNA(dsRNA)能够耐受RNase I的酶切作用的特性。首先提取Hp 26695的总RNA,RNase If酶切去除单链RNA;通过随机引物和逆转录酶的作用生成cDNA;然后在cDNA分子的5′端加上接头(adapter),在3′端加上poly(A)尾,PCR构建NATs的cDNA克隆文库。
3. Hp中NATs文库的序列分析及鉴定。
对提取的文库质粒进行SmaⅠ/XhoⅠ双酶切鉴定后,将所有阳性克隆进行测序。测序结果与Hp 26695基因组进行BLAST分析,剔除无关序列和冗长序列后,分析候选NATs的基因组定位,并利用Northern Blot和RT-PCR进行鉴定。
4. Hp感染相关miRNAs的筛选与鉴定。
以Hp感染细胞模型和Hp感染患者的胃粘膜组织为研究对象,利用miRNAs芯片、real-time PCR和Northern杂交技术,筛选Hp感染引起胃上皮细胞差异表达的miRNAs。
5. miR-155在Hp感染中负向调控炎症反应的作用研究。
在筛选出的Hp感染相关miRNAs中,我们以miR-155为深入研究的对象,通过启动子分析、荧光素酶实验、信号通路抑制剂实验等方法,探讨Hp感染诱导miR-155高表达的分子机制;结合生物信息学预测、荧光素酶实验、Western blot等方法鉴定miR-155在胃上皮细胞中的靶基因;同时,通过miR-155体外过表达或抑制表达,探讨miR-155负向调控炎症反应的作用及机制。
结果:
1. Hp中sRNAs的生物信息学预测与鉴定。
通过生物信息学预测,在Hp 26695基因组筛选出了6个候选sRNAs,经Northern Blot和RT-PCR鉴定,发现了Hp基因组中的两个新sRNAs,即IG-443和IG-524。基因组定位分析表明,IG-443和IG-524属于自然反义转录子(NATs),分别与相对链上的fliM基因和fumC基因互补,可能以碱基配对的方式调控互补基因的表达。
2.基于RNase I保护实验筛选和鉴定Hp中NATs。
通过基于RNase I保护实验的文库构建策略,我们成功构建了一个含68个克隆的cDNA文库,经Sma I和Xho I双酶切、测序和BLAST分析后,筛选出了6个能在Hp 26695基因组准确定位的候选NATs;经Northern Blot和poly(A)-tailed RT-PCR鉴定,发现了两个新的Hp NATs (NAT-39和NAT-67)。这两个NATs分别与frpB基因和ceuE基因互补,可能与Hp的铁代谢调节有关。
3. Hp感染相关miRNAs的筛选与鉴定。
Hp 26695感染胃上皮细胞GES-1 24h后,miRNAs芯片表明,Hp感染引起了一系列的miRNAs的表达改变,如:miR-155、miR-146a、miR-16等的表达上调,miR-181b、miR-324的表达下调。real-time PCR结果与芯片结果一致,表明miR-155, miR-16和miR-146a分别上调了3.0, 2.1和2.5倍。同时,与Hp阴性的正常胃粘膜组织相比,在Hp感染的慢性胃炎病人的胃粘膜组织中,miR-155的表达量也上调了3.9倍(P<0.05)。
4. Hp感染诱导miR-155高表达的信号通路。
启动子预测表明, miR-155基因的启动子序列中含有NF-κB和AP-1的结合位点。启动子分析、荧光素酶实验、信号通路抑制剂实验表明,NF-κB和AP-1信号通路参与了miR-155的诱导表达,其中AP-1可能在miR-155的诱导表达中起着更为关键的作用。
5. miR-155靶基因的预测与鉴定。
利用miRNAs靶基因预测软件,筛选出IKK-ε、SMAD2和FADD是miR-155的潜在作用靶点;构建靶基因荧光素酶报告载体和GFP报告载体,证实了miR-155能与靶基因的3′UTR结合;Western blot和real-time PCR结果表明,miR-155可以抑制IKK-ε、SMAD2和FADD靶基因的蛋白表达,而且作用机制各不相同,如直接降解mRNA或抑制蛋白的翻译。
6. miR-155抑制Hp感染中炎症因子的表达。
体外过表达miR-155后,能够显著减少Hp感染引起的炎症因子(IL-8、GRO-α)的mRNA和蛋白水平(P<0.05),而且这种抑制作用可能是通过降低NF-κB的活性引起的次级效应,这表明miR-155参与了Hp感染中炎症反应的负反馈调节。
结论:
1.建立了生物信息学预测Hp中sRNAs的方法,且成功筛选出了IG-443和IG-524两个sRNAs,这表明Hp中也存在着sRNAs,它们可能在Hp的基因表达调控中发挥调节作用。
2.通过基于RNase I保护实验的文库构建策略,成功构建Hp的NATs文库,且鉴定出两个新的Hp NATs(NAT-39和NAT-67),它们以碱基配对的方式调控互补基因的表达,可能与Hp的铁代谢调节有关。
3. Hp感染能够引起胃上皮细胞株和胃粘膜组织中miR-155的表达上调,且miR-155作为一类新的负反馈调节因子,参与了Hp感染中炎症反应的调节过程,这为进一步阐明Hp感染的免疫调控机制及Hp的致病性研究提供新方向。
Recently, small non-coding RNAs, which are a group of regulatory RNA molecules normally without a protein-coding function, have attracted great interest as key regulators in both eukaryotic and prokaryotic life.
In bacteria, these regulatory RNAs are also termed as small RNAs or sRNAs. The majority of sRNAs can control gene expression at the post-transcriptional level via base pairing with complementary sequences in target transcripts. Many sRNAs have been identified as crucial regulatory elements in bacterial stress responses, bacterial virulence, quorum sensing and bacterial homeostasis. As a result of recent systematic searches, over 70 sRNAs are now known in Escherichia coli, and more and more sRNAs are found in other pathogens, for example, Salmonella, Yersinia and Vibrio harveyi, etc.
microRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. Mature miRNAs can specifically bind to 3′UTRs of target cellular mRNA in turn triggering mRNA degradation or inhibition of translation. miRNAs can act as key regulators in a wide variety of biological processes and various diseases, including development, cell differentiation, apoptosis, metabolism, and cancer.
Helicobacter pylori (H. pylori) is a major human pathogen which is associated with gastric diseases like chronic active gastritis, peptic ulcer, and gastric carcinoma. A remarkable feature of H. pylori is its ability to survive in the extremely acidic environment of the stomach. It requires regulation of bacterial gene expression to cope with the environmental fluctuations. Despite the limited number of transcriptional regulators, evidence is accumulating for the existence of new and complex circuits regulating gene transcription. Another remarkable feature of the H. pylori infection is its complex immune response. Though strong cellular and humoral immunity is induced during H. pylori infection, the immune and inflammatory response is unable to clear the bacterium, resulting in lifelong bacterial persistence. During its long co-existence with humans, H. pylori, host and environmental factors consist of a complex network to precisely regulate the immune response, however, the regulatory mechanism of this complex system is not clear.
To date, little is known about sRNAs and H. pylori, and the potential role of miRNAs in the immune response to H. pylori infection has not been investigated. Here we first systematically identified sRNAs in H. pylori genome by a computational approach and a novel experimental strategy based on RNase I protection assay. The expression profile of cellular miRNAs during H. pylori infection was analyzed by using microarray, quantitative RT-PCR and Northern blot. Then we choose miR-155 for detailed investigation, defined the underlying mechanism leading to the miR-155 up-regulation by H. pylori, and identified the potential target genes of miR-155, and investigated the possible role of miR-155 as novel negative regulator that help to fine-tune the inflammation response of H. pylori infection. Our results provide insight into the knowledge of regulatory network of H. pylori. Furthermore, the altered miR-155 expression may identify a potential link between miRNAs and immune regulation during H. pylori infection.
Methods
1. Identification of sRNAs in H. pylori by a bioinformatics based approach. A file of all H. pylori Intergenic regions (IGRs) sequences was created by a Perl package based on the gene annotations. Then we systematically identified sRNAs in H. pylori based on gene location, sequence conservation, promoter and terminator search, and secondary structure. The candidate sRNAs were confirmed by Northern blot and RT-PCR.
2. Screening of natural antisense transcripts(NATs) in H. pylori by a novel approach based on RNase I protection assay. Total RNA H. pylori was digested by RNase I to remove single-stranded RNA. Synthesis of the first strand of cDNA was performed with random primer and reverse transcriptase. Then cDNA was tailed with poly(A) at the 3′-end, and a 5′adapter was ligated to 5' phosphorylated cDNA, and a cDNA library of NATs was constructed by PCR.
3. Identification of NATs in H. pylori. After the identification by SmaⅠ/XhoⅠrestriction endonuclease, insert-containing clones were subsequently characterized by DNA sequencing and BLAST analysis. The expression of NATs was confirmed by Northern blot and poly(A)-tailed RT-PCR.
4. The expression profile of miRNAs in gastric epithelial cell lines and gastric mucosal tissues during H. pylori infection was analyzed by using microarray, quantitative RT-PCR and Northern blot.
5. The negative regulatory role of miR-155 in the inflammatory response.
The potential target of miR-155 was identified by luciferase assay and western blot. Promoter analysis and inhibitor experiment were used to investigate the pathway involved in the induction of miR-155. Examination of miR-155 function was performed by overexpression and inhibition of miR-155.
Results:
1. Identification of sRNAs in H. pylori by a bioinformatics based approach.
Among a total of 6 candidate sRNAs initially predicted, two novel sRNAs (IG-443 and IG-524) were confirmed by Northern blot and RT-PCR. Virtually, they were a class of natural antisense transcripts (NATs), which were complementary to partial sequences of the following genes: flagellar motor switch gene (fliM) and fumarase (fumC).
2. Screening and identification of NATs in H. pylori by a novel approach.
We successfully constructed a cDNA library of NATs containing 68 clones. After the identification by SmaⅠ/XhoⅠrestriction endonuclease, a total of 33 insert-containing clones were subsequently characterized by DNA sequencing and BLAST analysis. 6 of all inserts were partially homologous to H. pylori genomic sequences and corresponded to the opposite strands of ORFs. Two novel NATs (NAT-39 and NAT-67) were confirmed by Northern blot and poly(A)-tailed RT-PCR. They were respectively complementary to the following genes: iron-regulated outer membrane protein (frpB) and periplasmic iron-binding protein (ceuE). NAT-39 and NAT-67 may participate in the regulation of iron homeostasis in H. pylori in a sequence complementary manner.
3. Screening and identification of obviously altered miRNAs in response to H. pylori infection.
The expression of miRNAs could be significantly altered during H. pylori infection, including the up-regulation of miR-155, miR-16, and miR-146a, and the down-regulation of miR-181b and miR-324. Consistent with the microarray findings, the results of qRT-PCR showed that miR-155, miR-16, and miR-146a was increased, 3.0, 2.1 and 2.5 fold change, respectively. miR-155 was highly up-regulated in H.pylori-positive patients, with a 3.9-fold increase as compared to the control (P<0.05).
4. The promoter region of miR-155 contained putative NF-κB and AP-1 binding sites. The results of promoter analysis and inhibitor experiment showed that both NF-κB and AP-1 pathways are required for the up-regulation of miR-155 in response to H. pylori, and AP-1 plays a central role in the induction of miR-155.
5. IKK-ε, SMAD2, and FADD are potential targets of miR-155, and miR-155 might down-regulate the target protein through different mechanism, either mRNA degradation or inhibition of translation.
6. miR-155 mimics significantly attenuated the mRNA and protein levels of IL-8 and GRO-α, and the effect of miR-155 in modulating the inflammation may be as a secondary effect through diminishing NF-κB activity. miR-155 may be involved in the negative feedback regulation of inflammation.
Conclusion:
1. We developed a computational approach to identify novel sRNAs in H. pylori, and identified two sRNAs (IG-443 and IG-524). The results indicate that there exist novel sRNAs in H. pylori and these RNAs may play potential role in regulating gene expression.
2. We developed a novel approach based on RNase I protection assay to identify NATs in H. pylori, and identified two NATs (NAT-39 and NAT-67). They may participate in the regulation of iron homeostasis in H. pylori in a sequence complementary manner.
3. H. pylori can stimulate the expression of miR-155 in gastric epithelial cells as well as in gastric mucosal tissues. miR-155 may function as novel negative regulator that help to fine-tune the inflammation response of H. pylori infection. Furthermore, the altered miR-155 expression may identify a potential link between miRNAs and pathogenesis of H. pylori related diseases.
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