1、肝癌差异表达miRNAs阵列分析及miR-138、miR-483-5p与肝癌发生机理的研究 2、HBx诱发anti-URGs用于肝硬化肝癌高危人群预警的实验研究
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摘要
第一部分肝癌差异表达miRNAs阵列分析及miR-138和miR-483-5p与肝癌发生机理的研究
肝细胞肝癌（hepatocellular carcinoma，HCC）是全球肿瘤病人死亡的主要原因之一。肝癌5年生存率不足5％，肝癌病人确诊越晚，其预后效果越差。晚期诊断严重限制了肝癌治疗手段的选择，因此肝癌的早期发现非常重要，阐明肝癌发生机制将有助于寻找早期诊断肝癌的肿瘤标记物。
MicroRNA(miRNA)是一类可调控基因表达的非编码RNA家族，能通过与mRNA分子相互作用导致RNA降解或阻遏蛋白质翻译。成熟的miRNA是一种约20-22nt长的单链RNA分子，可通过完全或不完全碱基配对与mRNA分子3’UTR相结合，从而抑制靶基因的翻译，参与细胞一系列的重要过程，包括细胞分化、细胞增殖与细胞凋亡。越来越多的研究表明肿瘤细胞与癌旁组织来源细胞间miRNA表达谱具有明显差异，miRNA表达谱可将肝癌、乳腺癌、肺癌、胰腺癌及白血病与临近正常组织分开。半数以上的miRNA定位于与肿瘤发生相关的染色体区域和脆性位点，提示miRNA在肿瘤形成过程中可能扮演着重要的角色。因此研究与肝癌发生、发展相关的miRNA表达谱的变化，探索肝癌发生发展的分子机制，这对肝癌的早期诊断和及时防治具有非常重要的意义。
在本研究中通过检测临床肝癌标本与癌旁组织标本中miRNA的表达谱，对筛选出的差异表达miRNA，以定量RT-PCR和Western blotting分析其相对应靶蛋白的表达状况，探讨其在HCC发生发展中的作用机制。
一、肝癌差异表达miRNAs阵列分析
miRNA与肿瘤的发生密切相关，目前已经发现miRNA在许多肿瘤中差异表达，但miRNA在肝癌发生发展中的研究尚不多。本部分通过Taqman低密度miRNA阵列（Taqman low density miRNA array，TLDA）分析了3对肝癌癌组织与其癌旁组织中667种miRNA表达谱差异。应用Hierarchical Cluster软件将24种差异表达的miRNA进行聚类分析，利用Real time RT-PCR在另外18对肝癌癌组织中验证出了20种miRNA的差异表达，进一步对差异表达miRNA的靶蛋白进行预测，并分析其参与的细胞功能和信号通路，绘制miRNA-靶蛋白mRNA网络图。
结果如下：
在本实验中，Taqman低密度miRNA阵列检测结果显示24种miRNA在肝癌组织与非肝癌组织间呈现差异表达，每种miRNA表达数值均与U6表达值相比进行标化。其中11种miRNA在癌组织中相对于在癌旁组织中上调表达，13种miRNA在癌组织中下调表达。应用Hierarchical Cluster软件将24种差异表达miRNA进行聚类时，可基本将肝癌组织与毗邻癌旁组织分开。利用Realtime RT-PCR检测上述24种miRNA在另外18例肝癌病人癌组织与非癌组织中的表达情况，证实了10种上调表达的miRNA(miR-217, miR-518b, miR-517c, miR-520g, miR-519a, miR-522, miR-518e,miR-525-3p, miR-512-3p, and miR-518a-3p)和10种下调表达的miRNA(miR-138,miR-214, miR-214#, miR-27a#, miR-199a-5p, miR-433, miR-511, miR-592,miR-483-5p andmiR-483-3p)。
预测上述差异表达miRNA的靶基因，并进行靶基因的功能分类（GO）和信号通路分析（KEGG Pathway)可知，signal transduction是这些miRNA调控的最重要的细胞功能，而regulation of actin skeleton和panthway in cancer是这些miRNA调控的最重要的信号通路。绘制miRNA与靶基因调控网络图，可以得知：miR-519a和miR-199a-5p可能是起到主导作用的miRNA，而RPS6KA3、SMAD4、ACVR2A等基因可能是与肝癌发生发展相关的重要基因。基于上述研究结果，选取miR-519a、miR-199a-5p、miR-138、miR-483-5p和miR-520g进一步深入研究。
二、miR-138调控细胞周期蛋白D3（CCND3）引起G1期阻滞
miR-138在肝组织中表达量较高，且在肝癌中呈现明显下调表达，推测miR-138与HCC的发生发展密切相关，但是它在HCC中的作用机制尚不清楚。本研究对miR-138调控靶基因及其在肿瘤发生中的功能研究进行更深入的研究。应用miRNA靶标预测软件TargetScan和miRnada预测miR-138靶基因。常规培养人肝癌细胞HepG2和Huh7，进行脂质体瞬时转染后，定量RT-PCR检测CCND3的表达量， Celltiter blue检测肝癌细胞活性，应用流式细胞技术检测细胞周期的改变，WesternBlotting用于检测CCND3蛋白水平的改变。
结果如下：
采用TargetScan和miRanda软件预测到CCND3为miR-138的靶基因。WesternBlotting结果显示，miR-138在肝癌中发生不同程度的下调，而CCND3是上调表达，两者呈负相关。在HepG2和Huh7细胞中转染miR-138模拟物，发现CCND3蛋白表达被下调，而转染miR-138抑制物，CCND3蛋白表达上调。外源表达miR-138抑制CCND3蛋白的表达，同时减弱了细胞的生长速度。与之相反的是，抑制细胞内miR-138表达则促进细胞的生长速度。应用流式细胞仪检miR-138对细胞周期及凋亡的影响，结果发现转染miR-138模拟物，细胞G0/Gl期延长，S期相应缩短；与之相反的是转染miR-138抑制物，细胞G0/Gl期缩短，S期相应延长。在裸鼠模型中，皮下注射转染miR-138模拟物或抑制物的HepG2细胞，连续观察5周，从第3周开始注射了转染miR-138模拟物HepG2细胞的肿瘤显著小于未转染HepG2对照组，而注射了转染miR-138抑制物HepG2细胞的肿瘤显著大于未转染HepG2对照组，此动物体内的肿瘤抑制实验再次验证了miR-138的抑制肿瘤细胞生长的作用。
三、miR-483-5p调控的维甲酸诱导蛋白16（RAI16）是一种新的癌蛋白和肝癌标志物
miR-483-5p在肝癌中呈现明显下调表达，推测miR-483-5p与HCC的发生发展密切相关，但是它在HCC中的作用机制尚不清楚。本研究对miR-483-5p调控靶基因及其在肿瘤发生中的功能研究进行更深入的研究。应用miRNA靶标预测软件TargetScan和miRnada预测miR-483-5p靶基因。常规培养人肝癌细胞HepG2和Huh7，进行脂质体瞬时转染后，定量RT-PCR检测CCND3的表达量， Cell titer blue检测肝癌细胞活性，应用流式细胞技术检测细胞周期的改变，Western Blotting用于检测RAI16蛋白水平的改变。
结果如下：
采用TargetScan和miRanda软件预测到RAI16为miR-483-5p的靶蛋白。WesternBlotting结果显示，miR-483-5p在肝癌中发生不同程度的下调，而RAI16是上调表达，两者呈负相关。在HepG2和Huh7细胞中转染miR-483-5p模拟物，发现RAI16蛋白表达被下调，而转染miR-483-5p抑制物，RAI16蛋白表达上调。外源表达miR-483-5p抑制RAI16蛋白的表达，同时减弱了细胞的生长速度。与之相反的是，抑制细胞内miR-483-5p表达则促进细胞的生长速度。应用流式细胞仪检miR-483-5p对细胞周期及凋亡的影响，结果发现转染miR-483-5p模拟物，细胞G0/Gl期延长，S期相应缩短；与之相反的是转染miR-483-5p抑制物，细胞G0/Gl期缩短，S期相应延长。在裸鼠模型中，皮下注射转染pcDNA3.1-RAI16或siRNA-RAI16的HepG2细胞，连续观察5周，从第3周开始注射了转染pcDNA3.1-RAI16HepG2细胞的肿瘤显著大于未转染HepG2对照组，而注射了转染siRNA-RAI16HepG2细胞的肿瘤显著小于未转染HepG2对照组，此动物体内的肿瘤抑制实验再次验证了RAI16的促进瘤细胞生长的作用。
四、结论
本研究在配对肝癌样本中筛选出20个差异表达miRNA，包括10个上调的miRNA和10个下调的miRNA。上述差异表达miRNA的靶基因的功能分类（GO）和信号通路（KEGG Pathway)分析可知，signal transduction是这些miRNA调控的最重要的细胞功能，而regulation of actin skeleton和panthway in cancer是这些miRNA调控的最重要的信号通路。而miR-519a、miR-199a-5p、miR-138、miR-483-5p和miR-520g在这些差异表达miRNAs中起主导作用，值得进一步深入研究。
对miR-138深入的研究表明：miR-138调控CCND3的表达，诱导细胞周期在G0/Gl期停滞，进而抑制肝癌细胞的生长。在HCC组织中，miR-138下调表达，减弱了对CCND3的调控，CCND3呈上调表达，失去对细胞周期控制，肿瘤细胞生长速度加快。综上说明miR-138在HCC的发生发展中发挥抑癌基因的作用，这将为HCC的诊断与治疗带来新的曙光。
对miR-483-5p及其靶蛋白RAI16的深入研究表明：miR-483-5p调控RAI16的表达，诱导细胞周期在G0/Gl期停滞，进而抑制肝癌细胞的生长。在HCC组织中，miR-483-5p下调表达，减弱了对RAI16的调控，RAI16呈上调表达，失去对细胞周期控制，肿瘤细胞生长速度加快。综上说明miR-483-5p和RAI16在HCC的发生发展中发挥重要作用，这深入对HCC致病机理的认识提供新的思路。
第二部分
HBx诱发anti-URGs用于肝硬化肝癌高危人群预警的实验研究
肝细胞肝癌（hepatocellular carcinoma，HCC）是目前最为常见的癌症之一。在我国，肝癌的发病率高与乙型肝炎病毒（HBV）慢性感染密不可分，相当一部分的慢性乙肝迁延不愈，并经肝纤维化、肝硬化进程向肝癌转化。但对慢乙肝发生和转归的分子机制认知严重不足，缺少有效的早期诊断和干预、治疗手段，难以阻断大量病患从慢性肝炎向肝硬化和肝癌发展。
目前，甲胎蛋白（AFP）用于诊断HCC已被广泛认可，但实属无奈之选。越来越多的文献报道了AFP在诊断和监测HCC的局限性。事实上，按照目前的HCC诊断标准，当AFP和影像学检查出现异常时，肝癌往往已发展到晚期，多数患者已失去了宝贵的早期干预治疗的机会。此外，有3个最有可能用于HCC辅助诊断的标志物：AFP的异构体(AFP-L3%)、人磷脂酰肌醇蛋白聚糖3（GPC3）及去γ羟基凝血素(DCP)。其他已报道的标志物还有肝癌癌蛋白p28GANK、高尔基磷蛋白(GOLPH2/GP73)、鳞状上皮细胞癌抗原(SCCA)、α-L-岩藻糖苷酶(AFU)和肝细胞生长因子(HGF)等，但上述潜力标志物，即使联合应用，对于HCC诊断的敏感度仍不能达到作为理想标志物的标准。因此，探究肝炎发展为肝癌的致病机制，寻找理想的肝癌标志物，最终实现阻断或减缓肝炎向肝硬化肝癌转化，降低肝癌的发病率和死亡率，仍需不懈的努力。
课题组前期工作发现了5种HBx诱导的高表达蛋白(URG4, URG7, URG11,URG12和Sui1)，URG4和URG11是癌基因，能够促进肿瘤生长；URG7具有抵抗Fas介导细胞凋亡的作用；URG12是核糖体蛋白，而Sui1是转录启始因子，两者对于蛋白翻译的完整性非常重要。这5种高表达蛋白都具有刺激肝细胞生长和抵抗TNF作用的特性，其抗体与肝炎发展至肝癌的进程密切相关，上述蛋白的抗体（anti-URGs）是否可作为肝癌标志物，监测肝炎发展为肝硬化、肝癌的进程，用于HCC的早期诊断和高危人群的筛查值得进一步研究。
一、合成多肽抗原检测anti-URGs ELISA方法的建立
针对5种URGs的蛋白序列，设计并合成14-22氨基酸长度的多肽作为包被抗原，建立检测血清中anti-URGs抗体的间接ELISA方法。在包被液、多肽抗原包被浓度、封闭剂、封闭时间、血清稀释度、血清反应时间和显色时间方面对检测方法进行优化，确定Cut-off值，并评估检测方法的敏感性、特异性、精确性和稳定性。
结果如下：
1、建立了合成多肽抗原检测anti-URGs的ELISA方法，检测流程为：多肽抗原以10ug/ml的浓度用pH9.6的碳酸盐缓冲液4℃包被过夜，含3%BSA的Tris缓冲液37℃封闭2小时以上，按照1：10稀释加入血清4℃过夜，充分洗涤后加入HRP标记山羊抗人IgG,37℃孵育30分钟，充分洗涤后加入TMB，避光显色10分钟，2M硫酸终止反应后测OD值。
2、确定了ELISA检测anti-URG4、anti-URG7、anti-URG11、anti-URG12和anti-Sui1的Cut-off值分别为：0.103、0.098、0.101、0.107和0.113。
3、ELISA方法具有较好的敏感性（稀释倍数20-640倍）和特异性（多肽抗原可阻断76.9-89.6%的反应），检测试剂批内差3.2-4.5%，批间差7.4-10.3%，在4℃可保存至少5个月。
二、anti-URGs在乙型肝炎发展为肝癌进程中预警作用的研究
采用自行建立的多肽抗原检测anti-URGs的ELISA方法，大规模检测肝炎、肝硬化和肝癌患者血清中的5种anti-URGs，进行了下列研究：
1、anti-URGs与肝炎发展为肝癌进程的相关性研究：检测全部血清样本中5种anti-URGs，OD值大于或等于Cut off值的判定为阳性，反之为阴性。利用统计学分析软件对实验数据进行分析，确定anti-URGs阳性数在健康献血员组、肝炎组、肝硬化组和肝癌组之间是否有统计学差异，即anti-URGs与肝炎发展为肝硬化、肝癌进展是否相关，并进一步评估anti-URGs用于HCC高危人群的筛查与小肝癌的早期诊断的可靠性。
2、anti-URGs检查与AFP及其他标志物的对照研究：在肝炎、肝硬化、肝癌患者组中，对比anti-URGs检查与现有的AFP结合影像学检查的敏感度、特异度、阳性率和符合率。单独使用anti-URGs及其他已报道的标志物，包括AFP-L3%、GPC3和GP73等，对比其敏感度、特异度、阳性率和符合率；以多种方式联合使用anti-URGs及其他已报道的标志物，是否可提高检测的敏感度、特异度、阳性率和符合率。
3、anti-URGs对于原发性肝癌发生、复发等的前瞻性研究：A在乙肝肝炎患者组中，追踪anti-URGs的动态变化，对比3种以上（包括3种）anti-URGs阳性和3种以下anti-URGs阳性的患者病情发展、转归的差异；B在肝硬化、肝癌患者组中，追踪治疗前后anti-URGs的变化，对比anti-URGs持续阳性和anti-URGs转阴的患者癌症复发及生存时间的差异。
4、anti-URGs对于肝癌高危人群筛查的前瞻性研究：在乙肝病毒携带者与慢乙肝患者组中，对比3种或3种以上anti-URGs阳性、2种anti-URGs阳性、1种anti-URGs阳性和anti-URGs阳性全阴性的患者病情发展的差异。
结果如下：
1、发现全部558份患者血清和108份健康献血员血清中anti-URGs阳性个数在HBV相关的肝炎、肝硬化、肝癌及健康献血员组中有明显差异（P<0.001），在健康献血员组，91.7%小于等于1个anti-URGs阳性，在HBV相关的肝炎、肝硬化、肝癌组中，大于等于3个anti-URGs阳性的比例分别为：35.8%、60.7%和55.2%，此结果提示anti-URGs与肝炎发展为肝癌进程相关。
2、按照在5种anti-URGs中有3个或3个以上阳性则判定为anti-URGs阳性的标准，在肝硬化和肝癌患者中检测敏感度为58.3%，在健康献血员中的特异度为99.1%，在HBV感染无症状携带者中的特异度为87.7%，在其他癌症患者中的特异度为90.0%。
3、为全面评价anti-URGs作为肝硬化肝癌高危人群预警的肿瘤标志物的可靠性，本研究还同时检测了血清中的AFP、AFP-L3%、GPC3和GP73含量。在HBV相关肝硬化和肝癌组中，anti-URGs的敏感度为60.0%和53.3%；AFP的敏感度为33.3%和30.0%； AFP-L3%的敏感度为40.0%和33.3%；GPC3的敏感度为36.3%和23.3%；GP73的敏感度为53.3%和33.3%；在健康献血员组中, anti-URGs的特异度为100%，AFP、AFP-L3%、GPC3、GP73的特异度分别为93.4%，96.7%，93.4%和93.4%。将anti-URGs分别与AFP、AFP-L3%、GPC3和GP73联合检测，可部分提高敏感度，在肝硬化组为76.6%，73.3%，73.3%和76.6%，在肝癌组为60.3%，60.3%，60.0%和60.0%。
4、对10例肝硬化患者的进一步追踪研究显示anti-URGs阳性的肝硬化患者24个月内发生肝癌的几率大幅升高（anti-URGs阳性组：6/10人，anti-URGs阴性组：1/10人），提示anti-URGs与肝硬化肝癌发生的风险性相关，anti-URGs阳性的患者应给予适当的预防性治疗及定期随访。
5、对于10例HBV相关肝癌患者治疗前后系列血清中anti-URGs进行检测发现，anti-URGs在肝癌临床诊断前即可出现，在治疗后发生转阴，而在个别癌症复发的患者血清中持续阳性。
三、结论
本课题建立了多肽抗原检测anti-URGs的ELISA方法，并对HBV相关急、慢性肝炎、肝硬化和肝癌患者血清，其他癌症患者、HCV肝炎肝硬化患者以及健康献血员血清进行规模化检测，结果显示：anti-URGs在HBV相关的肝炎、肝硬化、肝癌组中的检出率分别为21.7%、60.2%和55.1%，存在明显统计学差异（P<0.001），anti-URGs的确与HBV相关的肝炎、肝硬化和肝癌的病情发展相关，anti-URGs在肝硬化和肝癌患者中检测敏感度为58.3%，在健康献血员中的特异度为99.1%。因此，anti-URGs可以作为肝硬化、肝癌高危人群预警的血清标志物。
本课题研究的创新点在于：（1）本课题首次将anti-URGs作为监测肝炎发展为肝癌进程的血清标志物，具有较好的敏感度和特异度，具有先进性。（2）anti-URGs与肝癌发展的多个方面相关，采用anti-URGs与AFP、AFP-L3%或GP73等组合检测时可提高检测敏感度15%。（3）本课题着眼于HBV感染后病情由肝炎发展为肝硬化、肝癌的整个进程的监测以及相应的肝硬化、肝癌高危人群预警的血清标志物的筛选和评估。在肝炎和肝硬化阶段血清中即已出现3个或3个以上anti-URGs阳性的患者，具有发展成为肝癌患者的高风险性，应例行血清学和影像学检查，尽早进行干预和有效的治疗。本课题的完成将有助于原发性肝癌的预警、诊断和预后，阻断肝炎病患向肝硬化肝癌转化，降低肝癌的发病率和死亡率。
Part I
Taqman array analysis of miRNAs in hepatocellularcarcinoma and the roles of miR-138and miR-483-5p intumorigenesis
Hepatocellular carcinoma (HCC) is the leading cause of cancer deaths in theworld．More than half of those people are in China．The overall5-year survival rate forHCC patients is less than5％．Early detection of HCC is of supreme importance becausethe treatment options are severely limited by a late diagnosis．Late clinical presentationshave also led to poor prognosis for HCC patients．Thus，it is necessary to el ucidate themolecular mechanisms underlying HCC and identify novel therapeutic targets andbiomarkers for the early detection of HCC．Although serum a-fetoprotein(AFP)is a usefultumor marker for the detection and monitoring of HCC development，the false-negativerate by AFP level alone may be as high as40％for patients with a small HCC．So，if a newtumor marker has more sensitivity and can be detected in the primary stage of HCC，it ispossible to extrapolate the likelihood of a malignant liver tumor．Thus，discovery of newtarget molecules that are critically involved in the majority of cases and expressedspecifically in tumors will be essential to understand the mechanisms and improvetherapeutic intervention and prognosis of HCC．
MicroRNA (miRNA) represent a class of small noncoding RNA that control geneexpression by targeting mRNA and triggering either translation repression or RNAdegradation．Mature miRNA are19-25nucleotides that can silence genes through eitherconsummate or defective binding to the3’ untranslated region (3’UTR) of thetranscript．miRNA have been involved in the modulation of diverse cellular processesincluding cell differentiation, proliferation and apoptosis．An increasing number of studiessuggest that there all differential miRNA expression profiles between those in cancertissues and adjacent nontumorous tissue，these studies also show that miRNA profilescould distinguish malignant tumor of liver，breast，lung，pancreas and leukemia fromadjacent nontumorous tissue．The importance of miRNA in cancer is highlighted by the observation that50％of miRNA genes are located in cancer associated genomic regions orfragile sites．Importantly，miRNA expression is frequently deregulated in several cancersincluding B-cell chronic lymphocytic leukemia Burkitt’ S lymphoma，colorectal cancer,lung cancer and HCC．Additionally，differential expression of miRNA have been found tobe associated with postoperativesurvival in lung cancer patients and all diagnostic andprognostic markers of lung cancer．miRNA have been implicated to play both tumorsuppressor and oncogenic roles．These studies indicate that miRNA profiling studies couldbe used for defining clinical phenotypes，as well as potentially useful molecular diagnosticmarkers．An understanding of the molecular mechanism by which mi RNA regulate HCCdevelopment potentially may provide new avenues of research that could aid earlydiagnosis and treatment of this highly malignant tumor．
In this present study，we examined the expression profiles of667miRNA in3pairs ofHCC and adjacent non-tumorous tissue by a Taqman low density miRNA array(TLDA)．The biological functions and targets of some differentially expressed miRNAshave been detected by Real-time RT-PCR and Western-blotting analysis．This study mayhelp clarify the molecular mechanisms involved in the progression of HCC．
1、Deregulated miRNAs in hepatocellular carcinoma
667microRNA expression profiles of was performed on3pairs of surgically removedHCC and adjacent non-tumorous tissues by using TLDA, then23deregulated miRNAswere validated in an extended samples set of18paired HCC and adjacent non-tumorousliver tissues by realtime reverse transcription polymerase chain reaction (RT-PCR)analysis．
Results：
Only miRNAs altered by at least3-fold in all three pairs of the samples wereconsidered significant candidates. Under these strict criteria,11up-regulated miRNAs and12down-regulated miRNAs were identified. To validate the miRNA array data, qRT-PCRwas performed in18pairs of HCC tissues.10up-regulated and10down-regulatedmiRNAs showed consistent changes in more than50%tumorous tissues, of whichmiR-217, miR-520g, miR-522and miR-525-3p were up-regulated in more than70%tumorous tissues, and miR-199a-5p, miR-138, miR-483-5p and miR-511weredown-regulated in more than70%tumorous tissues.
The targets of the above20deregulated miRNAs were predicted by TargetScan. Tojudge the most significant candidates and investigate the cellular function, the signaling pathway and GOs of these target genes were analyzed. The results showed that a widevariety of cellular processes were featured significantly in signaling pathways. Many ofthese signaling pathways, such as insulin, MAPK, TGF-beta and Wnt signaling pathway,have been shown to participate in the tumorigenesis. However, some other signalingpathways have never been reported to play a role in tumorigenesis, e.g. axon guidance.Among all these differentially regulated signaling pathways,“regulation of actincytoskeleton” and “pathway in cancer” appeared to be the most enriched two in bothup-regulated and down-regulated miRNA groups. A similar phenomenon was observed inGOs analysis. Many cellular functions were featured significantly, of which the “signaltransduction” appeared to be the most enriched one. Based on these results, the miRNAswhich were involved in both “signal transduction” and “the regulation of actincytoskeleton or pathway in cancer” might be the most significant candidates. MiR-520g,miR-483-5p, miR-138, miR-199a-5p, miR-217and miR-518e were selected under thecriteria for further studies. Here, the studies on miRNA-138were presented.
2、miR-138induces cell cycle arrest by targeting cycline D3
The significant reduction of miR-138expression in HCC tissues indicated possiblebiological significance in tumorgenesis. At first, the effect of miR-138on cell growth wasevaluated in HepG2and Huh7cells transfected with or not, miR-138mimic, miR-138inhibitor or NC duplex. From2day (HepG2) or3day (Huh7) after the transfection, theviability of cells transfected with miR-138mimic significantly decreased compared withthat of NC duplex transfected or nontransfected cells, but the viability of cells transfectedwith miR-138inhibitor significantly increased. These results indicate miR-138couldinhibit cell growth. To validate the inhibitor effect of miR-138on cell growth, the colonyformation assay was performed in HepG2and Huh7transfected with or not, miR-138mimic, miR-138inhibitor or NC duplex. HepG2and Huh7cells transfected with20nMmiR-138mimic displayed much fewer and smaller colonies (218or161colonies)compared with NC duplex transfected (783or729colonies) and nontransfected cells (756or692colonies), but cells transfected with20nM miR-138inhibitor displayed much moreand larger colonies (1238or1349colonies).
To further confirm the above findings, an in vivo mouse model was used. For theduration of the treatment with miR-138mimic or miR-138inhibitor for5weeks, tumorvolume curves revealed a significant decrease in growth rates at the3rd,4th and5th weekafter treatment with miR-138mimic and a significant increase in growth rates at the4th and5th week after treatment with miR-138inhibitor whereas no significant differences intumor growth rates were observed between the NC group and the ctrl group. These resultsindicate that introduction of miR-138significantly inhibits tumorigenicity of HepG2cellsin xenograft nude mouse model.
To investigate the mechanism of inhibitor effect of miR-138, flow cytometry assayshowed that the percentages of miR-138mimic transfected HepG2and Huh7cells in theG0-G1phase were18%(HepG2) and11%(Huh7) higher than that of NC duplextransfected or nontransfected cells, which paralleled with a44%(HepG2) and38%(Huh7)decrease in the S phase. In miR-138inhibitor transfected cells, the percentages of cells inthe G0-G1phase were8%(HepG2) and6%(Huh7) less than that of NC duplex transfectedor nontransfected cells, which paralleled with a14%(HepG2) and19%(Huh7) increase inthe S phase. These results indicate miR-138could inhibit HepG2and Huh7proliferationby inducing cell cycle arrest at G1/S phase.
It is generally accepted that miRNAs exert their function through regulating theexpression of their downstream target genes. Cycline D3(CCND3) was predicted as apotential target of miR-138by TargetScan and Miranda. To validate whether CCND3is adirect target of miR-138, a human CCND33’-UTR fragment containing wild-type ormutant miR-138binding sequence was cloned downstream of the firefly luciferase reportergene in pGL3. In HEK293cells cotransfected with the reporter plasmids and miR-138mimic or NC duplex, the luciferase activity of the reporter that contained wild-type3’-UTR was significantly suppressed by miR-138mimic, but the luciferase activity ofmutant reporter was unaffected, indicating that miR-138may suppress gene expressionthrough miR-138binding sequence at the3’-UTR of CCND3. Furthermore, transfection ofmiR-138mimic decreased CCND3expression and transfection of miR-138inhibitorincreased CCND3expression in HepG2cells at protein but not mRNA level, suggestingthat CCND3expression could be inhibited by miR-138at posttranscriptional level.Together, the results show that miR-138could regulate the expression of endogenoushuman CCND3by directly targeting the3’-UTR of CCND3mRNA and human CCND3isa new target of miR-138.
To identify whether inhibition of CCND3, just like miR-138restoration, also resultedin HCC repression, the effects of knockdown of CCND3on cell growth were examined.Firstly, HepG2cells were transfected with or not, CCND3siRNA or control siRNA.Seventy two hours after transfection, a dose dependent knockdown of CCND3was observed in HepG2cells. In cell viability assay and cell cycle analysis, in vitro knockdownof CCND3repressed cell viability, induced cell cycle arrest and inhibited the colonyformation. The similar data were obtained in Huh7cells transfected with CCND3siRNA.These results indicate that CCND3is most likely involved in the induction of cell cyclearrest by miR-138.
3、Retinoic acid induced16, regulated by miR-483-5p, is a novel oncogene and tumormarker for hepatocellular carcinoma
The significant reduction of miR-483-5p expression in HCC tissues indicatedpossible biological significance in tumorgenesis. At first, the effect of miR-483-5p on cellgrowth was evaluated in HepG2and Huh7cells transfected with or not, miR-483-5p mimic,miR-483-5p inhibitor or NC duplex. From2day (HepG2) or3day (Huh7) after thetransfection, the viability of cells transfected with miR-138mimic significantly decreasedcompared with that of NC duplex transfected or nontransfected cells, but the viability ofcells transfected with miR-483-5p inhibitor significantly increased. These results indicatemiR-138could inhibit cell growth.
To investigate the mechanism of inhibitor effect of miR-483-5p, flow cytometry assayshowed that the percentages of miR-483-5p mimic transfected HepG2and Huh7cells inthe G0-G1phase were18%(HepG2) and11%(Huh7) higher than that of NC duplextransfected or nontransfected cells, which paralleled with a44%(HepG2) and38%(Huh7)decrease in the S phase. In miR-483-5p inhibitor transfected cells, the percentages of cellsin the G0-G1phase were8%(HepG2) and6%(Huh7) less than that of NC duplextransfected or nontransfected cells, which paralleled with a14%(HepG2) and19%(Huh7)increase in the S phase. These results indicate miR-483-5p could inhibit HepG2and Huh7proliferation by inducing cell cycle arrest at G1/S phase.
Retinoic acid induced16(RAI16) was predicted as a potential target of miR-483-5pby TargetScan and Miranda. To validate whether RAI16is a direct target of miR-483-5p, ahuman RAI163’-UTR fragment containing wild-type or mutant miR-483-5p bindingsequence was cloned downstream of the firefly luciferase reporter gene in pGL3. InHEK293cells cotransfected with the reporter plasmids and miR-483-5p mimic or NCduplex, the luciferase activity of the reporter that contained wild-type3’-UTR wassignificantly suppressed by miR-483-5p mimic, but the luciferase activity of mutantreporter was unaffected, indicating that miR-483-5p may suppress gene expression throughmiR-483-5p binding sequence at the3’-UTR of RAI16. Furthermore, transfection of miR-483-5p mimic decreased RAI16expression and transfection of miR-138inhibitorincreased RAI16expression in HepG2cells at protein but not mRNA level, suggesting thatRAI16expression could be inhibited by miR-483-5p at posttranscriptional level. Together,the results show that miR-483-5p could regulate the expression of endogenous humanRAI16by directly targeting the3’-UTR of RAI16mRNA and human RAI16is a newtarget of miR-483-5p.
In commercial HCC tissue arrays, most HCC tumor tissues showed moderate (score2) or strong (score3) RAI16expression (89of126cases). In contrast, most nontumortissues showed negative (score0) or weak (score1) RAI16expression (118of126cases).The sensitivity and specificity of RAI16expression for the diagnosis of HCC were70.6%and93.6%, respectively. Strikingly, the sensitivity and specificity increased to80.9%and92.0%when glypican-3was used with RAI16. These results indicate that RAI16may bepotential tumor marker for HCC diagnosis, especially combined with GPC3.
The significant up-regulation of RAI16expression in HCC tissues indicated possiblebiological significance in tumorgenesis. At first, the viability of cells transfected withpcDNA3.1-RAI16significantly increased compared with that of empty vector pcDNA3.1transfected or nontransfected cells, but the viability of cells transfected with RAI16siRNAsignificantly decreased. HepG2and Huh7cells transfected with pcDNA3.1-RAI16displayed much more and larger colonies (1238or1349colonies) compared with emptyvector pcDNA3.1transfected (783or729colonies) and nontransfected cells (756or692colonies). Cell cycle analysis showed that the percentages of pcDNA3.1-RAI16transfectedHepG2and Huh7cells in the G0-G1phase were11.6%(HepG2) and9.4%(Huh7) lowerthan that of pcDNA3.1transfected or nontransfected cells, which paralleled with a35.6%(HepG2) and27.0%(Huh7) increase in the S phase. In RAI16siRNA transfected cells, thepercentages of cells in the G0-G1phase were10.9%(HepG2) and14.1%(Huh7) higherthan that of NC siRNA transfected or nontransfected cells, which paralleled with a25.8%(HepG2) and19.4%(Huh7) decrease in the S phase. These results indicate RAI16couldenhance HepG2and Huh7proliferation by promoting cell cycle from G0/G1to S phase.Furthermore, the percentage of apoptotic cells in pcDNA3.1-RAI16transfected HepG2andHuh7cells were36.4%(HepG2) and33.5%(Huh7) lower than that of pcDNA3.1transfected or nontransfected cells. In RAI16siRNA transfected cells, the percentages ofapoptotic cells were21.5%(HepG2) and26.3%(Huh7) higher than that of NC siRNAtransfected or nontransfected cells.
To further confirm the above findings, an in vivo mouse model was used. For theduration of the treatment with HepG2cells stably expressing RAI16or transfected withRAI16siRNA, tumor volume curves revealed a significant increase in growth rates at the3rd,4th and5th week after treatment with HepG2cells stable expressing RAI16and asignificant decrease in growth rates at the4th and5th week after treatment with HepG2cells transfected with RAI16siRNA whereas no significant differences in tumor growthrates were observed between the NC siRNA transfected or non-transfected HepG2cellsgroups. These results indicate that introduction of RAI16significantly promotestumorigenesis in xenograft nude mouse model.
To investigate the mechanism of RAI16enhancement of tumor growth in HCC,10major cancer related pathways were analyzed in transfected HepG2cells using adual-luciferase reporter system (Promega). The most affected pathways were transforminggrowth factor-β (TGF-β), mitogen-activated protein kinase (MAPK)/extracellularsignal-regulated kinase (ERK) and Wnt. The most affected pathways were TGF-β,MAPK/ERK and Nuclear factor κB (NFκB). The up-regulated MAPK/ERK and TGF-βpathways in RAI16overexpressed cells is consistent with the down-regulated MAPK/ERKand TGF-β pathways in RAI16knockdown cells These results indicate MAPK/ERK andTGF-β pathway may be involved in RAI16induction of tumorigenesis.
To verify above findings, the key molecules in MAPK/ERK and TGF-β pathway weredetected by immunoblotting. RAI16overexpession increased the phosphorylation of MEKand ERK1/2significantly, the total ERK but not total MEK expression. Similar phenomenawere observed in TGF-β pathway, the phosphorylation of SMAD2/3and total SMAD2/3,together with SMAD4were increased by RAI16overexpression. RAI16knockdown ladeto the reverse results. Interesting, phosph-SMAD2/3and SAMD4in HCC tumor tissueswere increased much more significantly than that in RAI16transfected cell modal, butphosphorylation of MEK and ERK1/2seemed more significant in RAI16transfected cellmodal. These results suggest it is possible that MAPK/ERK and TGF-β pathway play therole in different stage of RAI16induction of tumorigenesis.
Part II
Application of HBx-induced anti-URGs as early warningbiomarker of liver cirrhosis and hepatocellularcarcinoma
Hepatocellular carcinoma (HCC) is one of the most common cancers, with morethan500,000deaths with over600,000new cases yearly worldwide. The clinical course ofHCC is mostly asymptomatic. Suspected liver changes are too large and too advanced forthe tumor to be subjected to potentially effective and radical therapy when they aredetected incidentally. Because of serious limitations of the surgical and oncologicaltreatment available, it seems necessary to concentrate on the earliest possible diagnosis,particularly sensitive detection of resectable focal liver changes-preferably when tumorsare less than2cm in diameter. Surveillance with immaging techniques and serumα-fetoprotein (AFP) analyses is recommended for all cirrhotic patients and other specificrisk groups every6months.
AFP has to be considered “the golden standard” for HCC serum markers, however,the usefulness of AFP testing for the population at risk should be seriously questioned. AFPdiagnostic values for this assay are undoubtedly poor. AFP specificity varies from about76%to96%and increases with elevated cut-off value. Simultaneous sensitivity decreasesmuch more from about25%for potentially resectable tumors of less than3cm in diameterto about50%for lesions of>3cm in diameter. Other serum biomarkers such as AFP-L3%,DCP, AFU, GGT, GP-73, MUC-1, SCCA and GPC-3have significant diagnosticlimitations, and in fact they are not particularly precise for the early diagnosis of HCC.Simultaneous determination of these markers in various combinations could improve theaccuracy in differentiating HCC from nonmalignant hepatopathy, but there still exists theunresolved problem of tiny ‘grey’ nodules in the ‘black and white’ diagnostic perspective.
Previous studies have found that HBx contributes to the development of HCC byaltering patterns of host gene expression.5up-regulated genes (URGs: URG4, URG7,URG11, URG12and Sui1) associated with HBx triggered corresponding antibodies(anti-URGs) in the sera of patients prior to tumor development and/or at early stagetumorigenesis.
1、Establish of ELISA assay for anti-URGs detection using synthesized peptides
To establish an ELISA method to detect the anti-URGs in the serum of patients withHCC,5groups of peptides (L4A/L4B, L7B, L11-1/L11-3/L11-4, L12A/L12B andSui1A/Sui1B) were synthesized. The sensitivity, specificity and precision of this assaywere evaluated.Results:
（1）The procure of ELISA assay is as following:10ug/mlpeptides were coatedby0.01M NaHCO3buffer at4℃overnight, blocking by Tri-HCl buffer with3%BSA at37℃for more than2hours, diluted serum samples at1:10were incubatedat4℃overnight, after washing, HRP Goat anti-human IgG incubation at37℃for30minutes,washing, TMB development for10minutes，read the OD value after stopping reaction with2M H2SO4.
（2）The Cut-off value of ELISA assay to detect anti-URG4、anti-URG7、anti-URG11、 anti-URG12and anti-Sui1is:0.103,0.098,0.101,0.107and0.113respectively.
（3）The developed ELISAmethod showed good sensitivity, specificity and precision.Intra-division is3.2-4.5%, inter-division is7.4-10.3%.
2、Detection of anti-URGs in serum samples by ELISA
To evaluate the anti-URGs as the potecial serological markers of HCC and thecorresponding ELISA assay. The anti-URGs were detected in serum samples from558patients and108blood donors by ELISA assay.Results:
（1） The number of anti-URGs showed significantly difference among the558patient with HBV associated hepatitis, cirrhosis, HCC and108blood donor serum samples.In HBV associated live disease groups, the sensitivity of anti-URGs is35.8%,60.7%and55.2%respectively.
（2）The spececificity of anti-URGs is99.1%,87.7%and90.0%in blood donorsgroup, HBV asymptomatic carriers and other tumors respectively.
（3）In HBV associated cirrhosis and HCC groups, the sensitivity of anti-URGs is60.0%and53.3%; the sensitivity of AFP is33.3%and30.0%; the sensitivity of AFP-L3%is40.0%and33.3%; the sensitivity of GPC3is36.3%and23.3%; the sensitivity of GP73is53.3%and33.3%; In blood donors group, the spececificity of anti-URGs is100%, thespececificity of AFP, AFP-L3%, GPC3and GP73is93.4%,96.7%,93.4%and93.4% respectively. Combination anti-URGs with AFP, AFP-L3%, GPC3and GP73respectivelycould improve the sensitivity by10%.
（4） Longitudinal studies in20HBV-cirrhosis patients with more than3anti-URGsor with less than1anti-URGs showed that patients with more anti-URGs were more likelyto develope tumor compared with those with fewer anti-URGs.
3、Conclusion
In this study, ELISA to detect the anti-URGs in serum was established and used inHBV infected patients and blood donors. It was found that anti-URGs were predominantlypresent among patients with HBV-associated HCC (55.2%) and cirrhosis (60.7%), and at alower frequency among patients with chronic hepatitis (35.8%), with significant difference(P<0.001). The sensitivity of anti-URGs in patients with HBV-associated HCC andcirrhosis was58.3%and the specificity of anti-URGs in blood donors was99.1%.
The innovation point of this study lies on:(1) anti-URGs were firstly as the earlywarning biomarker for liver cirrhosis and HCC with much better sensitivity and specificity.(2) The combination of anti-URGs with AFP, AFP-L3or GP73improved the sensitivity ofdetection at15%.(3) This study was focused on the whole progress of HBV infection fromhepatitis to HCC, the HBV hepatitis and cirrhosis patients with3or more than3anti-URGs would have more risk to develop HCC, and should be paid more attention. Asearly warning biomarker of HCC, anti-URGs may be useful in the identification of HBVpatients with chronic liver disease who are most likely to develop HCC, which wouldprovide the help to diagnosis of human small HCC, early-stage asymptomatic HCC and thescreening of HCC in high risk population.
肝细胞肝癌（hepatocellular carcinoma，HCC）是全球肿瘤病人死亡的主要原因之一。肝癌5年生存率不足5％，肝癌病人确诊越晚，其预后效果越差。晚期诊断严重限制了肝癌治疗手段的选择，因此肝癌的早期发现非常重要，阐明肝癌发生机制将有助于寻找早期诊断肝癌的肿瘤标记物。
MicroRNA(miRNA)是一类可调控基因表达的非编码RNA家族，能通过与mRNA分子相互作用导致RNA降解或阻遏蛋白质翻译。成熟的miRNA是一种约20-22nt长的单链RNA分子，可通过完全或不完全碱基配对与mRNA分子3’UTR相结合，从而抑制靶基因的翻译，参与细胞一系列的重要过程，包括细胞分化、细胞增殖与细胞凋亡。越来越多的研究表明肿瘤细胞与癌旁组织来源细胞间miRNA表达谱具有明显差异，miRNA表达谱可将肝癌、乳腺癌、肺癌、胰腺癌及白血病与临近正常组织分开。半数以上的miRNA定位于与肿瘤发生相关的染色体区域和脆性位点，提示miRNA在肿瘤形成过程中可能扮演着重要的角色。因此研究与肝癌发生、发展相关的miRNA表达谱的变化，探索肝癌发生发展的分子机制，这对肝癌的早期诊断和及时防治具有非常重要的意义。
在本研究中通过检测临床肝癌标本与癌旁组织标本中miRNA的表达谱，对筛选出的差异表达miRNA，以定量RT-PCR和Western blotting分析其相对应靶蛋白的表达状况，探讨其在HCC发生发展中的作用机制。
一、肝癌差异表达miRNAs阵列分析
miRNA与肿瘤的发生密切相关，目前已经发现miRNA在许多肿瘤中差异表达，但miRNA在肝癌发生发展中的研究尚不多。本部分通过Taqman低密度miRNA阵列（Taqman low density miRNA array，TLDA）分析了3对肝癌癌组织与其癌旁组织中667种miRNA表达谱差异。应用Hierarchical Cluster软件将24种差异表达的miRNA进行聚类分析，利用Real time RT-PCR在另外18对肝癌癌组织中验证出了20种miRNA的差异表达，进一步对差异表达miRNA的靶蛋白进行预测，并分析其参与的细胞功能和信号通路，绘制miRNA-靶蛋白mRNA网络图。
结果如下：
在本实验中，Taqman低密度miRNA阵列检测结果显示24种miRNA在肝癌组织与非肝癌组织间呈现差异表达，每种miRNA表达数值均与U6表达值相比进行标化。其中11种miRNA在癌组织中相对于在癌旁组织中上调表达，13种miRNA在癌组织中下调表达。应用Hierarchical Cluster软件将24种差异表达miRNA进行聚类时，可基本将肝癌组织与毗邻癌旁组织分开。利用Realtime RT-PCR检测上述24种miRNA在另外18例肝癌病人癌组织与非癌组织中的表达情况，证实了10种上调表达的miRNA(miR-217, miR-518b, miR-517c, miR-520g, miR-519a, miR-522, miR-518e,miR-525-3p, miR-512-3p, and miR-518a-3p)和10种下调表达的miRNA(miR-138,miR-214, miR-214#, miR-27a#, miR-199a-5p, miR-433, miR-511, miR-592,miR-483-5p andmiR-483-3p)。
预测上述差异表达miRNA的靶基因，并进行靶基因的功能分类（GO）和信号通路分析（KEGG Pathway)可知，signal transduction是这些miRNA调控的最重要的细胞功能，而regulation of actin skeleton和panthway in cancer是这些miRNA调控的最重要的信号通路。绘制miRNA与靶基因调控网络图，可以得知：miR-519a和miR-199a-5p可能是起到主导作用的miRNA，而RPS6KA3、SMAD4、ACVR2A等基因可能是与肝癌发生发展相关的重要基因。基于上述研究结果，选取miR-519a、miR-199a-5p、miR-138、miR-483-5p和miR-520g进一步深入研究。
二、miR-138调控细胞周期蛋白D3（CCND3）引起G1期阻滞
miR-138在肝组织中表达量较高，且在肝癌中呈现明显下调表达，推测miR-138与HCC的发生发展密切相关，但是它在HCC中的作用机制尚不清楚。本研究对miR-138调控靶基因及其在肿瘤发生中的功能研究进行更深入的研究。应用miRNA靶标预测软件TargetScan和miRnada预测miR-138靶基因。常规培养人肝癌细胞HepG2和Huh7，进行脂质体瞬时转染后，定量RT-PCR检测CCND3的表达量， Celltiter blue检测肝癌细胞活性，应用流式细胞技术检测细胞周期的改变，WesternBlotting用于检测CCND3蛋白水平的改变。
结果如下：
采用TargetScan和miRanda软件预测到CCND3为miR-138的靶基因。WesternBlotting结果显示，miR-138在肝癌中发生不同程度的下调，而CCND3是上调表达，两者呈负相关。在HepG2和Huh7细胞中转染miR-138模拟物，发现CCND3蛋白表达被下调，而转染miR-138抑制物，CCND3蛋白表达上调。外源表达miR-138抑制CCND3蛋白的表达，同时减弱了细胞的生长速度。与之相反的是，抑制细胞内miR-138表达则促进细胞的生长速度。应用流式细胞仪检miR-138对细胞周期及凋亡的影响，结果发现转染miR-138模拟物，细胞G0/Gl期延长，S期相应缩短；与之相反的是转染miR-138抑制物，细胞G0/Gl期缩短，S期相应延长。在裸鼠模型中，皮下注射转染miR-138模拟物或抑制物的HepG2细胞，连续观察5周，从第3周开始注射了转染miR-138模拟物HepG2细胞的肿瘤显著小于未转染HepG2对照组，而注射了转染miR-138抑制物HepG2细胞的肿瘤显著大于未转染HepG2对照组，此动物体内的肿瘤抑制实验再次验证了miR-138的抑制肿瘤细胞生长的作用。
三、miR-483-5p调控的维甲酸诱导蛋白16（RAI16）是一种新的癌蛋白和肝癌标志物
miR-483-5p在肝癌中呈现明显下调表达，推测miR-483-5p与HCC的发生发展密切相关，但是它在HCC中的作用机制尚不清楚。本研究对miR-483-5p调控靶基因及其在肿瘤发生中的功能研究进行更深入的研究。应用miRNA靶标预测软件TargetScan和miRnada预测miR-483-5p靶基因。常规培养人肝癌细胞HepG2和Huh7，进行脂质体瞬时转染后，定量RT-PCR检测CCND3的表达量， Cell titer blue检测肝癌细胞活性，应用流式细胞技术检测细胞周期的改变，Western Blotting用于检测RAI16蛋白水平的改变。
结果如下：
采用TargetScan和miRanda软件预测到RAI16为miR-483-5p的靶蛋白。WesternBlotting结果显示，miR-483-5p在肝癌中发生不同程度的下调，而RAI16是上调表达，两者呈负相关。在HepG2和Huh7细胞中转染miR-483-5p模拟物，发现RAI16蛋白表达被下调，而转染miR-483-5p抑制物，RAI16蛋白表达上调。外源表达miR-483-5p抑制RAI16蛋白的表达，同时减弱了细胞的生长速度。与之相反的是，抑制细胞内miR-483-5p表达则促进细胞的生长速度。应用流式细胞仪检miR-483-5p对细胞周期及凋亡的影响，结果发现转染miR-483-5p模拟物，细胞G0/Gl期延长，S期相应缩短；与之相反的是转染miR-483-5p抑制物，细胞G0/Gl期缩短，S期相应延长。在裸鼠模型中，皮下注射转染pcDNA3.1-RAI16或siRNA-RAI16的HepG2细胞，连续观察5周，从第3周开始注射了转染pcDNA3.1-RAI16HepG2细胞的肿瘤显著大于未转染HepG2对照组，而注射了转染siRNA-RAI16HepG2细胞的肿瘤显著小于未转染HepG2对照组，此动物体内的肿瘤抑制实验再次验证了RAI16的促进瘤细胞生长的作用。
四、结论
本研究在配对肝癌样本中筛选出20个差异表达miRNA，包括10个上调的miRNA和10个下调的miRNA。上述差异表达miRNA的靶基因的功能分类（GO）和信号通路（KEGG Pathway)分析可知，signal transduction是这些miRNA调控的最重要的细胞功能，而regulation of actin skeleton和panthway in cancer是这些miRNA调控的最重要的信号通路。而miR-519a、miR-199a-5p、miR-138、miR-483-5p和miR-520g在这些差异表达miRNAs中起主导作用，值得进一步深入研究。
对miR-138深入的研究表明：miR-138调控CCND3的表达，诱导细胞周期在G0/Gl期停滞，进而抑制肝癌细胞的生长。在HCC组织中，miR-138下调表达，减弱了对CCND3的调控，CCND3呈上调表达，失去对细胞周期控制，肿瘤细胞生长速度加快。综上说明miR-138在HCC的发生发展中发挥抑癌基因的作用，这将为HCC的诊断与治疗带来新的曙光。
对miR-483-5p及其靶蛋白RAI16的深入研究表明：miR-483-5p调控RAI16的表达，诱导细胞周期在G0/Gl期停滞，进而抑制肝癌细胞的生长。在HCC组织中，miR-483-5p下调表达，减弱了对RAI16的调控，RAI16呈上调表达，失去对细胞周期控制，肿瘤细胞生长速度加快。综上说明miR-483-5p和RAI16在HCC的发生发展中发挥重要作用，这深入对HCC致病机理的认识提供新的思路。
第二部分
HBx诱发anti-URGs用于肝硬化肝癌高危人群预警的实验研究
肝细胞肝癌（hepatocellular carcinoma，HCC）是目前最为常见的癌症之一。在我国，肝癌的发病率高与乙型肝炎病毒（HBV）慢性感染密不可分，相当一部分的慢性乙肝迁延不愈，并经肝纤维化、肝硬化进程向肝癌转化。但对慢乙肝发生和转归的分子机制认知严重不足，缺少有效的早期诊断和干预、治疗手段，难以阻断大量病患从慢性肝炎向肝硬化和肝癌发展。
目前，甲胎蛋白（AFP）用于诊断HCC已被广泛认可，但实属无奈之选。越来越多的文献报道了AFP在诊断和监测HCC的局限性。事实上，按照目前的HCC诊断标准，当AFP和影像学检查出现异常时，肝癌往往已发展到晚期，多数患者已失去了宝贵的早期干预治疗的机会。此外，有3个最有可能用于HCC辅助诊断的标志物：AFP的异构体(AFP-L3%)、人磷脂酰肌醇蛋白聚糖3（GPC3）及去γ羟基凝血素(DCP)。其他已报道的标志物还有肝癌癌蛋白p28GANK、高尔基磷蛋白(GOLPH2/GP73)、鳞状上皮细胞癌抗原(SCCA)、α-L-岩藻糖苷酶(AFU)和肝细胞生长因子(HGF)等，但上述潜力标志物，即使联合应用，对于HCC诊断的敏感度仍不能达到作为理想标志物的标准。因此，探究肝炎发展为肝癌的致病机制，寻找理想的肝癌标志物，最终实现阻断或减缓肝炎向肝硬化肝癌转化，降低肝癌的发病率和死亡率，仍需不懈的努力。
课题组前期工作发现了5种HBx诱导的高表达蛋白(URG4, URG7, URG11,URG12和Sui1)，URG4和URG11是癌基因，能够促进肿瘤生长；URG7具有抵抗Fas介导细胞凋亡的作用；URG12是核糖体蛋白，而Sui1是转录启始因子，两者对于蛋白翻译的完整性非常重要。这5种高表达蛋白都具有刺激肝细胞生长和抵抗TNF作用的特性，其抗体与肝炎发展至肝癌的进程密切相关，上述蛋白的抗体（anti-URGs）是否可作为肝癌标志物，监测肝炎发展为肝硬化、肝癌的进程，用于HCC的早期诊断和高危人群的筛查值得进一步研究。
一、合成多肽抗原检测anti-URGs ELISA方法的建立
针对5种URGs的蛋白序列，设计并合成14-22氨基酸长度的多肽作为包被抗原，建立检测血清中anti-URGs抗体的间接ELISA方法。在包被液、多肽抗原包被浓度、封闭剂、封闭时间、血清稀释度、血清反应时间和显色时间方面对检测方法进行优化，确定Cut-off值，并评估检测方法的敏感性、特异性、精确性和稳定性。
结果如下：
1、建立了合成多肽抗原检测anti-URGs的ELISA方法，检测流程为：多肽抗原以10ug/ml的浓度用pH9.6的碳酸盐缓冲液4℃包被过夜，含3%BSA的Tris缓冲液37℃封闭2小时以上，按照1：10稀释加入血清4℃过夜，充分洗涤后加入HRP标记山羊抗人IgG,37℃孵育30分钟，充分洗涤后加入TMB，避光显色10分钟，2M硫酸终止反应后测OD值。
2、确定了ELISA检测anti-URG4、anti-URG7、anti-URG11、anti-URG12和anti-Sui1的Cut-off值分别为：0.103、0.098、0.101、0.107和0.113。
3、ELISA方法具有较好的敏感性（稀释倍数20-640倍）和特异性（多肽抗原可阻断76.9-89.6%的反应），检测试剂批内差3.2-4.5%，批间差7.4-10.3%，在4℃可保存至少5个月。
二、anti-URGs在乙型肝炎发展为肝癌进程中预警作用的研究
采用自行建立的多肽抗原检测anti-URGs的ELISA方法，大规模检测肝炎、肝硬化和肝癌患者血清中的5种anti-URGs，进行了下列研究：
1、anti-URGs与肝炎发展为肝癌进程的相关性研究：检测全部血清样本中5种anti-URGs，OD值大于或等于Cut off值的判定为阳性，反之为阴性。利用统计学分析软件对实验数据进行分析，确定anti-URGs阳性数在健康献血员组、肝炎组、肝硬化组和肝癌组之间是否有统计学差异，即anti-URGs与肝炎发展为肝硬化、肝癌进展是否相关，并进一步评估anti-URGs用于HCC高危人群的筛查与小肝癌的早期诊断的可靠性。
2、anti-URGs检查与AFP及其他标志物的对照研究：在肝炎、肝硬化、肝癌患者组中，对比anti-URGs检查与现有的AFP结合影像学检查的敏感度、特异度、阳性率和符合率。单独使用anti-URGs及其他已报道的标志物，包括AFP-L3%、GPC3和GP73等，对比其敏感度、特异度、阳性率和符合率；以多种方式联合使用anti-URGs及其他已报道的标志物，是否可提高检测的敏感度、特异度、阳性率和符合率。
3、anti-URGs对于原发性肝癌发生、复发等的前瞻性研究：A在乙肝肝炎患者组中，追踪anti-URGs的动态变化，对比3种以上（包括3种）anti-URGs阳性和3种以下anti-URGs阳性的患者病情发展、转归的差异；B在肝硬化、肝癌患者组中，追踪治疗前后anti-URGs的变化，对比anti-URGs持续阳性和anti-URGs转阴的患者癌症复发及生存时间的差异。
4、anti-URGs对于肝癌高危人群筛查的前瞻性研究：在乙肝病毒携带者与慢乙肝患者组中，对比3种或3种以上anti-URGs阳性、2种anti-URGs阳性、1种anti-URGs阳性和anti-URGs阳性全阴性的患者病情发展的差异。
结果如下：
1、发现全部558份患者血清和108份健康献血员血清中anti-URGs阳性个数在HBV相关的肝炎、肝硬化、肝癌及健康献血员组中有明显差异（P<0.001），在健康献血员组，91.7%小于等于1个anti-URGs阳性，在HBV相关的肝炎、肝硬化、肝癌组中，大于等于3个anti-URGs阳性的比例分别为：35.8%、60.7%和55.2%，此结果提示anti-URGs与肝炎发展为肝癌进程相关。
2、按照在5种anti-URGs中有3个或3个以上阳性则判定为anti-URGs阳性的标准，在肝硬化和肝癌患者中检测敏感度为58.3%，在健康献血员中的特异度为99.1%，在HBV感染无症状携带者中的特异度为87.7%，在其他癌症患者中的特异度为90.0%。
3、为全面评价anti-URGs作为肝硬化肝癌高危人群预警的肿瘤标志物的可靠性，本研究还同时检测了血清中的AFP、AFP-L3%、GPC3和GP73含量。在HBV相关肝硬化和肝癌组中，anti-URGs的敏感度为60.0%和53.3%；AFP的敏感度为33.3%和30.0%； AFP-L3%的敏感度为40.0%和33.3%；GPC3的敏感度为36.3%和23.3%；GP73的敏感度为53.3%和33.3%；在健康献血员组中, anti-URGs的特异度为100%，AFP、AFP-L3%、GPC3、GP73的特异度分别为93.4%，96.7%，93.4%和93.4%。将anti-URGs分别与AFP、AFP-L3%、GPC3和GP73联合检测，可部分提高敏感度，在肝硬化组为76.6%，73.3%，73.3%和76.6%，在肝癌组为60.3%，60.3%，60.0%和60.0%。
4、对10例肝硬化患者的进一步追踪研究显示anti-URGs阳性的肝硬化患者24个月内发生肝癌的几率大幅升高（anti-URGs阳性组：6/10人，anti-URGs阴性组：1/10人），提示anti-URGs与肝硬化肝癌发生的风险性相关，anti-URGs阳性的患者应给予适当的预防性治疗及定期随访。
5、对于10例HBV相关肝癌患者治疗前后系列血清中anti-URGs进行检测发现，anti-URGs在肝癌临床诊断前即可出现，在治疗后发生转阴，而在个别癌症复发的患者血清中持续阳性。
三、结论
本课题建立了多肽抗原检测anti-URGs的ELISA方法，并对HBV相关急、慢性肝炎、肝硬化和肝癌患者血清，其他癌症患者、HCV肝炎肝硬化患者以及健康献血员血清进行规模化检测，结果显示：anti-URGs在HBV相关的肝炎、肝硬化、肝癌组中的检出率分别为21.7%、60.2%和55.1%，存在明显统计学差异（P<0.001），anti-URGs的确与HBV相关的肝炎、肝硬化和肝癌的病情发展相关，anti-URGs在肝硬化和肝癌患者中检测敏感度为58.3%，在健康献血员中的特异度为99.1%。因此，anti-URGs可以作为肝硬化、肝癌高危人群预警的血清标志物。
本课题研究的创新点在于：（1）本课题首次将anti-URGs作为监测肝炎发展为肝癌进程的血清标志物，具有较好的敏感度和特异度，具有先进性。（2）anti-URGs与肝癌发展的多个方面相关，采用anti-URGs与AFP、AFP-L3%或GP73等组合检测时可提高检测敏感度15%。（3）本课题着眼于HBV感染后病情由肝炎发展为肝硬化、肝癌的整个进程的监测以及相应的肝硬化、肝癌高危人群预警的血清标志物的筛选和评估。在肝炎和肝硬化阶段血清中即已出现3个或3个以上anti-URGs阳性的患者，具有发展成为肝癌患者的高风险性，应例行血清学和影像学检查，尽早进行干预和有效的治疗。本课题的完成将有助于原发性肝癌的预警、诊断和预后，阻断肝炎病患向肝硬化肝癌转化，降低肝癌的发病率和死亡率。
Part I
Taqman array analysis of miRNAs in hepatocellularcarcinoma and the roles of miR-138and miR-483-5p intumorigenesis
Hepatocellular carcinoma (HCC) is the leading cause of cancer deaths in theworld．More than half of those people are in China．The overall5-year survival rate forHCC patients is less than5％．Early detection of HCC is of supreme importance becausethe treatment options are severely limited by a late diagnosis．Late clinical presentationshave also led to poor prognosis for HCC patients．Thus，it is necessary to el ucidate themolecular mechanisms underlying HCC and identify novel therapeutic targets andbiomarkers for the early detection of HCC．Although serum a-fetoprotein(AFP)is a usefultumor marker for the detection and monitoring of HCC development，the false-negativerate by AFP level alone may be as high as40％for patients with a small HCC．So，if a newtumor marker has more sensitivity and can be detected in the primary stage of HCC，it ispossible to extrapolate the likelihood of a malignant liver tumor．Thus，discovery of newtarget molecules that are critically involved in the majority of cases and expressedspecifically in tumors will be essential to understand the mechanisms and improvetherapeutic intervention and prognosis of HCC．
MicroRNA (miRNA) represent a class of small noncoding RNA that control geneexpression by targeting mRNA and triggering either translation repression or RNAdegradation．Mature miRNA are19-25nucleotides that can silence genes through eitherconsummate or defective binding to the3’ untranslated region (3’UTR) of thetranscript．miRNA have been involved in the modulation of diverse cellular processesincluding cell differentiation, proliferation and apoptosis．An increasing number of studiessuggest that there all differential miRNA expression profiles between those in cancertissues and adjacent nontumorous tissue，these studies also show that miRNA profilescould distinguish malignant tumor of liver，breast，lung，pancreas and leukemia fromadjacent nontumorous tissue．The importance of miRNA in cancer is highlighted by the observation that50％of miRNA genes are located in cancer associated genomic regions orfragile sites．Importantly，miRNA expression is frequently deregulated in several cancersincluding B-cell chronic lymphocytic leukemia Burkitt’ S lymphoma，colorectal cancer,lung cancer and HCC．Additionally，differential expression of miRNA have been found tobe associated with postoperativesurvival in lung cancer patients and all diagnostic andprognostic markers of lung cancer．miRNA have been implicated to play both tumorsuppressor and oncogenic roles．These studies indicate that miRNA profiling studies couldbe used for defining clinical phenotypes，as well as potentially useful molecular diagnosticmarkers．An understanding of the molecular mechanism by which mi RNA regulate HCCdevelopment potentially may provide new avenues of research that could aid earlydiagnosis and treatment of this highly malignant tumor．
In this present study，we examined the expression profiles of667miRNA in3pairs ofHCC and adjacent non-tumorous tissue by a Taqman low density miRNA array(TLDA)．The biological functions and targets of some differentially expressed miRNAshave been detected by Real-time RT-PCR and Western-blotting analysis．This study mayhelp clarify the molecular mechanisms involved in the progression of HCC．
1、Deregulated miRNAs in hepatocellular carcinoma
667microRNA expression profiles of was performed on3pairs of surgically removedHCC and adjacent non-tumorous tissues by using TLDA, then23deregulated miRNAswere validated in an extended samples set of18paired HCC and adjacent non-tumorousliver tissues by realtime reverse transcription polymerase chain reaction (RT-PCR)analysis．
Results：
Only miRNAs altered by at least3-fold in all three pairs of the samples wereconsidered significant candidates. Under these strict criteria,11up-regulated miRNAs and12down-regulated miRNAs were identified. To validate the miRNA array data, qRT-PCRwas performed in18pairs of HCC tissues.10up-regulated and10down-regulatedmiRNAs showed consistent changes in more than50%tumorous tissues, of whichmiR-217, miR-520g, miR-522and miR-525-3p were up-regulated in more than70%tumorous tissues, and miR-199a-5p, miR-138, miR-483-5p and miR-511weredown-regulated in more than70%tumorous tissues.
The targets of the above20deregulated miRNAs were predicted by TargetScan. Tojudge the most significant candidates and investigate the cellular function, the signaling pathway and GOs of these target genes were analyzed. The results showed that a widevariety of cellular processes were featured significantly in signaling pathways. Many ofthese signaling pathways, such as insulin, MAPK, TGF-beta and Wnt signaling pathway,have been shown to participate in the tumorigenesis. However, some other signalingpathways have never been reported to play a role in tumorigenesis, e.g. axon guidance.Among all these differentially regulated signaling pathways,“regulation of actincytoskeleton” and “pathway in cancer” appeared to be the most enriched two in bothup-regulated and down-regulated miRNA groups. A similar phenomenon was observed inGOs analysis. Many cellular functions were featured significantly, of which the “signaltransduction” appeared to be the most enriched one. Based on these results, the miRNAswhich were involved in both “signal transduction” and “the regulation of actincytoskeleton or pathway in cancer” might be the most significant candidates. MiR-520g,miR-483-5p, miR-138, miR-199a-5p, miR-217and miR-518e were selected under thecriteria for further studies. Here, the studies on miRNA-138were presented.
2、miR-138induces cell cycle arrest by targeting cycline D3
The significant reduction of miR-138expression in HCC tissues indicated possiblebiological significance in tumorgenesis. At first, the effect of miR-138on cell growth wasevaluated in HepG2and Huh7cells transfected with or not, miR-138mimic, miR-138inhibitor or NC duplex. From2day (HepG2) or3day (Huh7) after the transfection, theviability of cells transfected with miR-138mimic significantly decreased compared withthat of NC duplex transfected or nontransfected cells, but the viability of cells transfectedwith miR-138inhibitor significantly increased. These results indicate miR-138couldinhibit cell growth. To validate the inhibitor effect of miR-138on cell growth, the colonyformation assay was performed in HepG2and Huh7transfected with or not, miR-138mimic, miR-138inhibitor or NC duplex. HepG2and Huh7cells transfected with20nMmiR-138mimic displayed much fewer and smaller colonies (218or161colonies)compared with NC duplex transfected (783or729colonies) and nontransfected cells (756or692colonies), but cells transfected with20nM miR-138inhibitor displayed much moreand larger colonies (1238or1349colonies).
To further confirm the above findings, an in vivo mouse model was used. For theduration of the treatment with miR-138mimic or miR-138inhibitor for5weeks, tumorvolume curves revealed a significant decrease in growth rates at the3rd,4th and5th weekafter treatment with miR-138mimic and a significant increase in growth rates at the4th and5th week after treatment with miR-138inhibitor whereas no significant differences intumor growth rates were observed between the NC group and the ctrl group. These resultsindicate that introduction of miR-138significantly inhibits tumorigenicity of HepG2cellsin xenograft nude mouse model.
To investigate the mechanism of inhibitor effect of miR-138, flow cytometry assayshowed that the percentages of miR-138mimic transfected HepG2and Huh7cells in theG0-G1phase were18%(HepG2) and11%(Huh7) higher than that of NC duplextransfected or nontransfected cells, which paralleled with a44%(HepG2) and38%(Huh7)decrease in the S phase. In miR-138inhibitor transfected cells, the percentages of cells inthe G0-G1phase were8%(HepG2) and6%(Huh7) less than that of NC duplex transfectedor nontransfected cells, which paralleled with a14%(HepG2) and19%(Huh7) increase inthe S phase. These results indicate miR-138could inhibit HepG2and Huh7proliferationby inducing cell cycle arrest at G1/S phase.
It is generally accepted that miRNAs exert their function through regulating theexpression of their downstream target genes. Cycline D3(CCND3) was predicted as apotential target of miR-138by TargetScan and Miranda. To validate whether CCND3is adirect target of miR-138, a human CCND33’-UTR fragment containing wild-type ormutant miR-138binding sequence was cloned downstream of the firefly luciferase reportergene in pGL3. In HEK293cells cotransfected with the reporter plasmids and miR-138mimic or NC duplex, the luciferase activity of the reporter that contained wild-type3’-UTR was significantly suppressed by miR-138mimic, but the luciferase activity ofmutant reporter was unaffected, indicating that miR-138may suppress gene expressionthrough miR-138binding sequence at the3’-UTR of CCND3. Furthermore, transfection ofmiR-138mimic decreased CCND3expression and transfection of miR-138inhibitorincreased CCND3expression in HepG2cells at protein but not mRNA level, suggestingthat CCND3expression could be inhibited by miR-138at posttranscriptional level.Together, the results show that miR-138could regulate the expression of endogenoushuman CCND3by directly targeting the3’-UTR of CCND3mRNA and human CCND3isa new target of miR-138.
To identify whether inhibition of CCND3, just like miR-138restoration, also resultedin HCC repression, the effects of knockdown of CCND3on cell growth were examined.Firstly, HepG2cells were transfected with or not, CCND3siRNA or control siRNA.Seventy two hours after transfection, a dose dependent knockdown of CCND3was observed in HepG2cells. In cell viability assay and cell cycle analysis, in vitro knockdownof CCND3repressed cell viability, induced cell cycle arrest and inhibited the colonyformation. The similar data were obtained in Huh7cells transfected with CCND3siRNA.These results indicate that CCND3is most likely involved in the induction of cell cyclearrest by miR-138.
3、Retinoic acid induced16, regulated by miR-483-5p, is a novel oncogene and tumormarker for hepatocellular carcinoma
The significant reduction of miR-483-5p expression in HCC tissues indicatedpossible biological significance in tumorgenesis. At first, the effect of miR-483-5p on cellgrowth was evaluated in HepG2and Huh7cells transfected with or not, miR-483-5p mimic,miR-483-5p inhibitor or NC duplex. From2day (HepG2) or3day (Huh7) after thetransfection, the viability of cells transfected with miR-138mimic significantly decreasedcompared with that of NC duplex transfected or nontransfected cells, but the viability ofcells transfected with miR-483-5p inhibitor significantly increased. These results indicatemiR-138could inhibit cell growth.
To investigate the mechanism of inhibitor effect of miR-483-5p, flow cytometry assayshowed that the percentages of miR-483-5p mimic transfected HepG2and Huh7cells inthe G0-G1phase were18%(HepG2) and11%(Huh7) higher than that of NC duplextransfected or nontransfected cells, which paralleled with a44%(HepG2) and38%(Huh7)decrease in the S phase. In miR-483-5p inhibitor transfected cells, the percentages of cellsin the G0-G1phase were8%(HepG2) and6%(Huh7) less than that of NC duplextransfected or nontransfected cells, which paralleled with a14%(HepG2) and19%(Huh7)increase in the S phase. These results indicate miR-483-5p could inhibit HepG2and Huh7proliferation by inducing cell cycle arrest at G1/S phase.
Retinoic acid induced16(RAI16) was predicted as a potential target of miR-483-5pby TargetScan and Miranda. To validate whether RAI16is a direct target of miR-483-5p, ahuman RAI163’-UTR fragment containing wild-type or mutant miR-483-5p bindingsequence was cloned downstream of the firefly luciferase reporter gene in pGL3. InHEK293cells cotransfected with the reporter plasmids and miR-483-5p mimic or NCduplex, the luciferase activity of the reporter that contained wild-type3’-UTR wassignificantly suppressed by miR-483-5p mimic, but the luciferase activity of mutantreporter was unaffected, indicating that miR-483-5p may suppress gene expression throughmiR-483-5p binding sequence at the3’-UTR of RAI16. Furthermore, transfection of miR-483-5p mimic decreased RAI16expression and transfection of miR-138inhibitorincreased RAI16expression in HepG2cells at protein but not mRNA level, suggesting thatRAI16expression could be inhibited by miR-483-5p at posttranscriptional level. Together,the results show that miR-483-5p could regulate the expression of endogenous humanRAI16by directly targeting the3’-UTR of RAI16mRNA and human RAI16is a newtarget of miR-483-5p.
In commercial HCC tissue arrays, most HCC tumor tissues showed moderate (score2) or strong (score3) RAI16expression (89of126cases). In contrast, most nontumortissues showed negative (score0) or weak (score1) RAI16expression (118of126cases).The sensitivity and specificity of RAI16expression for the diagnosis of HCC were70.6%and93.6%, respectively. Strikingly, the sensitivity and specificity increased to80.9%and92.0%when glypican-3was used with RAI16. These results indicate that RAI16may bepotential tumor marker for HCC diagnosis, especially combined with GPC3.
The significant up-regulation of RAI16expression in HCC tissues indicated possiblebiological significance in tumorgenesis. At first, the viability of cells transfected withpcDNA3.1-RAI16significantly increased compared with that of empty vector pcDNA3.1transfected or nontransfected cells, but the viability of cells transfected with RAI16siRNAsignificantly decreased. HepG2and Huh7cells transfected with pcDNA3.1-RAI16displayed much more and larger colonies (1238or1349colonies) compared with emptyvector pcDNA3.1transfected (783or729colonies) and nontransfected cells (756or692colonies). Cell cycle analysis showed that the percentages of pcDNA3.1-RAI16transfectedHepG2and Huh7cells in the G0-G1phase were11.6%(HepG2) and9.4%(Huh7) lowerthan that of pcDNA3.1transfected or nontransfected cells, which paralleled with a35.6%(HepG2) and27.0%(Huh7) increase in the S phase. In RAI16siRNA transfected cells, thepercentages of cells in the G0-G1phase were10.9%(HepG2) and14.1%(Huh7) higherthan that of NC siRNA transfected or nontransfected cells, which paralleled with a25.8%(HepG2) and19.4%(Huh7) decrease in the S phase. These results indicate RAI16couldenhance HepG2and Huh7proliferation by promoting cell cycle from G0/G1to S phase.Furthermore, the percentage of apoptotic cells in pcDNA3.1-RAI16transfected HepG2andHuh7cells were36.4%(HepG2) and33.5%(Huh7) lower than that of pcDNA3.1transfected or nontransfected cells. In RAI16siRNA transfected cells, the percentages ofapoptotic cells were21.5%(HepG2) and26.3%(Huh7) higher than that of NC siRNAtransfected or nontransfected cells.
To further confirm the above findings, an in vivo mouse model was used. For theduration of the treatment with HepG2cells stably expressing RAI16or transfected withRAI16siRNA, tumor volume curves revealed a significant increase in growth rates at the3rd,4th and5th week after treatment with HepG2cells stable expressing RAI16and asignificant decrease in growth rates at the4th and5th week after treatment with HepG2cells transfected with RAI16siRNA whereas no significant differences in tumor growthrates were observed between the NC siRNA transfected or non-transfected HepG2cellsgroups. These results indicate that introduction of RAI16significantly promotestumorigenesis in xenograft nude mouse model.
To investigate the mechanism of RAI16enhancement of tumor growth in HCC,10major cancer related pathways were analyzed in transfected HepG2cells using adual-luciferase reporter system (Promega). The most affected pathways were transforminggrowth factor-β (TGF-β), mitogen-activated protein kinase (MAPK)/extracellularsignal-regulated kinase (ERK) and Wnt. The most affected pathways were TGF-β,MAPK/ERK and Nuclear factor κB (NFκB). The up-regulated MAPK/ERK and TGF-βpathways in RAI16overexpressed cells is consistent with the down-regulated MAPK/ERKand TGF-β pathways in RAI16knockdown cells These results indicate MAPK/ERK andTGF-β pathway may be involved in RAI16induction of tumorigenesis.
To verify above findings, the key molecules in MAPK/ERK and TGF-β pathway weredetected by immunoblotting. RAI16overexpession increased the phosphorylation of MEKand ERK1/2significantly, the total ERK but not total MEK expression. Similar phenomenawere observed in TGF-β pathway, the phosphorylation of SMAD2/3and total SMAD2/3,together with SMAD4were increased by RAI16overexpression. RAI16knockdown ladeto the reverse results. Interesting, phosph-SMAD2/3and SAMD4in HCC tumor tissueswere increased much more significantly than that in RAI16transfected cell modal, butphosphorylation of MEK and ERK1/2seemed more significant in RAI16transfected cellmodal. These results suggest it is possible that MAPK/ERK and TGF-β pathway play therole in different stage of RAI16induction of tumorigenesis.
Part II
Application of HBx-induced anti-URGs as early warningbiomarker of liver cirrhosis and hepatocellularcarcinoma
Hepatocellular carcinoma (HCC) is one of the most common cancers, with morethan500,000deaths with over600,000new cases yearly worldwide. The clinical course ofHCC is mostly asymptomatic. Suspected liver changes are too large and too advanced forthe tumor to be subjected to potentially effective and radical therapy when they aredetected incidentally. Because of serious limitations of the surgical and oncologicaltreatment available, it seems necessary to concentrate on the earliest possible diagnosis,particularly sensitive detection of resectable focal liver changes-preferably when tumorsare less than2cm in diameter. Surveillance with immaging techniques and serumα-fetoprotein (AFP) analyses is recommended for all cirrhotic patients and other specificrisk groups every6months.
AFP has to be considered “the golden standard” for HCC serum markers, however,the usefulness of AFP testing for the population at risk should be seriously questioned. AFPdiagnostic values for this assay are undoubtedly poor. AFP specificity varies from about76%to96%and increases with elevated cut-off value. Simultaneous sensitivity decreasesmuch more from about25%for potentially resectable tumors of less than3cm in diameterto about50%for lesions of>3cm in diameter. Other serum biomarkers such as AFP-L3%,DCP, AFU, GGT, GP-73, MUC-1, SCCA and GPC-3have significant diagnosticlimitations, and in fact they are not particularly precise for the early diagnosis of HCC.Simultaneous determination of these markers in various combinations could improve theaccuracy in differentiating HCC from nonmalignant hepatopathy, but there still exists theunresolved problem of tiny ‘grey’ nodules in the ‘black and white’ diagnostic perspective.
Previous studies have found that HBx contributes to the development of HCC byaltering patterns of host gene expression.5up-regulated genes (URGs: URG4, URG7,URG11, URG12and Sui1) associated with HBx triggered corresponding antibodies(anti-URGs) in the sera of patients prior to tumor development and/or at early stagetumorigenesis.
1、Establish of ELISA assay for anti-URGs detection using synthesized peptides
To establish an ELISA method to detect the anti-URGs in the serum of patients withHCC,5groups of peptides (L4A/L4B, L7B, L11-1/L11-3/L11-4, L12A/L12B andSui1A/Sui1B) were synthesized. The sensitivity, specificity and precision of this assaywere evaluated.Results:
（1）The procure of ELISA assay is as following:10ug/mlpeptides were coatedby0.01M NaHCO3buffer at4℃overnight, blocking by Tri-HCl buffer with3%BSA at37℃for more than2hours, diluted serum samples at1:10were incubatedat4℃overnight, after washing, HRP Goat anti-human IgG incubation at37℃for30minutes,washing, TMB development for10minutes，read the OD value after stopping reaction with2M H2SO4.
（2）The Cut-off value of ELISA assay to detect anti-URG4、anti-URG7、anti-URG11、 anti-URG12and anti-Sui1is:0.103,0.098,0.101,0.107and0.113respectively.
（3）The developed ELISAmethod showed good sensitivity, specificity and precision.Intra-division is3.2-4.5%, inter-division is7.4-10.3%.
2、Detection of anti-URGs in serum samples by ELISA
To evaluate the anti-URGs as the potecial serological markers of HCC and thecorresponding ELISA assay. The anti-URGs were detected in serum samples from558patients and108blood donors by ELISA assay.Results:
（1） The number of anti-URGs showed significantly difference among the558patient with HBV associated hepatitis, cirrhosis, HCC and108blood donor serum samples.In HBV associated live disease groups, the sensitivity of anti-URGs is35.8%,60.7%and55.2%respectively.
（2）The spececificity of anti-URGs is99.1%,87.7%and90.0%in blood donorsgroup, HBV asymptomatic carriers and other tumors respectively.
（3）In HBV associated cirrhosis and HCC groups, the sensitivity of anti-URGs is60.0%and53.3%; the sensitivity of AFP is33.3%and30.0%; the sensitivity of AFP-L3%is40.0%and33.3%; the sensitivity of GPC3is36.3%and23.3%; the sensitivity of GP73is53.3%and33.3%; In blood donors group, the spececificity of anti-URGs is100%, thespececificity of AFP, AFP-L3%, GPC3and GP73is93.4%,96.7%,93.4%and93.4% respectively. Combination anti-URGs with AFP, AFP-L3%, GPC3and GP73respectivelycould improve the sensitivity by10%.
（4） Longitudinal studies in20HBV-cirrhosis patients with more than3anti-URGsor with less than1anti-URGs showed that patients with more anti-URGs were more likelyto develope tumor compared with those with fewer anti-URGs.
3、Conclusion
In this study, ELISA to detect the anti-URGs in serum was established and used inHBV infected patients and blood donors. It was found that anti-URGs were predominantlypresent among patients with HBV-associated HCC (55.2%) and cirrhosis (60.7%), and at alower frequency among patients with chronic hepatitis (35.8%), with significant difference(P<0.001). The sensitivity of anti-URGs in patients with HBV-associated HCC andcirrhosis was58.3%and the specificity of anti-URGs in blood donors was99.1%.
The innovation point of this study lies on:(1) anti-URGs were firstly as the earlywarning biomarker for liver cirrhosis and HCC with much better sensitivity and specificity.(2) The combination of anti-URGs with AFP, AFP-L3or GP73improved the sensitivity ofdetection at15%.(3) This study was focused on the whole progress of HBV infection fromhepatitis to HCC, the HBV hepatitis and cirrhosis patients with3or more than3anti-URGs would have more risk to develop HCC, and should be paid more attention. Asearly warning biomarker of HCC, anti-URGs may be useful in the identification of HBVpatients with chronic liver disease who are most likely to develop HCC, which wouldprovide the help to diagnosis of human small HCC, early-stage asymptomatic HCC and thescreening of HCC in high risk population.
引文
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