【作者】 符小玉；

【导师】 谭德明；

【作者基本信息】 中南大学 ， 临床医学， 2012， 博士

【摘要】 乙型肝炎病毒(HBV)的慢性感染是世界范围内原发性肝细胞癌(HCC)的主要发病原因之一,其中HBV X片段被认为是导致肝细胞恶性转化的主要因素。HBV-DNA,尤其是HBx基因频繁整合于宿主基因组,导致染色体的不稳定性和插入突变的发生,与HCC的发生密切相关。MicroRNA (miRNA)是一类内源性的,长度约22nt非编码小分子单链RNA。它通过与靶mRNA的3’UTR结合,参与转录后水平调控基因的表达。近来的研究结果证明,miRNA突变和异常表达与各种人体肿瘤发生、发展相关,miRNAs与肿瘤相关性的研究已成为近年来肿瘤研究领域中的一个热点,它被认为是一组新的癌或抑癌基因,具有特异性的肿瘤组织表达谱。研究认为miRNA水平的变化常导致其靶基因的表达异常,从而影响了肿瘤的进程,其表达的模式可作为肿瘤诊断／预后的分子标记。深入研究miRNA在人体肿瘤中的表达状况及其功能,对研究肿瘤的发生、发展和转移的机制具有重要意义,并将有利于肿瘤的诊断、预后及治疗。但现在很少有文献系统的研究与HBx特异相关的miRNA在HBx导致的HCC发生、发展中的作用。我们前期研究已成功构建了稳定表达HBx的肝细胞株(L02/HBx),并通过裸鼠成瘤实验证实HBx可引起肝细胞癌变,同时发现转染HBx基因后L02细胞的增殖能力与非锚定依赖生长能力明显增强,我们分析HBx促L02细胞恶性转化作用可能与其增强G1期调控相关基因如CyclinD1、CyclinG1、E2F1的表达相关。本研究首先采用Agilent公司生产的miRNA芯片检测转染野生型HBx基因前后L02细胞miRNA表达谱,并筛选出部分差异表达的miRNA,接着阐述通过改变特异性的miRNA的表达是否调节了细胞增殖,特别是对Cyclin基因的调控是本研究的主要内容。本研究从HBx与miRNA入手,探索它们在HBV相关性肝癌的发生发展中的作用及其机制。为进一步理解受HBx影响的纷繁复杂的信号通路提供一定的帮助,试图探讨HBx致癌变新的发病机制。第一章利用芯片技术检测HBx基因对肝细胞miRNA表达谱的影响—下调miR-338-3p的表达目的：研究HBx基因影响L02细胞的miRNA表达谱,筛选出与HBx相关的miRNA。方法：以LO2/pcDNA3.0细胞为对照,采用Agilent公司生产的miRNA芯片技术检测转染了HBx基因后L02细胞miRNA表达谱的变化,并通过Real-time荧光定量PCR (qRT-PCR)验证芯片所得结果,筛选出在LO2/HBx细胞中下调倍数最大的miR-338-3p进行下一步的功能研究。结果：芯片结果显示,与LO2/pcDNA3.0细胞相比,LO2/HBx细胞有4个miRNA表达上调,分别为miR-7、miR-1274a、miR-137、 miR-663;有11个miRNA表达下调,分别为miR-338-3p、miR-551b、miR-24-1*、miR-29c、miR-744、miR-455-3p、miR-324-5p、miR-340、 miR-660、miR-193a-3p、miR-590-5p,其中miR-338-3p下调最为明显,与qRT-PCR验证的结果相一致。结论：①HBx能影响LO2细胞的miRNA表达谱,如下调miR-338-3p的表达；②miRNA芯片技术检测结果可靠,可用于筛选组织、细胞间的miRNA表达谱。第二章miR-338-3p对稳定表达HBx细胞的增殖能力影响目的：第一部分研究已发现HBx可下调miR-338-3p的表达,本部分研究miR-338-3p在HBx致人肝细胞异常增殖中的作用及其相关机制,试图探讨HBx致癌变新的机制。方法：合成特异的miR-338-3p模拟物(mimics)及抑制剂(inhibitor),以相应的阴性对照(NC)作为对照组,通过脂质体转染miR-338-3p mimics/inhibitor到LO2/HBx和LO2/pcDNA3.0细胞中,MTT、Edu检测细胞增殖,流式细胞仪检测细胞周期。结果：流式细胞仪发现,转染niR-338-3p增加细胞内miR-338-3p表达之后,G1期延长,S期缩短,而与之相反的是转染miR-338-3p inhibitor降低细胞内miR-338-3p的表达后,G1期缩短,S期延长；MTT结果显示,miR-338-3p可以明显抑制细胞增殖,抑制率分别为40%±9.41和34%±8.3；而抑制miR-338-3p在L02/HBx和LO2/pcDNA3.0细胞中的表达,则促进细胞增殖(53%士8.1和38%士7.6)。而Edu结果也表明,miR-338-3p可抑制肝细胞DNA的合成,缩短S期细胞比率。结论：①miR-338-3p可抑制细胞增殖;②miR-338-3p抑制细胞增殖的机制可能是通过延长细胞G1期,抑制肝细胞DNA的合成,缩短S期细胞比率,从而抑制肝细胞的增殖。第三章miR-338-3p靶向调节CyclinDl的机制研究目的：根据课题组前期研究及在第二部分所得结果,我们发现miR-338-3p可调节肝细胞增殖,且在L02/HBx细胞中呈现低表达,与CyclinD1的表达水平成负相关,结合生物信息学分析,发现miR-338-3p可与CyclinD1的3’UTR结合,因此推测CyclinD1极可能为miR-338-3p的目的基因。本部分研究miR-338-3p调节CyclinD1的作用机制。方法：①通过Lipofectamine转染miR-338-3p-mimics/inhibitor到细胞中,Real-time定量PCR、Western Blot检测细胞中CyclinD1mRNA、蛋白表达的改变；②利用PCR方法,扩增CyclinD1-3’UTR序列,同时设计引物突变掉两个miR-338-3p的靶标序列,将其克隆到pmirGLO双荧光素酶报告载体中,构建报告质粒CyclinD1-3’UTR野生型及突变型(分别命名为pCyclinD1-3’UTR-WT、pCyclinD1-3’UTR-Mut);同时为进一步明确具体的调控位点,将上述两个靶标序列分别进行突变,构建报告质粒CyclinD1-3’UTR突变型1和2,分别命名为pCyclinD1-3’UTR-Mutl(突变907-913nt)和pCyclinD1-3’UTR-Mut2(突变2397-2403nt)。将以上报告质粒与miR-338-3p mimics及miR-NC共转染到L02/HBx细胞中,检测荧光素酶表达的改变。结果：①与阴性对照组比较,模拟miR-338-3p表达后,L02/HBx和LO2/pcDNA3.0细胞中CyclinD1蛋白表达下调,反之抑制miR-338-3p表达后,细胞中CyclinD1蛋白表达显著上调,而miR-338-3p对细胞CyclinD1mRNA表达无明显影响。②经PCR扩增、双酶切及测序鉴定pCyclinD1-3’UTR-WT、pCyclinD1-3’UTR-Mut、 pCyclinD1-3’UTR-Mut1和pCyclinD1-3’UTR-Mut2重组质粒构建成功。③与miR-NC相比,miR-338-3p与pCyclinD1-3’UTR-WT共转染可使海肾荧光素酶活性下降,荧光素酶相对比值下降；虽miR-338-3p均可调控两个位点,但以对pCyclinD1-3’UTR-Mutl的作用更为显著。结论：①miR-338-3p可负性调控CyclinD1蛋白表达；②成功构建了CyclinD1-3’UTR报告质粒；③miR-338-3p与CyclinD1-3’UTR结合,靶向调控CyclinD1,这种作用以CyclinD1-3’UTR的2397-2403nt位点最为显著。HBx可通过下调miR-338-3p的表达而调控了肝细胞恶性增殖,建立了HBx致癌变新的发病机制。第四章干扰HBx表达后miR-338-3p及CyclinD1表达的改变目的：我们的研究发现,在稳定表达HBx基因的L02细胞中,HBx通过下调niR-338-3p的表达调控CyclinD1,从而促进肝细胞增殖。那么,抑制了HBx表达后miR-338-3p与CyclinD1的表达是否也发生相应的改变?本研究为进一步证实,miR-338-3p与CyclinD1在肝细胞中的表达依赖于HBx基因的改变。方法：设计并化学合成针对HBx的3对siRNA分子序列(HBx-siRNA1、HBx-siRNA2、HBx-siRNA3),同时设阴性对照组(转染siRNA阴性对照)。脂质体法瞬时转染L02/HBx细胞,通过RT-PCR和Western Blot检测HBx基因在mRNA和蛋白水平的变化以验证HBx-siRNA的抑制效果；Real-time PCR检测细胞中miR-338-3p的表达,Western Blot检测细胞中CyclinD1表达的变化。结果：3对HBx-siRNAs均能降低HBx mRNA的表达,分别降低52.3±2.3%、45.1±3.4%、70.6±4.3%,与阴性对照组相比,差异有显著性(P均<0.01)；3对特异性HBx-siRNAs转染L02/HBx细胞后,与阴性对照组相比,均能抑制]HBx白表达,抑制率分别为54.6±1.8%,45.8士2.2%,78±3.5%(P均<0.01)。转染HBx-siRNA到L02/HBx细胞后,以U6作为内参,Real-time PCR显示细胞中miR-338-3p表达上调(P=0.013)；而Western Blot结果显示CyclinD1的表达水平则显著下降(P<0.001)。结论：干扰HBx基因表达后LO2/HBx细胞中miR-338-3p表达上调,而CyclinD1表达则下降,进一步验证了miR-338-3p与CyclinD1在细胞中的表达依赖于HBx基因的改变,也证实了miR-338-3p直接受HBx基因的影响,通路HBx/miR-338-3p/CyclinD1参与了HBx介导的HCC肿瘤形成的过程。第五章肝癌组织中miR-338-3p、HBx及CyclinD1的表达及相互间关系目的：体外实验已证实并建立了]HBx/miR-338-3p/CyclinD1通路,本部分研究肝癌病人的癌组织与癌旁组织miR-338-3p、HBx及CyclinD1的表达情况,结合临床探讨miR-338-3p在HBx介导细胞增殖方面的作用。方法：收集23例肝癌病人癌组织及癌旁组织,采用qRT-PCR检测23例肝癌病人miR-338-3p的表达,RT-PCR、Western Blot、免疫组化分别检测HBx mRNA、CyclinD1及HBx蛋白表达情况,根据Pearson’s相关性分析研究HBx、miR-338-3p及CyclinD1三者的关系；同时行HE染色,评估肝组织的病理学改变。结果：(1)17例癌组织(17/23,74%)及5例相应的癌旁组织可检测到HBx mRNA表达,miR-338-3p的表达与肝癌分化程度相关,且在癌组织中的表达量明显低于癌旁组织,然而这种差异只表现在HBx阳性的癌组织中p<0.001)；尽管无统计学差异,miR-338-3p在HBx阴性的癌组织中表达略高于癌旁组织(p>0.05)。(2)肝癌组织中HBx蛋白的总体阳性检出率为19/23(82.6%),其中5例相应的癌旁组织检测到HBx蛋白弱表达,miR-338-3p与HBx的表达呈负相关,即HBx积分越高者miR-338-3p的mRNA表达越低。(3)与癌旁组织相比,肝癌组织中CyclinD1蛋白表达明显增强,HBx可上调CyclinD1的表达,且CyclinD1与miR-338-3p的表达呈显著负相关,miR-338-3p表达越高者,CyclinD1表达则越低。结论：①miR-338-3p在肝癌组织中表达下调,然而仅在HBx表达阳性的癌组织中下调,而在HBx表达阴性的癌组织中表达略有上调,但无统计学意义。②miR-338-3p与HBx表达呈显著的负相关。③HBx可上调肝癌组织中CyclinD1的表达,且CyclinD1蛋白表达水平与miR-338-3p呈负相关。更多还原

【Abstract】 Chronic Hepatitis B virus (HBV) infection is one of the main reason contributing to primary hepatocellular carcinoma (HCC) worldwide,of which HBV X fragment has been recognized as a leading cause for liver cells malignant transformation. HBV-DNA, especially HBx gene frequently integrates into the host genome, leading to the chromosome instability and insert mutations, is closely related with hepatocar-cinogenesis.MicroRNAs (miRNAs) are a class of endogenous noncoding small (-22nucleotide) RNAs which regulate gene expression at post-transcriptional level through binding to the mRNA3’UTR region of target gene. A number of studies in recent years have shown that dysregulation of miRNA expression is associated with a variety of tumors.The relevance of miRNAs and tumours is of great interest to researchers in the tumourous reseacrch field. These miRNAs may play a role of tumor suppressor gene or oncogene,they have the specific expression profiles in different tumor tissues. Multiple studies have indicated that changes in the expression of miRNAs frequently influence their targets gene expression,therefore infect the process of oncogenesis. The miRNA expression patterns are molecular markers of cancer diagnose/prognosis. It is significant that further research in the expression and function of miRNA in human tumours,which is helpful for the mechanism of tumourous occurrence, development and metastasis and therefore is beneficial to cancer diagnose,prognosis and theapy.However,there are few studies about the miRNA and their function in the HBx-related hepatocarcinogenesis.We have successfully established a stably expressing HBx liver cell line (L02/HBx) in previous study, confirming that HBx gene induce hepatocellular carcinoma in nude mice, and found that HBx enhanced the proliferation and anchorage-independent growth ability of LO2cells. We suggested the mechanisms of HBx influence vicious transformation of LO2cells was to promote G1phase of cell cycle regulator gene such as expression of CyclinD1,CyclinG1or E2F1. In the present studies, firstly we demonstrated the miRNAs expression profiles in the LO2cells stably transfected with HBx gene using a Agilent mammalian miRNA microarray and selected out differential expression miRNA. Then, further studies focus on regulating cell proliferation,especially regulating Cyclin gene through altered differential expression miRNA.Our studies investigate altered expression profling of miRNA mediated by HBx and the function of miRNA in HBx-related HCC may sheds new insight into elucidating the complicated molecular mechanism of the development and progression of HBx-HCC and discuss new mechanisms in hepatocarcinogenesis related with HBx. Part I MiRNA microarray analysis on the effect of HBx on miRNA expression profile in liver cells-downregulate miR-338-3pAim:To investigate the effect of HBx gene on the miRNAs expression profile in LO2cells and find out the different miRNAs related with HBx.Methods:We demonstrated the miRNAs expression profiles in the LO2cells stably transfected with HBx gene using a Agilent mammalian miRNA microarray compared with LO2/pcDNA3.0cells and confirmed the results by real-time quantitative PCR.miR-338-3p was selected out for the further function study as the most down-regualted in the L02/HBx cells.Results:According to the microarray, compared with the LO2/pcDNA3.0cells,4miRNAs were exhibited higher expression and11miRNAs were demonstrated lower expression in the LO2/HBx cells, the4upregulated miRNAs were miR-7, miR-1274a, miR-137, miR-663while the11downregulated miRNAs were miR-338-3p, miR-551b, miR-24-1*,miR-29c,miR-744,miR-455-3p,miR-324-5p,miR-340,miR-660,miR-193a-3p,miR-590-5p. miR-338-3p was the most down-regulated in LO2/HBx cells confirmed by real-time PCR.Conclusion:Hepatitis B virus X gene can influence the microRNA expression profile in LO2cells. MiRNA chip results are reliable and can be used for screening miRNA expression patterns between tissue or cells. Part Ⅱ The influence of miR-338-3p on cell proliferation in cells stably transfected HBx geneAim:On the basis of HBx down-regulating miR-338-3p in part one, the second part is to investigate function of miR-338-3p on cell proliferation induced by HBx in liver cells and its mechanism, trying to discuss the new mechanism of HBx-mediated hepatocarcinogenesis.Methods:We synthetized specific miR-338-3p mimics/inhibitor and transfected mimics/inhibitor into L02/HBx and LO2/pcDNA3.0cells by lipofectamine compared with correspondoing negative control(NC). The effect of miR-338-3p on cellular proliferation was studied using MTT, EdU and cell cycle assay.Results:While the cells transfected with related negative control RNAs progressed through different phases of the cell cycle, the cells transfected with the miR-338-3p mimics displayed a significantly higher frequency of cells in the G1phase and a lower frequency of cells in S phase. In contrast, the cells transfected with the miR-338-3p inhibitor showed a significant decrease in the proportion of cells in the G1phase and an increase in the proportion of cells in S phase. According to MTT assay,miR-338-3p inhibited cell proliferation in both of L02/HBx and LO2/pcDNA3.0cells and the inhibiting rate was40%±9.41,34%±8.3, respectively,while knock-downing miR-338-3p expression in cells could promote cell proliferation.Moreover, we found that the number of EdU-positive cells among the cells transfected with the miR-338-3p mimics was significantly reduced, while the number of EdU cells was significantly increased in the miR-338-3p inhibitor group compared with the cells transfected with control oligos. miR-338-3p inhibited DNA synthesis and shortened S phase in liver cells.Conclusion:miR-338-3p could inhibit cell proliferation and the mechanisms was to prolong G1phase, inhibit DNA synthesis and shorten S phase in liver cells. Part Ⅲ miR-338-3p targets CyclinD1Aim:According to the previous studies and the results in second part,we found that miR-338-3p could inhibit cell proliferation,which was down-regulated in L02/HBx cells negatively related with CyclinD1expression. As predicted by several in silico methods, miR-338-3p may combine with CyclinDl-3’UTR,suggesting that cyclinDl was one of the candidate genes potentially regulated by miR-338-3p.Hence,in this part,we investigate the effect of miR-338-3p on the expression of CyclinD1. Methods:①miR-338-3p-mimics/inhibitor was transfected into LO2/HBx cells, quantitative Real-time PCR and Western Blot were used to examine the CyclinDl mRNA and protein expression.②We amplified CyclinDl-3’UTR sequences by PCR method, mutated two putative sites of CyclinD13’UTR and cloned into pmirGLO dual luciferase reporter vector,and finally constructed plasmids wild-type and mutant of CyclinD1-3’UTR(termed pCyclinD1-3’UTR-WT and pCyclinD1-3’UTR-Mut, separately). Moreover,to examine which is the more important position of miR-338-3p target-sites, we further to mutate each of putative miR-338-3p binding sequence separately and the constructed plasmid termed pCyclinDl-3’UTR-Mut1(mutated907-913nt) and pCyclinD1-3’UTR-Mut2(mutated2397-2403nt). Cotransfected these plasmids and miR-338-3p mimics into LO2/HBx cells and detected changes of luciferase activity using miR-NC as controls.Results:①Relative to the control, the overexpression of miR-338-3p down-regulated the endogenous CyclinDl protein, while the inhibition of miR-338-3p up-regulated the CyclinDl protein. In contrast, no change in the CyclinD1mRNA level was noted.②It confirmed that plasmids pCyclinD1-3’UTR-WT, pCyclinD1-3’UTR-Mut, pCyclinD1-3’UTR-Mut1and pCyclinD1-3’UTR-Mut2were successfully constructed by PCR amplification,double enzyme cut and sequencing.③Overex-pression of miR-338-3p suppressed the luciferase activity of the WT construct of CyclinD13’-UTR with respect to the negative control, whereas the Mut of both two putative sites construct of CyclinD13’-UTR abolished this suppression; although both two sites have functional role, it is the pCyclinD1-3’UTR-Mut1mainly displayed the function.Conclusion:①miR-338-3p negative regulated CyclinD1protein expression.②We have successfully constructed CyclinD1-3’UTR plas-mids.③miR-338-3p regulated CyclinD1and mainly at the position2397-2403nt of CyclinD1-3’UTR. We establish a novel mechanism of HBx-related HCC that HBx inhibits liver cell malignant proliferation by down-regulating miR-338-3p. Part Ⅳ Knocking-down HBx expression affect miR-338-3p and CyclinDl expression in transfected LO2cellsObjective:According to our studies,miR-338-3p is down-regulated by HBx and targets CyclinD1in high level HBx-expressing LO2cells, promoting cell proliferation. So we wondered whether the reduction of HBx could also lead to an different levels of miR-338-3p and CyclinDl expression. This study is to confirm expression of miR-338-3p and CyclinD1in liver cells depends on the change of HBx genes.Methods:Three specific siRNAs of HBx were designed and synthesized (HBx-siRNA1,HBx-siRNA2and HBx-siRNA3),and transiently transfected into L02/HBx cells by Lipofectamine compared with negative control. RT-PCR and Western Blot was used to detect the HBx mRNA/protein expression and test the inhibition effect,respectively. Real-time PCR was used to examine the miR-338-3p expression while Western Blot was used to detect CyclinD1expression.Results:Compared with negative control, all of three specific HBx-siRNAs reduced HBx mRNA expression and the inhibition rate was52.3±2.3%,45.1±3.4%and70.6±4.3%,respectively(P<0.01). Correspondingly, all of three specific HBx-siRNAs, transfecting into LO2/HBx cells, could efficiently and specifically inhibit the HBx protein level and the inhibition rate was54.6±1.8%,45.8±2.2%and78±3.5%,respectively (P<0.01).We measured the miR-338-3p expression normalized against U6by Real-time PCR after48hours of transfection and found that there was a significant increase of miR-338-3p expression after knocking-down HBx in comparison with the negative controls (P=0.013).While,the western blot showed that CyclinDl protein levels was significantly down-reguletd after the reduction of HBx(P<0.001).Conclusion:Knocking-down HBx expression lead to an increase of miR-338-3p expression and a reduction of CyclinDl expression,which suggesting that HBx is necessary for cyclinD1upregulation through miR-338-3p. The data confirms that miR-338-3p is affected by HBx and the HBx/miR-338-3p/CyclinDl module induces hepatocarcinogenesis in the HBx-mediated HCC process. Part Ⅴ Expression of miR-338-3p、HBx and CyclinDl and mutual relationship in HCC tissuesAim:We have established a newly identified miR-338-3p/CyclinDl interaction in vitro.Now,in present study,we investigate the miR-338-3p, HBx and CyclinD1expression in HCC-tumour and adjacent non-tumourous liver tissues,discussing the function of miR-338-3p and its role in HBx-mediated hepatocarcinogenesis integrated with clinical data.Methods:The miR-338-3p in23paired HCC-tumour and adjacent non-tumourous liver tissues were measured using qRT-PCR, HBx and CyclinD1expression were measured using RT-PCR, immunohistoche-mistry or Western blotting. Mutual relationship of HBx、miR-338-3p and CyclinD1was measured by Person’s correlation. The identities of both the tumour and noncancerous samples were confirmed histologically.Results:(1)HBx mRNA expression was detectable in17of23(74%) tumour tissues and also in5adjacent non-tumors among that17cases. miR-338-3p expression was related with tumour grade and reduced in all23HCC tumour tissues compared to the adjacent non-tumourous tissues. However, the expression of miR-338-3p was significantly down-regulated only in HBx-positive tumour tissues compared to adjacent non-tumour tissues with low or no-HBx mRNA positive (p<0.001). In contrast, the expression of miR-338-3p was slightly higher in the tumour tissues with undetectable HBx mRNA compared to the non-tumour tissues, although this difference was not statistically significant(p>0.05).(2) There were19tumor tissues with higher HBx protein expression(19/23,82.6%) and5cases among that adjacent non-tumor tissues with lower HBx protein expression.There was a significant negative correlation between miR-338-3p expression and HBx protein expression in the19paired HBx-positive tumours and non-tumour tissues,suggesting that the higher scores of HBx protein,the lower expression of miR-338-3p mRNA.(3)CyclinDl protein level was enhanced in HCC-tumour compared with non-tumourous tissues. HBx up-regulated CyclinD1expression and CyclinD1expression was inversly correlated to miR-338-3p in23paired HCC samples subjected to a Person correlation analysis,that is the higher expression of miR-338-3p,the lower expression of CyclinD1protein.Conclusion:①miR-338-3p was down-regulated in HCC-tumour tissues and down-regulated only in HBx-positive tumour tissues, although this difference was not statistically significant, the expression of miR-338-3p was slightly higher in the tumour tissues with undetectable HBx mRNA.③HBx expression was found to have an inverse correlation with miR-338-3p expression.③HBx up-regulated CyclinD1expression and CyclinDl protein expression was inversly correlated to miR-338-3p.更多还原