【作者】 朱亚珍；

【导师】 朱虹光； 朱荣；

【作者基本信息】 复旦大学 ， 病理学， 2010， 博士

【摘要】 前言肝细胞性肝癌(HCC)是威胁全世界人民尤其是中国人民身体健康的最常见的恶性肿瘤之一。研究发现,HCC的发生与黄曲霉素B1的污染、酒精性肝硬化、某些遗传性肝脏疾病、特别是乙型或丙型肝炎病毒(HBV或HCV)的感染密切相关。HBV感染的人群中,HCC的发生率比普通人群高200倍至300倍左右,我国HCC病人HBV标志物阳性率更高达90%左右。HCC的发生是一个多因子、多步骤的过程,但其具体机制仍不清楚。有研究表明,抑癌基因如GSTP、SOCS-1、APC、E-cadherin、RAR-β、p14、p15、p16和p73启动子区高甲基化普遍存在于肝癌和其它人类恶性肿瘤中。HBV的感染能增加抑癌基因启动子区的甲基化而使其表达下调,该机制可能是HBV相关HCC中病毒致癌的一个重要途径。HCC中普遍存在p16INK4A启动子区的高甲基化,而且HBV感染能显著增加肝组织p16INK4A启动子区的甲基化频率,从而使p16蛋白低表达或不表达,在HCC形成早期起了重要作用。本课题组前期研究结果提示,在组织学水平,HBV的X蛋白(HBx)与p16INK4A启动子区高甲基化相关,但其中的机制尚不清楚。本研究一方面选取了大量HBV相关HCC部分肝切除标本作为研究对象,同时辅以部分HBV阴性标本作为对照。通过对HCC的癌组织和相应的癌旁组织在mRNA水平和蛋白水平进行检测,对组织水平HBx与p16INK4A的启动子高甲基化相关性进行进一步确认,明确HBx与DNA甲基转移酶(DNMTs)和去甲基化酶的表达是否相关,DNMTs和去甲基化酶的表达是否与p16INK4A启动子区高甲基化相关。另一方面在体外细胞水平通过稳定转染pcDNA3.1-HBx质粒至肝癌细胞研究HBx促进p16INK4A启动子高甲基化的机制,明确DNA甲基转移酶DNMT1, DNMT3A, DNMT3B和去甲基化酶MBD2在HBx促进p16INK4A启动子高甲基化过程中各自的作用,为HBV相关HCC的形成机制提供新的研究方向。第一部分乙型肝炎病毒X蛋白与p16INK4A启动子甲基化及甲基转移酶的相关性的体内研究目的：通过对HBV相关HCC的癌组织和相应的癌旁组织标本中HBx与p16INK4A启动子高甲基化及甲基转移酶的相关性研究,明确HBx与p16INK4A启动子甲基化和DNMTs以及去甲基化酶的表达是否相关。方法：收集88例HBV相关HCC部分肝切除标本癌组织和癌旁组织进行分析,并收集部分HBV阴性标本作为对照。p16INK4A启动子甲基化状态的检测采用甲基化特异性聚合酶链反应(MSP)；荧光实时定量PCR检测HBx,DNMT1, DNMT3A, DNMT3B,去甲基化酶MBD2的表达和肝组织内HBVDNA的含量；HBV基因型采用巢式PCR和限制性片段长度多态性(RFLP)进行检测；Western blot和免疫组化En Vision二步法检测组织HBx,DNMT1, DNMT3A和p16蛋白的表达。结果：在HBV相关HCC病例的癌旁组织中,HBx的高表达与p16INK4A启动子高甲基化相关；无论是在mRNA水平还是蛋白水平,HBx的表达与DNMT1和DNMT3A的表达均呈正相关;而且HBx, DNMT1和DNMT3A的表达与p16蛋白的表达均呈负相关。在HBV相关HCC病例的癌组织中,HBx的表达与DNMT1和DNMT3A的表达在mRNA水平和蛋白水平均呈正相关；但HBx的高表达与p16INK4A启动子高甲基化和p16蛋白的表达均不相关。p16INK4A启动子甲基化状态与患者年龄,性别,血清中AFP, CEA,CA199水平无关,与血清AST, ALT水平,病毒基因型,HBV-DNA水平,肿瘤分化程度,癌旁组织Scheuer评分也无相关性。结论：p16INK4A启动子甲基化在肝癌中普遍存在,其表达量与HBx蛋白呈负相关。在HBV相关HCC形成的早期阶段,HBx可能通过上调甲基转移酶DNMT1和DNMT3A促进p16INK4A启动子甲基化从而使其表达下降,在HBV相关HCC的发生发展过程中起重要作用。第二部分乙型肝炎病毒X蛋白促p16INK4A启动子甲基化及其机制的体外研究目的：HBx可通过促进某些抑癌基因启动子甲基化下调抑癌基因的表达,但其具体机制却不清楚。本研究旨在细胞水平明确HBx促进p16INK4A启动子甲基化过程中甲基转移酶和去甲基化酶各自的作用,明确HBx介导的表观遗传修饰的可能机制。方法：将含有HBx基因的质粒pcDNA3.1 (pcDNA3.1-HBx质粒)稳定转染L02细胞,HepG2细胞和BEL-7404细胞；各组细胞中p16INK4A启动子甲基化状态的检测采用亚硫酸氢盐测序法；荧光实时定量PCR检测HBx, DNMT1, DNMT3A, DNMT3B和去甲基化酶MBD2的表达；Western blot检测HBx,DNMT1, DNMT3A和p16蛋白的表达。结果：在mRNA水平和蛋白水平,HBx均可上调DNMT1和DNMT3A的表达；HBx可通过上调DNMT1和DNMT3A的表达从而促进p16INK4A启动子甲基化；HBx可通过促进p16INK4A启动子甲基化从而下调p16蛋白的表达。结论：HBx通过上调DNMT1和DNMT3A的表达从而促进p16INK4A启动子甲基化,进而导致p16蛋白的表达降低。HBx-DNMTs-p16INK4A启动子甲基化-p16蛋白的表达降低可能是HBV相关HCC发生的机制之一。更多还原

【Abstract】 IntroductionIt is known that Hepatocellular carcinoma (HCC) is one of the leading cancers in the world, especially in China. So far, accumulating evidences have suggested that pathogenesis of HCC is multifactorial and heterogeneous among patients, and there are many risk factors for the development of HCC, including flavacin B1, alcoholic cirrhosis, and some genetic liver disease. Chronic and persistent infection of hepatitis B virus (HBV) is a major risk factor for the development of HCC. The incidence of HCC is 200-300 folds higher in patients with chronic HBV infection than normal people. In China, over 90% cases of HCC are HBV related. However, the exact mechanism of the development of HCC remains unclear.Recent studies showed that aberrant hypermethylation of CpG islands in the promoter regions of many tumor suppressor genes, which represses their transcription, such as GSTP、SOCS-1、APC、E-cadherin、RAR-β、p14、p15、p16 and p73, and is a common event in the development of HCC or other malignant tumors. Persistent HBV infection may induce the high frequency of p16INK4A promoter methylation, which in turn represses P16 expression and plays an important role in the early stage of HCC. Our previous study showed that p16INK4A promoter hypermethylation correlated closely with higher HBx expression in the precancerous lesions, which suggests that HBx plays an important role in the early stage of HBV-associated hepatocarcinogenesis via inducing hypermethylation of p16INK4A promoter. However, that HBx induced hypermethylation directly or indirectly, and whether other factors are related with this procedure is still unclear.In the first part of the present study, we investigated the associations among the expressions of HBx, DNA methyltransferase (DNMT)1, DNMT3A, DNMT3B and methyl-CpG binding domain protein 2 (MBD2) both in mRNA levels and protein levels, as well as the methylation status of p16INK4A promoter in fresh partial hepatectomy speciments of HBV-accociated HCC and their corresponding non-cancerous liver tissues, in order to identify whether the expression of HBx correlated with the expression of DNMT1, DNMT3A, DNMT3B or MBD2, and whether the expression of DNMT1, DNMT3A, DNMT3B or MBD2 correlated with the higher frequency of p16INK4A promoter methylation.In the second part of the present study, we investigated the expressions of HBx, DNMT1, DNMT3A, DNMT3B and MBD2 both in mRNA level and protein level in different cell lines with or without HBx-expressing vector, as well as the methylation status of p16INK4A promoter region, in order to identify the mechanism of HBx inducing hypermethylation of p16INK4A promoter, as well as the roles of DNMT1, DNMT3A, DNMT3B and MBD2 in the process of HBx inducing hypermethylation of p16INK4A promoter, which may provide new insights to the HBV-accociated hepatocarcinogenesis.PartⅠThe correlations among Hepatitis B Virus X Protein and hypermethylation of p16INK4A promoter as well as DNA methyltransferases in vivoPurpose:The aim of the present study was to authenticate the involvements of DNA methyltransferases (DNMTs) and methyl-CpG binding domain protein 2 (MBD2) in the process of HBx induced p16INK4A promoter hypermethylation in HBV-related hepatocellular carcinoma (HCC) and their corresponding non-cancerous liver tissues.Methods:Eighty-eight fresh tissue specimens of surgically resected HBV-associated HCC and their corresponding non-cancerous liver tissues as well as some HBV-negative tissues were studied. The methylation status of p16INK4A promoter was determined by methylation-specific polymerase chain reaction (MSP). Reverse transcription and real-time polymerase chain reaction (real-time RT-PCR) showed the expression of DNMTs, MBD2, and HBx. Western blot and immunohistochemistry were used for the protein analysis of HBx, DNMT1, DNMT3A and p16. Tissue HBV-DNA levels were determined by real-time PCR. HBV genotype was examined by nested PCR and restriction fragment length polymorphism (RFLP).Results:In the corresponding non-cancerous liver tissues of HBV-associated HCC, Higher HBx expression associated with hypermethylation of p16INK4A promoter. HBx was positively correlated with DNMTl, DNMT3A in both mRNA and protein levels. Furthermore, HBx, DNMT1 and DNMT3A protein expression were negtively correlated with p16 protein expression. In HBV-associated HCC tissues, HBx was positively correlated with DNMT1, DNMT3A in both mRNA and protein levels, however, HBx expression didn’t correlate with hypermethylation of p16INK4A promoter or p16 protein expression. Methylation status of p16INK4A promoter didn’t correlate with clinic-pathologic characteristics.Conclusions:DNMTl and DNMT3A may play important roles in the process of HBx inducing hypermethylation of p16INK4A promoter in the early stage of HBV-associated HCC. HBx-DNMTs-p16INK4A promoter hypermethylation-decreased expression of p16INK4A may suggest a mechanism for tumorigenesis during HBV-associated hepatocarcinogenesis.PartⅡThe mechanisms of Hepatitis B Virus X Protein promoting hypermethylation of p16INK4A promoter in vitroPurpose:The hepatitis B virus X protein (HBx) has been implicated as a potential trigger of the epigenetic deregulation of some genes, but the underlying mechanism remains unknown. The aim of this study is to identify underlying mechanisms involved in HBx-mediated epigenetic modification in the process of HBx induced p16INK4A promoter hypermethylation.Methods:Liver cell lines were stably transfected with HBx-expressing vector. The methylation status of p16INK4A was examined by bisulfite sequencing. Reverse transcription and real-time polymerase chain reaction (real-time RT-PCR), Western blot was used to analyze the expression of HBx, HBx-mediated DNA methylation abnormalities and p16INK4A.Results:HBx upregulates DNMT1 and DNMT3A expression in both mRNA level and protein level, and HBx represses p16INK4A expression through inducing hypermethylation of p16INK4A promoter. Moreover, HBx induces hypermethylation of p16INK4A promoter through DNMT1 and DNMT3A.Conclusions:Regulation of DNMT1 and DNMT3A by HBx promoted hypermethylation of p16INK4A promoter region. promoter hypermethylation-decreased expression of p16INK4A may suggest a mechanism for tumorigenesis during hepatocarcinogenesis.更多还原