乙型肝炎病毒X蛋白促p16~（INK4A）基因启动子甲基化及其机制的研究
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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。
Introduction
It 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 vivo
Purpose: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 vitro
Purpose: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.
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。
Introduction
It 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 vivo
Purpose: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 vitro
Purpose: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.
引文
1. 骆抗先主编.乙型肝炎基础和临床.第二版.北京：人民卫生出版社,2001.6-7
2. Zhang XD, Zhang H, Ye LH. Effects of hepatitis B virus X protein on the development of liver cancer. J Lab Clin Med.2006; 147(2):58-66.
3. Tang H ON, Murakami S, et al. Molecular functions and biological roles of hepatitis B virus x protein. Cancer Sci.2004; 97(10):977-83.
4. Park IY, Sohn BH, Yu E et al. Aberrant epigenetic modifications in-hepatocarcinogenesis induced by hepatitis B virus X protein. Gastroenterology 2007; 132(4):1476-1494.
5. Raptopoulou M, Papatheodoridis G, Antoniou A et al. Epidemiology, course and disease burden of chronic hepatitis B virus infection. HEPNET study for chronic hepatitis B:a multicentre Greek study. J Viral Hepat 2009;16(3): 195-202.
6. Schlaeger C, Longerich T, Schiller C et al. Etiology-dependent molecular mechanisms in human hepatocarcinogenesis. Hepatology 2008;47(2):511-520.
7. Santamaria E, Munoz J, Fernandez-Irigoyen J, Prieto J, Corrales FJ. Toward the discovery of new biomarkers of hepatocellular carcinoma by proteomics. Liver Int 2007;27(2):163-173.
8. Lavanchy D. Worldwide epidemiology of HBV infection, disease burden, and vaccine prevention. J Clin Virol 2005;34 (Suppl 1):S1-3.
9. Cha C, Dematteo RP. Molecular mechanisms in hepatocellular carcinoma development. Best Pract Res Clin. Gastroenterol 2005; 19(1):25-37
10. Gao W, Kondo Y, Shen L et al. Variable DNA methylation patterns associated with progression of disease in hepatocellular carcinomas. Carcinogenesis 2008; 29(10):1901-1910.
11. El-Serag HB, Rudolph KL. Hepatocellular carcinoma:epidemiology and molecular carcinogenesis. Gastroenterology 2007; 132(7):2557-2576.
12. Tan TL, Chen WN. A proteomics analysis of cellular proteins associated with HBV genotype-specific HBX:potential in identification of early diagnostic markers for HCC. J Clin Virol 2005; 33(4):293-298.
13. Severi T, Vander Borght S, Libbrecht L et al. HBx or HCV core gene expression in HepG2 human liver cells results in a survival benefit against oxidative stress with possible implications for HCC development. Chem Biol Interact 2007; 168(2):128-134.
14. Ou DP, Tao YM, Tang FQ, Yang LY. The hepatitis B virus X protein promotes hepatocellular carcinoma metastasis by upregulation of matrix metalloproteinases. Int J Cancer 2007; 120(6):1208-1214.
15. Calvisi DF, Ladu S, Gorden A et al. Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma. J Clin Invest 2007; 117(9):2713-2722.
16. Yang B, Guo M, Herman JG, Clark DP. Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. Am J Pathol 2003; 163(3):1101-1107.
17. Zhang JC, Yu ZT, Lu J, Li HP, Wu JM, Hu LH. Persistent infection of hepatitis B virus is involved in high rate of p16 methylation in hepatocellular carcinoma. Molecular Carcinogenesis 2006;45(7):530-536.
18. Li X, Hui AM, Sun L et al. p16(INK4A) hypermethylation is associated with hepatitis virus infection, age, and gender in hepatocellular carcinoma. Clinical Cancer Research 2004; 10(22):7484-7489.
19. Tamori A, Koh N, Kubo S et al. p16 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection. Intervirology 2004;47(1):26-31.
20. Lee JO, Kwun HJ, Jung JK, Choi KH, Min DS, Jang KL. Hepatitis B virus X protein represses E-cadherin expression via activation of DNA methyltransferase 1. Oncogene 2005; 24(44):6617-6625.
1. Motola-Kuba D, Zamora-Valdes D, Uribe M, Mendez-Sanchez N. Hepatocellular carcinoma. An overview. Ann Hepatol 2006;5(1):16-24.
2. Williams R. Global challenges in liver disease. Hepatology 2006;44(3):521-526.
3. Brechot C. Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms. Gastroenterology 2004; 127(5 Suppl 1):S56-61.
4. Lavanchy D. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat 2004;11(2):97-107.
5. GT Liu CS, QH Wang et al. Comments on the prevention and research of chronic hepatitis in China. Natl Med J China 2002;82:74-76.
6. Yeo W, Mo FK, Chan SL et al. Hepatitis B viral load predicts survival of HCC patients undergoing systemic chemotherapy. Hepatology 2007;45(6):1382-1389.
7. Calvisi DF, Ladu S, Gorden A et al. Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma. J Clin Invest 2007;117(9):2713-2722.
8. Yang B, Guo M, Herman JG, Clark DP. Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. Am J Pathol 2003;163(3):1101-1107.
9. Shim YH, Yoon GS, Choi HJ, Chung YH, Yu E. p16 Hypermethylation in the early stage of hepatitis B virus-associated hepatocarcinogenesis. Cancer Lett 2003;190(2):213-219.
10. Tamori A, Koh N, Kubo S et al. p16 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection. Intervirology 2004;47(1):26-31.
11. Kaneto H, Sasaki S, Yamamoto H et al. Detection of hypermethylation of the p16(INK4A) gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus. Gut 2001;48(3):372-377.
12. Li X, Hui AM, Sun L et al.p16(INK4A) hypermethylation is associated with hepatitis virus infection, age, and gender in hepatocellular carcinoma. Clinical Cancer Research 2004;10(22):7484-7489.
13. Zhu R, Li BZ, Li H et al. Association of p16INK4A hypermethylation with hepatitis B virus X protein expression in the early stage of HBV-associated hepatocarcinogenesis. Pathol Int 2007;57(6):328-336.
14. Bestor TH. Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain. EMBO J 1992; 11 (7):2611-2617.
15. Park HJ, Yu E, Shim YH. DNA methyltransferase expression and DNA hypermethylation in human hepatocellular carcinoma. Cancer Lett 2006;233(2):271-278.
16. Nagai M, Nakamura A, Makino R, Mitamura K. Expression of DNA (5-cytosin)-methyltransferases (DNMTs) in hepatocellular carcinomas. Hepatol Res 2003;26(3):186-191.
17. Saito Y, Kanai Y, Sakamoto M, Saito H, Ishii H, Hirohashi S. Expression of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and DNA methylation status on CpG islands and pericentromeric satellite regions during human hepatocarcinogenesis. Hepatology 2001;33(3):561-568.
18. Oh BK, Kim H, Park HJ et al. DNA methyltransferase expression and DNA methylation in human hepatocellular carcinoma and their clinicopathological correlation. International Journal of Molecular Medicine 2007;20(1):65-73.
19. Li HP, Leu YW, Chang YS. Epigenetic changes in virus-associated human cancers. Cell Research 2005;15(4):262-271.
20. Park IY, Sohn BH, Yu E et al. Aberrant epigenetic modifications in-hepatocarcinogenesis induced by hepatitis B virus X protein. Gastroenterology 2007; 132(4):1476-1494.
21. Lee JO, Kwun HJ, Jung JK, Choi KH, Min DS, Jang KL. Hepatitis B virus X protein represses E-cadherin expression via activation of DNA methyltransferase 1. Oncogene 2005;24(44):6617-6625.
22. Barr H, Hermann A, Berger J et al. Mbd2 contributes to DNA methylation-directed repression of the Xist gene. Molecular and Cellular Biology 2007;27(10):3750-3757.
23. Edmondson HA SP. Primary carcinoma of the liver:a study of 100 cases among 48900 necropsies. Cancer (Phila) 1954;7:462-503.
24. Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Scheuer PJ. Classification of Chronic Hepatitis-Diagnosis, Grading and Staging. Hepatology 1994; 19(6):1513-1520.
25. DT Leong AG, DW Hutmacher, et al. Absolute quantification of gene expression in biomaterials research using real-time PCR. Biomaterials 2007;28(2):203-210.
26. Macpherson AM, Archer DF, Leslie S, Charnock-Jones DS, Makkink WK, Smith SK. The effect of etonogestrel on VEGF, oestrogen and progesterone receptor immunoreactivity and endothelial cell number in human endometrium. Hum Reprod 1999;14(12):3080-3087.
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28. Yu AS, Keeffe EB. Management of hepatocellular carcinoma. Rev Gastroenterol Disord 2003;3(1):8-24.
29. Roncalli M, Bianchi P, Bruni B et al. Methylation framework of cell cycle gene inhibitors in cirrhosis and associated hepatocellular carcinoma. Hepatology 2002;36(2):427-432.
30. Lee S, Lee HJ, Kim JH, Lee HS, Jang JJ, Kang GH. Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. Am J Pathol 2003;163(4):1371-1378.
31. Fukai K, Yokosuka O, Imazeki F et al. Methylation status of p14ARF, p15INK4b, and p16INK4A genes in human hepatocellular carcinoma. Liver Int 2005;25(6):1209-1216.
32. Zheng DL, Zhang L, Cheng N et al. Epigenetic modification induced by hepatitis B virus X protein via interaction with de novo DNA methyltransferase DNMT3A. J Hepatol 2009;50(2):377-387.
33. Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003;349(21):2042-2054.
34. Jaenisch R, Bird A. Epigenetic regulation of gene expression:how the genome integrates intrinsic and environmental signals. Nature Genetics 2003;33:245-254.
35. Richardson B. Impact of aging on DNA methylation. Ageing Research Reviews 2003;2(3):245-261.
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1. Brechot C. Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms. Gastroenterology 2004;127(5 Suppl 1):S56-61.
2. Gao W, Kondo Y, Shen L et al. Variable DNA methylation patterns associated with progression of disease in hepatocellular carcinomas. Carcinogenesis 2008;29(10):1901-1910.
3. Zhu R, Li BZ, Li H et al. Association of p16INK4A hypermethylation with hepatitis B virus X protein expression in the early stage of HBV-associated hepatocarcinogenesis. Pathol Int 2007;57(6):328-336.
4. Lai M, Hyatt BJ, Nasser I, Curry M, Afdhal NH. The clinical significance of persistently normal ALT in chronic hepatitis B infection. J Hepatol 2007;47(6):760-767.
5. Chan DW, Ng IO. Knock-down of hepatitis B virus X protein reduces the tumorigenicity of hepatocellular carcinoma cells. J Pathol 2006;208(3):372-380.
6. Cheng AS, Wong N, Tse AM et al. RNA interference targeting HBx suppresses tumor growth and enhances cisplatin chemosensitivity in human hepatocellular carcinoma. Cancer Lett 2007;253(1):43-52.
7. Kim SY, Lee PY, Shin HJ et al. Proteomic analysis of liver tissue from HBx-transgenic mice at early stages of hepatocarcinogenesis. Proteomics 2009;9(22):5056-5066.
8. Zhang XD, Zhang H, Ye LH. Effects of hepatitis B virus X protein on the development of liver cancer. Journal of Laboratory and Clinical Medicine 2006;147(2):58-66.
9. Park IY, Sohn BH, Yu E et al. Aberrant epigenetic modifications in-hepatocarcinogenesis induced by hepatitis B virus X protein. Gastroenterology 2007;132(4):1476-1494.
10. Fukai K, Yokosuka O, Imazeki F et al. Methylation status of p14ARF, p15INK4b, and p16INK4a genes in human hepatocellular carcinoma. Liver Int 2005;25(6):1209-1216.
11. Lee JO, Kwun HJ, Jung JK, Choi KH, Min DS, Jang KL. Hepatitis B virus X protein represses E-cadherin expression via activation of DNA methyltransferase 1. Oncogene 2005;24(44):6617-6625.
12. Zheng DL, Zhang L, Cheng N et al. Epigenetic modification induced by hepatitis B virus X protein via interaction with de novo DNA methyltransferase DNMT3A. J Hepatol 2009;50(2):377-387
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16. Sun L, Zhao H, Xu Z et al. Phosphatidylinositol 3-kinase/protein kinase B pathway stabilizes DNA methyltransferase I protein and maintains DNA methylation. Cell Signal 2007;19(11):2255-2263.
17. Robertson KD, Uzvolgyi E, Liang G et al. The human DNA methyltransferases (DNMTs) 1,3a and 3b:coordinate mRNA expression in normal tissues and overexpression in tumors. Nucleic Acids Res 1999;27(11):2291-2298.
18. Wang J, Bhutani M, Pathak AK et al. Delta DNMT3B variants regulate DNA methylation in a promoter-specific manner. Cancer Res 2007;67(22):10647-10652.
19. Martin V, Jorgensen HF, Chaubert AS et al. MBD2-mediated transcriptional repression of the p14ARF tumor suppressor gene in human colon cancer cells. Pathobiology 2008;75(5):281-287.
1. Caldwell S, Park SH. The epidemiology of hepatocellular cancer:from the perspectives of public health problem to tumor biology. J Gastroenterol 2009;44(Suppl 19):96-101.
2. Lavanchy D. Worldwide epidemiology of HBV infection, disease burden, and vaccine prevention. J Clin Virol 2005;34(Suppl 1):S1-3.
3. Gao W, Kondo Y, Shen L et al. Variable DNA methylation patterns associated with progression of disease in hepatocellular carcinomas. Carcinogenesis 2008;29(10):1901-1910.
4. Yang B, Guo M, Herman JG, Clark DP. Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. Am J Pathol 2003;163(3):1101-1107.
5. Su PF, Lee TC, Lin PJ et al. Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma. Int J Cancer 2007;121(6):1257-1264.
6. Jones PA, Takai D. The role of DNA methylation in mammalian epigenetics. Science 2001;293(5532):1068-1070.
7. Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003;349(21):2042-2054.
8. Nagase H, Ghosh S. Epigenetics:differential DNA methylation in mammalian somatic tissues. FEBS J 2008;275(8):1617-1623.
9. Nishida N, Nagasaka T, Nishimura T, Ikai I, Boland CR, Goel A. Aberrant methylation of multiple tumor suppressor genes in aging liver, chronic hepatitis, and hepatocellular carcinoma. Hepatology 2008;47(3):908-918.
10. Feng Q, Stern JE, Hawes SE, Lu H, Jiang M, Kiviat NB. DNA methylation changes in normal liver tissues and hepatocellular carcinoma with different viral infection. Exp Mol Pathol;88(2):287-292.
11. Tamori A, Koh N, Kubo S et al. p16 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection. Intervirology 2004;47(1):26-31.
12. Zhang JC, Yu ZT, Lu J, Li HP, Wu JM, Hu LH. Persistent infection of hepatitis B virus is involved in high rate of p16 methylation in hepatocellular carcinoma. Molecular Carcinogenesis 2006;45(7):530-536.
13. Zhu R, Li BZ, Li H et al. Association of p16INK4A hypermethylation with hepatitis B virus X protein expression in the early stage of HBV-associated hepatocarcinogenesis. Pathol Int 2007;57(6):328-336.
14. Yeo W, Wong N, Wong WL, Lai PB, Zhong S, Johnson PJ. High frequency of promoter hypermethylation of RASSF1A in tumor and plasma of patients with hepatocellular carcinoma. Liver Int 2005;25(2):266-272.
15. Zhong S, Yeo W, Tang MW, Wong N, Lai PB, Johnson PJ. Intensive hypermethylation of the CpG island of Ras association domain family 1A in hepatitis B virus-associated hepatocellular carcinomas. Clin Cancer Res 2003;9(9):3376-3382.
16. Zhang YJ, Chen Y, Ahsan H et al. Silencing of glutathione S-transferase P1 by promoter hypermethylation and its relationship to environmental chemical carcinogens in hepatocellular carcinoma. Cancer Lett 2005;221(2):135-143.
17. Zhong S, Tang MW, Yeo W, Liu C, Lo YM, Johnson PJ. Silencing of GSTP1 gene by CpG island DNA hypermethylation in HBV-associated hepatocellular carcinomas. Clin Cancer Res 2002;8(4):1087-1092.
18. Niu D, Zhang J, Ren Y, Feng H, Chen WN. HBx genotype D represses GSTP1 expression and increases the oxidative level and apoptosis in HepG2 cells. Mol Oncol 2009;3(1):67-76.
19. Calvisi DF, Ladu S, Conner EA, Factor VM, Thorgeirsson SS. Disregulation of E-cadherin in transgenic mouse models of liver cancer. Lab Invest 2004;84(9):1137-1147.
20. Kwon GY, Yoo BC, Koh KC, Cho JW, Park WS, Park CK. Promoter methylation of E-cadherin in hepatocellular carcinomas and dysplastic nodules. J Korean Med Sci 2005;20(2):242-247.
21. Liu J, Lian Z, Han S et al. Downregulation of E-cadherin by hepatitis B virus X antigen in hepatocellullar carcinoma. Oncogene 2006;25(7):1008-1017.
22. Calvisi DF, Ladu S, Gorden A et al. Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology 2006; 130(4):1117-1128.
23. Okochi O, Hibi K, Sakai M et al. Methylation-mediated silencing of SOCS-1 gene in hepatocellular carcinoma derived from cirrhosis. Clin Cancer Res 2003;9(14):5295-5298.
24. Ko E, Kim SJ, Joh JW, Park CK, Park J, Kim DH. CpG island hypermethylation of SOCS-1 gene is inversely associated with HBV infection in hepatocellular carcinoma. Cancer Lett 2008;271(2):240-250.
25. Jung JK, Park SH, Jang KL. Hepatitis B virus X protein overcomes the growth-inhibitory potential of retinoic acid by downregulating retinoic acid receptor-beta2 expression via DNA methylation. J Gen Virol;91(Pt 2):493-500.
26. Vivekanandan P, Daniel HD, Kannangai R, Martinez-Murillo F, Torbenson M. Hepatitis B Viral Replication Induces Methylation of Both Host and Viral DNA. J Virol 2010;84(9):4321-4329.
27. Lee JO, Kwun HJ, Jung JK, Choi KH, Min DS, Jang KL. Hepatitis B virus X protein represses E-cadherin expression via activation of DNA methyltransferase 1. Oncogene 2005;24(44):6617-6625.
28. Park IY, Sohn BH, Yu E et al. Aberrant epigenetic modifications in hepatocarcinogenesis induced by hepatitis B virus X protein. Gastroenterology 2007;132(4):1476-1494.
29. Zheng DL, Zhang L, Cheng N et al. Epigenetic modification induced by hepatitis B virus X protein via interaction with de novo DNA methyltransferase DNMT3A. J Hepatol 2009;50(2):377-387.
30. Zhu YZ, Zhu R, Fan J et al. Hepatitis B virus X protein induces hypermethylation of p16(INK4A) promoter via DNA methyltransferases in the early stage of HBV-associated hepatocarcinogenesis. J Viral Hepat 2009;17(2):98-107.
31. Zhang YJ, Wu HC, Shen J et al. Predicting hepatocellular carcinoma by detection of aberrant promoter methylation in serum DNA. Clin Cancer Res 2007;13(8):2378-2384.
32. Di Gioia S, Bianchi P, Destro A et al. Quantitative evaluation of RASSF1A methylation in the non-lesional, regenerative and neoplastic liver. BMC Cancer 2006;10(6):89-101.
33. Wang J, Qin Y, Li B, Sun Z, Yang B. Detection of aberrant promoter methylation of GSTP1 in the tumor and serum of Chinese human primary hepatocellular carcinoma patients. Clin Biochem 2006;39(4):344-348.
34. Zhang C, Li Z, Cheng Y et al. CpG island methylator phenotype association with elevated serum alpha-fetoprotein level in hepatocellular carcinoma. Clin Cancer Res 2007;13(3):944-952.
35. Vucic EA, Brown CJ, Lam WL. Epigenetics of cancer progression. Pharmacogenomics 2008;9(2):215-234.
36. Ko E, Kim Y, Kim SJ et al. Promoter hypermethylation of the p16 gene is associated with poor prognosis in recurrent early-stage hepatocellular carcinoma. Cancer Epidemiology Biomarkers & Prevention 2008;17(9):2260-2267.
37. Lee S, Lee HJ, Kim JH, Lee HS, Jang JJ, Kang GH. Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. Am J Pathol 2003;163(4):1371-1378.
2. Zhang XD, Zhang H, Ye LH. Effects of hepatitis B virus X protein on the development of liver cancer. J Lab Clin Med.2006; 147(2):58-66.
3. Tang H ON, Murakami S, et al. Molecular functions and biological roles of hepatitis B virus x protein. Cancer Sci.2004; 97(10):977-83.
4. Park IY, Sohn BH, Yu E et al. Aberrant epigenetic modifications in-hepatocarcinogenesis induced by hepatitis B virus X protein. Gastroenterology 2007; 132(4):1476-1494.
5. Raptopoulou M, Papatheodoridis G, Antoniou A et al. Epidemiology, course and disease burden of chronic hepatitis B virus infection. HEPNET study for chronic hepatitis B:a multicentre Greek study. J Viral Hepat 2009;16(3): 195-202.
6. Schlaeger C, Longerich T, Schiller C et al. Etiology-dependent molecular mechanisms in human hepatocarcinogenesis. Hepatology 2008;47(2):511-520.
7. Santamaria E, Munoz J, Fernandez-Irigoyen J, Prieto J, Corrales FJ. Toward the discovery of new biomarkers of hepatocellular carcinoma by proteomics. Liver Int 2007;27(2):163-173.
8. Lavanchy D. Worldwide epidemiology of HBV infection, disease burden, and vaccine prevention. J Clin Virol 2005;34 (Suppl 1):S1-3.
9. Cha C, Dematteo RP. Molecular mechanisms in hepatocellular carcinoma development. Best Pract Res Clin. Gastroenterol 2005; 19(1):25-37
10. Gao W, Kondo Y, Shen L et al. Variable DNA methylation patterns associated with progression of disease in hepatocellular carcinomas. Carcinogenesis 2008; 29(10):1901-1910.
11. El-Serag HB, Rudolph KL. Hepatocellular carcinoma:epidemiology and molecular carcinogenesis. Gastroenterology 2007; 132(7):2557-2576.
12. Tan TL, Chen WN. A proteomics analysis of cellular proteins associated with HBV genotype-specific HBX:potential in identification of early diagnostic markers for HCC. J Clin Virol 2005; 33(4):293-298.
13. Severi T, Vander Borght S, Libbrecht L et al. HBx or HCV core gene expression in HepG2 human liver cells results in a survival benefit against oxidative stress with possible implications for HCC development. Chem Biol Interact 2007; 168(2):128-134.
14. Ou DP, Tao YM, Tang FQ, Yang LY. The hepatitis B virus X protein promotes hepatocellular carcinoma metastasis by upregulation of matrix metalloproteinases. Int J Cancer 2007; 120(6):1208-1214.
15. Calvisi DF, Ladu S, Gorden A et al. Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma. J Clin Invest 2007; 117(9):2713-2722.
16. Yang B, Guo M, Herman JG, Clark DP. Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. Am J Pathol 2003; 163(3):1101-1107.
17. Zhang JC, Yu ZT, Lu J, Li HP, Wu JM, Hu LH. Persistent infection of hepatitis B virus is involved in high rate of p16 methylation in hepatocellular carcinoma. Molecular Carcinogenesis 2006;45(7):530-536.
18. Li X, Hui AM, Sun L et al. p16(INK4A) hypermethylation is associated with hepatitis virus infection, age, and gender in hepatocellular carcinoma. Clinical Cancer Research 2004; 10(22):7484-7489.
19. Tamori A, Koh N, Kubo S et al. p16 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection. Intervirology 2004;47(1):26-31.
20. Lee JO, Kwun HJ, Jung JK, Choi KH, Min DS, Jang KL. Hepatitis B virus X protein represses E-cadherin expression via activation of DNA methyltransferase 1. Oncogene 2005; 24(44):6617-6625.
1. Motola-Kuba D, Zamora-Valdes D, Uribe M, Mendez-Sanchez N. Hepatocellular carcinoma. An overview. Ann Hepatol 2006;5(1):16-24.
2. Williams R. Global challenges in liver disease. Hepatology 2006;44(3):521-526.
3. Brechot C. Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms. Gastroenterology 2004; 127(5 Suppl 1):S56-61.
4. Lavanchy D. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat 2004;11(2):97-107.
5. GT Liu CS, QH Wang et al. Comments on the prevention and research of chronic hepatitis in China. Natl Med J China 2002;82:74-76.
6. Yeo W, Mo FK, Chan SL et al. Hepatitis B viral load predicts survival of HCC patients undergoing systemic chemotherapy. Hepatology 2007;45(6):1382-1389.
7. Calvisi DF, Ladu S, Gorden A et al. Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma. J Clin Invest 2007;117(9):2713-2722.
8. Yang B, Guo M, Herman JG, Clark DP. Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma. Am J Pathol 2003;163(3):1101-1107.
9. Shim YH, Yoon GS, Choi HJ, Chung YH, Yu E. p16 Hypermethylation in the early stage of hepatitis B virus-associated hepatocarcinogenesis. Cancer Lett 2003;190(2):213-219.
10. Tamori A, Koh N, Kubo S et al. p16 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection. Intervirology 2004;47(1):26-31.
11. Kaneto H, Sasaki S, Yamamoto H et al. Detection of hypermethylation of the p16(INK4A) gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus. Gut 2001;48(3):372-377.
12. Li X, Hui AM, Sun L et al.p16(INK4A) hypermethylation is associated with hepatitis virus infection, age, and gender in hepatocellular carcinoma. Clinical Cancer Research 2004;10(22):7484-7489.
13. Zhu R, Li BZ, Li H et al. Association of p16INK4A hypermethylation with hepatitis B virus X protein expression in the early stage of HBV-associated hepatocarcinogenesis. Pathol Int 2007;57(6):328-336.
14. Bestor TH. Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain. EMBO J 1992; 11 (7):2611-2617.
15. Park HJ, Yu E, Shim YH. DNA methyltransferase expression and DNA hypermethylation in human hepatocellular carcinoma. Cancer Lett 2006;233(2):271-278.
16. Nagai M, Nakamura A, Makino R, Mitamura K. Expression of DNA (5-cytosin)-methyltransferases (DNMTs) in hepatocellular carcinomas. Hepatol Res 2003;26(3):186-191.
17. Saito Y, Kanai Y, Sakamoto M, Saito H, Ishii H, Hirohashi S. Expression of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and DNA methylation status on CpG islands and pericentromeric satellite regions during human hepatocarcinogenesis. Hepatology 2001;33(3):561-568.
18. Oh BK, Kim H, Park HJ et al. DNA methyltransferase expression and DNA methylation in human hepatocellular carcinoma and their clinicopathological correlation. International Journal of Molecular Medicine 2007;20(1):65-73.
19. Li HP, Leu YW, Chang YS. Epigenetic changes in virus-associated human cancers. Cell Research 2005;15(4):262-271.
20. Park IY, Sohn BH, Yu E et al. Aberrant epigenetic modifications in-hepatocarcinogenesis induced by hepatitis B virus X protein. Gastroenterology 2007; 132(4):1476-1494.
21. Lee JO, Kwun HJ, Jung JK, Choi KH, Min DS, Jang KL. Hepatitis B virus X protein represses E-cadherin expression via activation of DNA methyltransferase 1. Oncogene 2005;24(44):6617-6625.
22. Barr H, Hermann A, Berger J et al. Mbd2 contributes to DNA methylation-directed repression of the Xist gene. Molecular and Cellular Biology 2007;27(10):3750-3757.
23. Edmondson HA SP. Primary carcinoma of the liver:a study of 100 cases among 48900 necropsies. Cancer (Phila) 1954;7:462-503.
24. Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Scheuer PJ. Classification of Chronic Hepatitis-Diagnosis, Grading and Staging. Hepatology 1994; 19(6):1513-1520.
25. DT Leong AG, DW Hutmacher, et al. Absolute quantification of gene expression in biomaterials research using real-time PCR. Biomaterials 2007;28(2):203-210.
26. Macpherson AM, Archer DF, Leslie S, Charnock-Jones DS, Makkink WK, Smith SK. The effect of etonogestrel on VEGF, oestrogen and progesterone receptor immunoreactivity and endothelial cell number in human endometrium. Hum Reprod 1999;14(12):3080-3087.
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