Research On Hepatitis B Virus X Protein Promoting Hypermethylation Of P16^INK4A Promoter

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.