Hepatitis B Virus-Induced Hypermethylation Of The Genes Associated With P16-Rb Signal Pathway And Its Mechanisms

Part One High rate of methylation of genes associated with p16^INK4A-RB signal pathway in HBV-Associated hepatocellular carcinomaObjective Both HBV and gene methylation play important role in hepatocarcinogenesis. However, their association between HBV infection and gene methylation is not fully understood. One of the main regulatory pathways were reported to be altered in hepatocellular carcinoma (HCC) is that of cell cycle control involving RB gene-related cell inhibitors. The purpose of this research is to assess the methylation status of p14^ARF and INK4 gene family (p14^ARF, p15^INK4B, p16^INK4A and p18INK4C) in HCC with HBV infection and HCC without it, and discuss possible role of HBV-induced hypermethylation in the mechanism of hepatocarcinogenesis.Methods Methylated and unmethylated products of tumor suppression genes were detected with methylation-specific polymerase chain reaction (PCR), its expression level of mRNA and HBV-DNA in tumor-associated tissues and blood were analyzed with realtime PCR, and HBV markers were examined with real-time PCR and immunologic method.Results p14^ARF, p15^INK4B, p16^INK4A and RB hypermethylation were observed in 34.1%, 56.8%, 70.5% and 27.3% of 44 hepatocellular carcinomas. Methylation frequencies of them between HCC with HBV infection and HCC without it were 43.8% vs. 8.3%(p14^ARF), 68.9% vs.25%(p15^INK4B), 90.6% vs.16.7%( p16^INK4A) and 28.1% vs.25%(RB), respectively. In HBV-associated HCC, the numbers of methylated genes were also more than HCC without virus infection, more than 2 methylated genes were seen in 75% cases; more than 3 methylated genes were found in 32 of 64(50%), correspondently, no one case has more than two gene methylated.In HBV and non-HBV associated cirrhotic tisses, Methylation frequencies of p14^ARF, p15^INK4B, p16^INK4A and RB were 20% vs.9.1%(p14^ARF), 30% vs.18.2%(p15^INK4B), 46.7% vs.9.1%( p16^INK4A) and10 % vs.0%(RB), respectively. 4 methylated production of p16^INK4A and 1 methylated p15^INK4B were also deteced in HBV-associated noncancerous tissues. Methylated products were also found in the blood of patients with cirrhotic liver and hepatitis, 23.9% and 5.5% (p16^INK4A), 17.4% and 5 % ( p15^INK4B), 6.5% and 0%(p14^ARF) .no methylation product were detected in normal control. Methylation frequencies of samples with HBV-DNA replication were significant higher than that without HBV-DNA replication.Conclusions: High rate of p15^INK4B and p16^INK4A in samples with HBV infection suggests that HBVinfection may induce hypermethylation of p15^INK4B and p16^INK4A; HBV-induced hypermthylation of tumor suppression genes may be one of the mechanisms of HBV involved in hepatocellular carcinogenesis. Part Two Overexpression of DNA Methyltransferase Associated with Hepatitis B Virus Infection in Hepatocellular CarcinogenesisObjective Higher rate of p16^INK4A methylation was detected in the cancerous and cirrhotic tissues of HCC associated with hepatitis B virus infection (HBV). These signify that HBV infection is a factor promoting methylation of p16^INK4A. But little is known about the mechanism HBV induces the methylation of p16^INK4A. It is known that the process of DNA methylation is governed by the interaction of trans-acting enzymes known as DNA methyltransferases. Thus, we hypothesized that HBV may promote the hypermethylation of p16^INK4A via inducing the expression of DNMT.Methods We assessed DNMT mRNA in 44 cases of cancerous tissues, and matched cirrhotic and non-cancerous liver tissues of HCC with different HBV infection status, tumor stage and differentiation by Cybr Green dye real time PCR. The relationship between the levels of DNMTs and hypermethylation of p16^INK4A gene was also evaluated. Data are mean±SEM. In the human studies, the Mann-Whitney unpaired test was used to compare groups. Otherwise, statistical significance was estimated with Student's t test.Results Mean level of four kinds of DNMTs mRNA were all elevated in cancerous and cirrhotic tissues than that in non-cancerous tissues, at least one kind of DNMTs was increased in every case of HCC.But, it was selectively increased in cirrhotic tissues. Some cases with increased DNMT1 or DNMT2, some with DNMT3A or 3B, and some may be with randomly increased DNMTs among the four kinds of DNMTs. The situations of individual DNMT were described in the following.The level of mRNA for DNMT1 was elevated in 24/44 (54.5%) of cancerous and 11/35 (31.4%) of cirrhotic tissues compared with the level in corresponding non-cancerous liver tissues. (p=0.009 for cancerous tissue, p=0.02 for cirrhotic tissue). The average level of mRNA for DNMT2 in cancerous and cirrhotic tissues of HCC was no significantly higher than in the corresponding non-cancerous liver tissues (p=0.12 for cancerous and p=0.35 for cirrhotic tissues). The status of DNMT3A was similar to that of DNMT1.It elevated in 30/44 (68.2%) of cancerous and 14/35(40%) of cirrhotic tissues compared with the level in the corresponding non-cancerous liver tissues (7/35, 20%)(p=0.005 for cancerous and 0.01 for non-cancerous tissues). The level of mRNA for DNMT3B was elevated in 17/44 (38.6%) of cancerous and 9/35(25.8%) of cirrhotic tissues compared with the level in the corresponding non-cancerous liver tissues (p=0.03 for cancerous and 0.036 for cirrhotic tissues). Generally, from non-cancerous tissues to cancerous tissues, not only the mRNA level of DNMTs was gradually increasing, the kind of DNMTs also increase from 1 or 2 kinds in noncancerous tissue to 2 or 3 kinds in cancerous tissue, and even more. No significant differences were detected in level of DNMTs mRNA among different gender, tumor staging and differentiation (p=0.23 for DNMT1, p=0.45 for DNMT2, p=0.32 for DNMT3A and p=0.36 for DNMT3B), an increasing tendency of DNMTs were seen in male, poorly differentiation and high stage of tumors.In HBV-associated cancerous tissues, mRNA level of DNMT1, 3A and 3B were more higher than in non-HBV-associated cancerous tissues (p= 0.001 for DNMT1, 0.006 for DNMT3A and 0.02 for DNMT3B); In the HBV-associated cirrhotic tissues, mRNA level of DNMT1, 3A and 3B were higher than non-HBV-associated cancerous tissues (p=0.007 for DNMT1, 0.008 for DNMT3A and 0.045 for DNMT3B); Furthermore, in non-cancerous tissues, mRNA level of DNMT1 and DNMT3A in HBV-associated samples were significantly higher than in the non-HBV-associated (p=0.01 for DNMT1, for 0.04 DNMT3A). mRNA level of DNMT2 in all tissue(p>0.05), DNMT3B in HBV-associated noncancerous tissues were not found markedly higher than in non-HBV-linked tissues (p= 0.76). Levels of mRNA for DNMT1, 2,3A and 3B in Hep3B cell were 2.0, 1.2, 3 and 1.8 fold higher than in HepG2 cell. Correlations analysis showed that there were significant associations between HBV infection and over overexpression of DNMTs and p16^INK4A methylation (p=0.009 for DNMT1, 0.006 for DNMT3A, 0.03 for DNMT3B).Conclsuion Overexpression of mRNA for DNMTs and high rate of p16^INK4A methylation were not only detected in the HBV-associated cancerous tissues, but also in HBV-associated cirrhotic tissues, and even in histologically normal tissues. Moreover, there was a close correlation between DNMTs and p16^INK4A methylation.all the data suggest that persistent HBV infection can stimulate the overexpression of DNMTs, especially DNMT1, 3A and 3B,which, in turn, may result in the hypermethylation/inactivation of p16^INK4A, then indirectly involve in the process of hepatocellular carcinogenesis. Part Three p16^INK4A promoter hypermethylation in the plasma DNA and its possible application in molecular diagnosis of hepatocellular carcinomaObjective To investigate the tumor-associated p16^INK4A promoter hypermethylation in the plasma of patients with hepatocellular carcinoma (HCC) and its possible clinical application in molecular diagnosis of HCC.Methods Tumor tissue DNA was isolated with phenol and chloroform method. The plasma DNA with Qiagen blood DNA isolation kit was manipulated according to the manufactory manual. The isolated DNA was converted with bisulfite, and purified again, then used as template for methylation specific PCR. The diagnosis efficiency of p16^INK4A promoter hypermethylation was compared with that ofα-fetoprotein.Results p16^INK4A promoter hypermethylation was found in 31 tissues of 44 HCCs (70.5%) using methylation-specific PCR. Among the 31 cases with aberrant methylation in their tumor tissues, similar changes were also detected in the plasma samples of 90.5% (29 of 31) of the cases. No methylated p16^INK4A products were detected in the peripheral plasma of the 13 cases without these changes in the tumor tissue.methylated product of p16^INK4A were found in blood of 5.5% patients with hepatititis and 23.9% with cirrhotic liver.The sensitivity, specifity, accurate rate, positive predictive value and negative predictive value of p16^INK4A methylation detection in HCC were higher than that of AFP .There was no correlation between p16^INK4A methylation and AFP.Conclusion The high rate of p16^INK4A promoter methylation in liver cancer tissue and corresponding plasma of the same group of patients indicates that the detection of p16^INK4A methylation in plasma may be a potential independent molecular diagnostic factor of HCC. Part Four Relations between gene hypermethylation ,hepatitis B virus infection and plasma homocysteine in liver diseasesObjective Homocysteine is an intermediate in methionine metabolism, which takes place mainly in the liver. Impaired liver function leads to altered methionine and homocysteine metabolism; the liver plays a central role in the synthesis and metabolism of homocysteine, the purpose of this study is to investigate the Relations between gene hypermethylation ,hepatitis B virus infection and plasma homocysteine in different liver diseases.Methods total serum Hcy (tHcy protein-bound and free Hcy) determinations were as detected with Axsym system of ABBOT company. Data are mean±SEM. the Mann-Whitney unpaired test was used to compare groups. Otherwise, statistical significance was estimated with Student's t test.Results: T-hcy levels were gradually increased in patients with HBV carrier, chronic liver disease, liver cirrhosis and HCC. With exception of chronic hepatitis(11.83±3.39) and HBV carrier(8.84±2.74),the mean value for serum Hcy was significantly higher in the cirrhotic(14.46±3.51)and liver cancer(16.78±4.40)patients,μmol /L than in healthy control group(8.32±2.25) (p=0.008 for liver cirrhosis,p=0.000 for HCC ).T-hcy level in HBV-associated cirrhotic and cancer patients was significant higher than in the corresponding non-HBV- associated groups (p=0.023 for liver cirrhosis,p=0.003 for HCC).Grouping based on the methylation status of the detected tumor suppression genes, T-hcy level cirrhotic and cancer patients with methylated products of tumor suppression genes was significant higher than in the corresponding the groups without methylated products of tumor suppression genes (p=0.004 for liver cirrhosis,p=0.000 for HCC).Conclusion our observations suggest that (1) impaired liver function could be a novel determinant in the development of hyperhomocysteinemia and (2) a role for elevated homocysteine levels in the development of liver isease.(3) gene methylation and heptatitis B virus may result in the increase of plasma total homocysteine.