Effects of HBV X protein on expression and promoter methylation of p16 tumor suppressor gene / 临床肝胆病杂志

ABSTRACT

ObjectiveTo explore the effects of hepatitis B virus X protein (HBx) on the expression and promoter methylation of the p16 tumor suppressor gene, and to investigate the epigenetic role of HBx in the development and progression of hepatitis B virus (HBV)-associated hepatocellular carcinomas (HCC). MethodsExperiments were performed in the human hepatoblastoma cell line HepG2, HepG2 cells expressing green fluorescent protein (HepG2/GFP), and HepG2 cells stably expressing GFP-HBx fusion protein (HepG2/GFP-HBx). Western blot was used to determine the expression levels of the p16 protein in HepG2 cells, HepG2/GFP cells, and HepG2/GFP-HBx cells. HepG2/GFP-HBx cells were treated with a universal inhibitor of DNA methyltransferase (DNMT), 5-aza-2'-deoxycytidine (5-aza-2'-dC). Methylation-specific polymerase chain reaction (MSP) was used to determine the promoter methylation of the p16 tumor suppressor gene in HepG2 cells, HepG2/GFP cells, and HepG2/GFP-HBx cells treated with or without 5-aza-2′-dC. Multiple-group comparison was made by analysis of variance. ResultsAccording to the results of Western blot, HepG2/GFP-HBx cells had a significantly lower expression level of the p16 protein than HepG2 cells and HepG2/GFP cells (P=0.0007; P=00014); there was no significant difference in the expression level of the p16 protein between HepG2/GFP and HepG2 cells (P＞0.05). The MSP assay revealed partial CpG methylation in the p16 promoter region in HepG2/GFP-HBx cells. No promoter methylation was detected in HepG2 cells or HepG2/GFP cells. Non-methylation in the p16 promoter region was restored in HepG2/GFP-HBx cells treated with 5-aza-2′-dC. ConclusionIn the hepatoblastoma cell line, HBx down-regulates the expression of the p16 tumor suppressor gene by inducing methylation in its promoter region. The DNMT inhibitor, 5-aza-2′-dC, restores non-methylation in the p16 promoter region. The reversible modification provides new insights for the treatment and prevention of HBV-associated HCC.