Effects of HBV X protein on expression and promoter methylation of p16 tumor suppressor gene

Objective To 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). Methods Experiments were performed in the human hepatoblastoma cell line Hep G2,Hep G2 cells expressing green fluorescent protein( Hep G2 / GFP),and Hep G2 cells stably expressing GFP- HBx fusion protein( Hep G2 / GFP- HBx).Western blot was used to determine the expression levels of the p16 protein in Hep G2 cells,Hep G2 / GFP cells,and Hep G2 / GFP- HBx cells. Hep G2 / GFP- HBx cells were treated with a universal inhibitor of DNA methyltransferase( DNMT),5- aza- 2'- deoxycytidine( 5-aza- 2'- d C). Methylation- specific polymerase chain reaction( MSP) was used to determine the promoter methylation of the p16 tumor suppressor gene in Hep G2 cells,Hep G2 / GFP cells,and Hep G2 / GFP- HBx cells treated with or without 5- aza- 2'- d C. Multiple- group comparison was made by analysis of variance. Results According to the results of Western blot,Hep G2 / GFP- HBx cells had a significantly lower expression level of the p16 protein than Hep G2 cells and Hep G2 / GFP cells( P = 0. 0007; P = 0. 0014); there was no significant difference in the expression level of the p16 protein between Hep G2 / GFP and Hep G2 cells( P > 0. 05). The MSP assay revealed partial Cp G methylation in the p16 promoter region in Hep G2 / GFP- HBx cells. No promoter methylation was detected in Hep G2 cells or Hep G2 / GFP cells. Non- methylation in the p16 promoter region was restored in Hep G2 / GFP- HBx cells treated with 5- aza- 2'- d C. Conclusion In 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'- d C,restores non- methylation in the p16 promoter region. The reversible modification provides new insights for the treatment and prevention of HBV- associated HCC.