OBJECTIVETo explore the relationship among HBV-associated histopathological indexes, x gene mutations and the methylation status of p16INK4A promoter in liver with chronic hepatitis B virus infection, in order to illustrate their role in p16INK4A hypermethylation and HCC progression.

METHODSTwenty-three cases of surgically resected HBV-associated hepatocellular carcinoma and twenty-five fine needle aspiration biopsy cases of chronic hepatitis B were chosen for this study. The methylation status of the p16INK4A promoter in tumors, their corresponding peritumoral samples and chronic hepatitis B cases was determined by methylation-specific polymerase chain reaction (MSP). EnVision two-step immunohistochemical staining showed the expression of viral antigens in situ. Tissue HBV DNA levels were determined by real-time fluorescence quantitative PCR. Polymerase chain reaction and the direct sequencing method was used for mutation analysis of HBV x gene.

RESULTSIn peritumoral samples (P = 0. 025) and chronic hepatitis B cases (P = 0.029), the expression of HBx protein in methylated groups was all significantly higher than that in unmethylated groups of p16INK4A gene. But in tumors, there was no such significant difference. Other HBV antigens including HBsAg and HBcAg, tissue HBV DNA levels and point mutations of HBV x gene did not show a relationship with the methylation status of p16INK4A gene.

CONCLUSIONThe data suggest that p16INK4A hypermethylation correlated closely with higher HBx expression in precancerous lesions. HBx may play an important role in the early stage of HBV-associated hepatocarcinogenesis via induction of hepermethylation of p16INK4A promoter.