Hepatitis B virus X protein promotes hypermethylation of p16(INK4A) promoter through upregulation of DNA methyltransferases in hepatocarcinogenesis.

Abstract The hepatitis B virus × 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 p16 INK4A promoter hypermethylation. Liver cell lines were stably transfected with HBx-expressing vector. The methylation status of p16 INK4A was examined by methyl-specific polymerase chain reaction (MSP) and bisulfite sequencing. Reverse transcription and real-time polymerase chain reaction (real-time RT-PCR), Western blot and immunohistochemistry were used to analyze the expression of HBx, HBx-mediated DNA methylation abnormalities and p16 INK4A . Some cases of HCC and corresponding noncancerous liver tissues were studied. HBx up-regulates DNMT1 and DNMT3A expression in both mRNA level and protein level, and HBx represses p16 INK4A expression through inducing hypermethylation of p16 INK4A promoter. Moreover, HBx induces hypermethylation of p16 INK4A promoter through DNMT1 and DNMT3A. Regulation of DNMT1 and DNMT3A by HBx promoted hypermethylation of p16 INK4A promoter region. HBx-DNMTs-p16 INK4A promoter hypermethylation may suggest a mechanism for tumorigenesis during hepatocarcinogenesis.