乙型肝炎病毒X蛋白与p16INK4A启动子甲基化及甲基转移酶的相关性的体内研究

Purpose: The aim of the present study was to authenticate the involvements of DNA methyltransferases (DNMTs) and methyl-CpG binding domain protein 2 (MBD2) in the process of HBx induced p16INK4A promoter hypermethylation in HBV-related hepatocellular carcinoma (HCC) and their corresponding non-cancerous liver tissues.Methods: Eighty-eight fresh tissue specimens of surgically resected HBV-associated HCC and their corresponding non-cancerous liver tissues as well as some HBV-negative tissues were studied. The methylation status of p16INK4A promoter was determined by methylation-specific polymerase chain reaction (MSP). Reverse transcription and real-time polymerase chain reaction (real-time RT-PCR) showed the expression of DNMTs, MBD2, and HBx. Western blot and immunohistochemistry were used for the protein analysis of HBx, DNMT1, DNMT3A and p16. Tissue HBV-DNA levels were determined by real-time PCR. HBV genotype was examined by nested PCR and restriction fragment length polymorphism (RFLP). Results: In the corresponding non-cancerous liver tissues of HBV-associated HCC, Higher HBx expression associated with hypermethylation of p16INK4A promoter. HBx was positively correlated with DNMT1, DNMT3A in both mRNA and protein levels. Furthermore, HBx, DNMT1 and DNMT3A protein expression were negtively correlated with p16 protein expression. In HBV-associated HCC tissues, HBx was positively correlated with DNMT1, DNMT3A in both mRNA and protein levels, however, HBx expression didn't correlate with hypermethylation of p16INK4A promoter or p16 protein expression. Methylation status of p16INK4A promoter didn't correlate with clinic-pathologic characteristics.Conclusions: DNMT1 and DNMT3A may play important roles in the process of HBx inducing hypermethylation of p16INK4A promoter in the early stage of HBV-associated HCC. HBx-DNMTs-p16INK4A promoter hypermethylation-decreased expression of p16INK4A may suggest a mechanism for tumorigenesis during HBV-associated hepatocarcinogenesis.