HBx诱导p16^(INK4a)高甲基化在Chang细胞株恶性转化倾向中的作用

Hepatitis B virus X protein induces malignant transformation tendency of Chang liver cells involving p16^(INK4a) hypermethylation

目的:探讨乙型肝炎病毒X(HBx)蛋白对p16INK4a表达和甲基化的影响,以及其在肝细胞癌早期发生中的作用机制。方法:采用脂质体法将真核表达载体pcDNA3.1B-HBx和pcDNA3.1B质粒分别稳定转染Chang细胞,构建表达HBx的细胞株Chang-HBx和不表达HBx的细胞株Chang-vector;半定量RT-PCR和Western blot法分别在mRNA和蛋白质水平上检测Chang-HBx、Chang-vector和Chang细胞株中的p16INK4a表达;甲基化特异性PCR检测HBx对细胞中p16INK4a启动子甲基化的影响;CCK-8溶剂检测3种细胞株的增殖曲线,流式细胞仪观察3种细胞的细胞周期和细胞凋亡率,重复3次。结果:成功构建了Chang-HBx和Chang-vector稳定细胞株,Chang-HBx细胞生长速度增快,Chang-HBx细胞的S期平均比率明显高于Chang-vector细胞和Chang细胞(28.96%vs.21.53%,P<0.001;28.96%vs.21.5%,P<0.001),而且Chang-HBx细胞的细胞凋亡率平均值明显低于Chang-vector细胞和Chang细胞(2.71%vs.3.69%,P<0.001;2.71%vs.3.36%,P<0.001)。初步机制研究证实HBx蛋白介导肝细胞的p16INK4a高甲基化,进而导致p16INK4a在mRNA和蛋白水平上表达均下降。结论:HBx通过诱导p16INK4a高甲基化下调后者的表达水平,可能在Chang细胞的恶性转化倾向中发挥作用。

Objective：To study the relationship between hepatitis B virus X（HBx） protein and DNA methylation of p16INK4a and the role of HBx in the carcinogenesis of hepatocellular carcinoma.Methods：Eukaryonic expression vectors pcDNA3.1B-HBx and pcDNA3.1B were transduced into Chang liver cells by using Lipofectamine 2000 to establish the Chang-HBx liver cell line（HBx expression） and Chang-vector liver cell line（non-HBx expression）.RT-PCR and Western blot were used to test the expression of p16INK4a in the two cell lines.The level of p16INK4a promoter methylation was tested by methylation specific PCR（MSP）.The proliferation curves were drew by CCK-8,and S phase in cell cycle and apoptosis were observed by flow cytometry.Results：Hypermethylation of p16 can be mediated by HBx,which decreases the expression of mRNA and protein of p16.Chang-HBx cells grow faster.Chang-HBx cells have much higher S-phase population（28.96% vs.21.53%,P0.001;28.96% vs.21.5%,P0.001） and lower apoptosis rate（2.71% vs.3.69%,P0.001;2.71% vs.3.36%,P0.001） than Chang-vector cells and Chang cells respectively.Conclusion：p16INK4aexpression was repressed by HBx protein via DNA methylation of p16INK4a,which can induce the malignant transformation tendency of Chang cells.