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目的:探讨丙型肝炎病毒(HCV)蛋白NS3、Core、NS5A对肝细胞RASSF2 mRNA表达及RASSF2A启动子甲基化的状态的影响。方法:用RT-PCR检测分别转染NS3、Core、NS5A表达质粒的肝QSG7701细胞(NS3/QSG7701、Core/QSG7701、NS5A/QSG7701)以及正常肝细胞L02中RASSF2 mRNA的表达;用甲基化特异性PCR检测NS3/QSG7701、Core/QSG7701及NS5A/QSG7701细胞中RASSF2A启动子甲基化的状态,以及去甲基化药物5-aza-d C处理后,各细胞RASSF2 mRNA表达情况与生物学行为的变化。结果:与正常肝细胞L02比较,NS3/QSG7701、Core/QSG7701、NS5A/QSG7701细胞中RASSF2 mRNA的表达均明显降低(均P<0.05);3种细胞的RASSF2A启动子均发生完全甲基化。经5-aza-d C处理后,RASSF2 mRNA的表达在NS3/QSG7701和Core/QSG7701细胞中明显上调(均P<0.05),但在NS5A/QSG7701细胞中无明显变化(P>0.05);NS3/QSG7701和Core/QSG770细胞经5-aza-d C处理后,增殖率下降、凋亡率增加(均P<0.05)。结论:NS3、Core、NS5A能通过RASSF2A启动子甲基化,且NS5A还通过其他机制降低RASSF2表达,该作用可能参与了HCV相关肝细胞癌的发生。
Objective: To investigate the effects of hepatitis C virus (HCV) proteins NS3, Core and NS5A on the expression of RASSF2 mRNA and the methylation of RASSF2A promoter in hepatocytes. Methods: The expression of RASSF2 mRNA in liver QSG7701 cells (NS3 / QSG7701, Core / QSG7701, NS5A / QSG7701) and normal liver cells L02 transfected with NS3, Core and NS5A plasmids respectively was detected by RT-PCR. The status of RASSF2A promoter methylation in NS3 / QSG7701, Core / QSG7701 and NS5A / QSG7701 cells was detected by sex PCR, and the expression of RASSF2 mRNA and the biological behavior of each cell after 5-aza-d C demethylation treatment The change. Results: Compared with normal L02 cells, the expression of RASSF2 mRNA in NS3 / QSG7701, Core / QSG7701 and NS5A / QSG7701 cells were significantly decreased (all P <0.05). The methylation status of RASSF2A promoter in all three cells was completely methylated. The expression of RASSF2 mRNA was significantly up-regulated in NS3 / QSG7701 and Core / QSG7701 cells after 5-aza-d C treatment (all P <0.05), but not in NS5A / QSG7701 cells (P> 0.05) After treated with 5-aza-d C, the proliferation rate and the apoptosis rate of QSG7701 and Core / QSG770 cells increased (all P <0.05). CONCLUSIONS: NS3, Core and NS5A can be methylated by RASSF2A promoter, and NS5A can reduce RASSF2 expression by other mechanisms, which may be involved in the development of HCV-related hepatocellular carcinoma.