【摘要】 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。更多还原

【Abstract】 Objective: To investigate the influences of hepatitis C virus(HCV) proteins NS3, Core and NS5 A on RASSF2 mRNA expression and methylation status of RASSF2 A promoter in hepatic cells.Methods: The RASSF2 mRNA expressions in hepatic cells respectively transfected with NS3-, Core-and NS5 Aexpression plasmids(NS3/QSG7701, Core/QSG7701 and NS5A/QSG7701) as well as normal hepatic cell line L02 were determined by RT-PCR method. The methylation statuses of RASSF2 A promoter in NS3/QSG7701, Core/QSG7701 and NS5A/QSG7701 cells were examined by methylation-specific PCR, and the changes in their RASSF2 mRNA expressions and biological behaviors after treatment with demethylation agent 5-aza-d C were observed. Results: The RASSF2 mRNA expressions were significantly decreased in all NS3/QSG7701, Core/QSG7701 and NS5A/QSG7701 cells compared with L02 cells(all P<0.05), and the RASSF2 A promoters were fully methylated in all the three types of cells. After 5-aza-d C treatment, the RASSF2 mRNA expression was upregulated in NS3/QSG7701 and Core/QSG7701 cells(both P<0.05), but did not change in NS5A/QSG7701 cells(P>0.05); the proliferation rate was decreased and apoptosis rate was increased in NS3/QSG7701 and Core/QSG7701 cells5-aza-d C treatment(both P<0.05).Conclusion: NS3, Core and NS5 A can reduce RASSF2 expression through RASSF2 A promoter methylation and also through other mechanism by NS5 A. This effect may probably contribute to the carcinogenesis of HCVassociated hepatocellular carcinoma.更多还原