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-dC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-dC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-dC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。
Influences of hepatitis C virus proteins on RASSF2 mRNA expression in hepatic cells and their mechanisms
Objective: To investigate the influences of hepatitis C virus (HCV) proteins NS3, Core and NS5A on RASSF2 mRNA expression and methylation status of RASSF2A promoter in hepatic cells. Methods: The RASSF2 mRNA expressions in hepatic cells respectively transfected with NS3-, Core- and NS5A-expression 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 RASSF2A 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-dC 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 RASSF2A promoters were fully methylated in all the three types of cells. After 5-aza-dC 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-dC treatment (both P<0.05). Conclusion: NS3, Core and NS5A can reduce RASSF2 expression through RASSF2A promoter methylation and also through other mechanism by NS5A. This effect may probably contribute to the carcinogenesis of HCV-associated hepatocellular carcinoma.