磷脂爬行酶1对干扰素抑制乙型肝炎病毒(HBV)作用的影响

Effect of PLSCR1 on the Antiviral Activity of IFN against HBV in HepG2 Cells

研究磷脂爬行酶1(Phospholipid scramblase 1,PLSCR1)对干扰素抑制HBV作用的影响。设计合成PLSCR1特异性小干扰RNA(siRNA),以完全随机序列的阴性小干扰(NCsiRNA)作为对照,转染HepG2细胞,于转染48h后分别检测PLSCR1mRNA和蛋白水平表达量的变化,筛选出对PLSCR1具有沉默作用的siRNA;将HepG2细胞分为正常对照组和干扰素处理组,将1.3倍乙型肝炎病毒(HBV)全基因真核细胞表达载体HBV1.3质粒分别与PLSCR1siRNA或NCsiRNA共同转染HepG2细胞或干扰素处理的HepG2细胞,转染48h后检测各组细胞中PLSCR1mRNA表达量及培养液上清中HBsAg表达水平。PLSCR1特异性小干扰RNA siRNA911转染后能够显著抑制HepG2细胞中PLSCR1基因在mRNA和蛋白水平的表达;与HepG2细胞对照组比较,干扰素处理组细胞转染HBV1.3质粒、NCsiRNA+HBV1.3质粒后,细胞培养液中HBsAg表达水平均显著降低(P<0.05);而PLSCR1siRNA与HBV1.3共转染IFN处理的HepG2细胞组与共转染HepG2细胞组相比较,细胞培养液中HBsAg的表达水平没有显著差异。提示抑制PLSCR1的siRNA可抑制干扰素的抗HBV活性,提示PLSCR1在干扰素抑制HBV复制中具有重要作用。

To study the effect of interferon（IFN）against hepatitis B virus（HBV）by silencing phospholipid scramblase（PLSCR）1in HepG2 cells.siRNA specific for PLSCR1 was designed and transfected in HepG2 cells.The inhibitory effect of siRNA was determined using semi-quantitative polymerase chain reaction（PCR）and western blotting 48hpost-transfection.HepG2 cells treated with IFN were co-transfected with plasmids expressing HBV1.3and siRNA targeting PLSCR1.Total RNA of HepG2 cells was isolated and the mRNA level of PLSCR1 measured by reverse-transcription semi-quantitative PCR.The expression of HBsAg in culture supernatants was determined by enzyme-linked immunosorbent assay.Expression of PLSCR1 was inhibited by siRNA911 in HepG2cells.Compared with the control,the level of HBsAg decreased in the cell supernatants of cells transfected with HBV1.3plasmid or NC-siRNA ＋ HBV1.3plasmid.Compared with cells not treated with IFN,the level of HBsAg did not change significantly in the supernatants of cells transfected with siRNA ＋ HBV1.3plasmid and treated with IFN.Inhibition of PLSCR1 could decrease the antiviral activity of IFN against HBV.These data suggest that PLSCR1 has an important role in the inhibition of HBV replication due to IFN.