X连锁凋亡抑制蛋白对肺癌细胞生长和细胞周期的影响

为了研究靶向X连锁凋亡抑制蛋白的发夹状RNA对肺癌细胞中抗肿瘤作用,构建XIAP基因的shRNA表达载体,并设计阴性对照(psiRNA-Con)质粒,分别转染A549细胞;实时定量PCR和免疫印记法分别检测XIAP的mR-NA和蛋白的表达;四甲基偶氮唑盐试验(MTT)检测A549细胞的增殖;流式细胞术检测细胞周期分布。实验结果表明:转染psiRNA-XIAP组细胞XIAP mRNA和蛋白水平均低于正常对照组,生长慢于正常组(t=16.82和t=12.13,均P<0.01),G2/M期细胞所占比例增高(t=3.78,P<0.05),并出现亚G1峰。而阴性对照质粒psiRNA-Con未能下调XIAP的表达水平,对A549细胞增殖和细胞周期亦无明显影响。结论:针对XIAP的RNA干扰质粒特异性地抑制了其RNA和蛋白水平,使肺癌细胞A549生长减慢,诱导凋亡,并使其发生G2/M期阻滞,有望发展为新的抗肿瘤药物。

Effects of XIAP shRNA Mediated by PsiRNA-hH1 Vector Plasmid in Lung Cancer Cell Proliferation and Cell Cycle Distribution

Objectve:To explore the anti-effects of shRNA targeting X-linked inhibitor-of-apoptosis protein(XIAP) in lung cancer cell.Methods:Two kinds of shRNA plasmid were constructed,one of which was designed targeting XIAP,named psiRNA-XIAP,the other targeting nothing as negative-control named psiRNA-Con.Both of them were transfected into A549 cells respectively.Real-time PCR and Western-blotting were applied to detect the expression of XIAP.The ability of cell proliferation was analyzed with MTT assay whereas cell cycles were observed by Flow Cytometry.Results: Compared with the normal group,either the XIAP mRNA or protein level of A549 cells expressing psiRNA-XIAP was significantly lower than that of the normal group(t=16.82 and t=12.13,both P<0.01).Cell proliferation ability was inhibited by shRNA generating by psiRNA-XIAP.In addition,A549 cell showed a strong G2/M arrest and apoptosis after transfected psiRNA-XIAP(t=3.78,P<0.05).However,this phenomenon was not observed in A549 cells transfected psiRNA-Con.Conclusion:shRNA targeting at XIAP efficiently decreases its expression,inhibits cell growth and induces cell cycle arrest and apoptosis.