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选择3个(S1,S2,S3)靶向存活素基因的siRNA序列,构建相应的RNA干涉载体pTet-U6-S1,pTet-U6-S2,pTet-U6-S3,将它们分别转染HeLa S3细胞,采用RT-PCR和W estern印迹检测分析转染对HeLa S3细胞内源存活素表达的影响,结果表明,针对存活素3’端非编码区序列的干涉载体pTet-U6-S3转染细胞后,存活素的mRNA水平和蛋白水平均明显下调,抑制水平高于S1序列.与对照相比,S1,S2和S3片段对存活素mRNA的抑制率分别是20%,12.5%和40%,对存活素蛋白的抑制率分别是39.2%,17.0%和58.6%.流式细胞术检测结果表明,抑制存活素表达后,HeLa S3细胞凋亡比例显著提高.
Three (S1, S2, S3) siRNA sequences targeting the survivin gene were selected to construct the corresponding RNA interference vectors pTet-U6-S1, pTet-U6-S2 and pTet-U6-S3, which were transfected into HeLa S3 Cells were detected by RT-PCR and Western blot analysis of transfected HeLa S3 cells endogenous survivin expression results show that the survivin 3 ’non-coding region of the interference vector pTet-U6-S3 transfected cells , Survivin mRNA and protein levels were significantly downregulated, the inhibition level is higher than S1 sequence.Compared with the control, S1, S2 and S3 fragments of survivin mRNA inhibition rates were 20%, 12.5% and 40%, respectively, The inhibitory rates of Survivin protein were 39.2%, 17.0% and 58.6%, respectively.The results of flow cytometry showed that the percentage of apoptotic cells in HeLa S3 cells was significantly increased after survivin expression was inhibited.