RNA干涉抑制存活素表达并诱导HeLa S3细胞凋亡的研究

Inhibition of Survivin Expression and Induction of Apoptosis of HeLa S3 Cells by RNA Interference

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.

To study the functions of survivin gene further, three siRNA segments （S1, S2, S3） specifically targeting survivin were selected. Accordingly RNAi plasmids pTet-U6-S1, pTet-U6-S2, pTet-U6-S3 were constructed. The changes of survivin expression were examined by semi-quantitative reverse transcription polymerase chain reaction （RT-PCR） and Western-blot analysis after the plasmids had been transfected into HeLaS3. The results show that survivin expression is inhibited at both mRNA and protein levels after the plasmid pTet-U6-S3 has been transfected, which tai＇gets on the 3＇ non-encoding region of survivin. The inhibition level is much higher than that of S1 sequence reported previously. Compared with that of the control, the inhibition ratios of S1, S2, S3 to mRNA of survivin are 20% , 12. 5% and 40% respectively, and 39.2%, 17.0% and 58.6% to its protein. And the effect of the plasmid pTet-U6-S3 on the cell apoptosis was analyzed by flow cytometry. The apoptosis of HeLaS3 cells was greatly increased after transfection for 48 h.