RNA干扰技术对肝癌细胞内源survivin基因表达的影响

RNA Interference-mediated Inhibition of Endogenous Survivin Expression in Hepatocarcinoma Cell

应用RNA干扰技术(RNAi)研究针对凋亡抑制因子survivin的siRNA抑制肝癌细胞株内源survivin基因的表达.转染重组质粒pshRNA survivin至肝癌细胞株SMMC 772 1,通过免疫荧光、蛋白质印迹和半定量RT PCR检测survivin蛋白表达及mRNA转录水平的变化.结果表明:构建的三种重组质粒pshRNA survivin1/2/3均能明显抑制survivin基因的表达;应用免疫荧光检测survivin基因的表达,转染重组质粒pshRNA survivin的实验组survivin荧光强度明显低于转染载体pTZU6 +1和pshRNA GFP对照组;蛋白质印迹结果表明,重组质粒pshRNA survivin明显抑制survivin蛋白的表达,抑制率为 6 2 %～ 78%,通过半定量RT PCR检测到survivin基因mRNA转录明显减少,抑制率为5 7%～ 6 4 %.由上述结果可以得出结论:重组质粒pshRNA survivin可明显抑制SMMC 772 1细胞内源survivin的表达和mRNA的转录。

To apply the small interfering RNAs targeting survivin to inhibit expression of endogenous survivin gene in human hepatocarcinoma cell SMMC-7721, the recombinant plasmid pshRNA-survivins were transfeetted into SMMC-7721. The expression level of survivin was determined by Western blot and immunofluorescence staining the transcription of survivin gene was detected by semi-quantitative RT-PCR. The introduction of plasmids pshRNA-survivin was showed to efficiently and specifically inhibit the expression of survivin according to results of Western blot and immunofluorescence staining, with inhibitory rates at 62% similar to 78% peaking 72 h. Semi-quantitative RT-PCR showed that mRNA transcription of survivin gene was reduced by nearly 57% similar to 64%. On the contrast, the control plasmid did exhibit no inhibitory effect on the protein expression and mRNA transcription of survivin gene. The results demonstrate that the small interfering RNA targeting survivin gene shows dramastic inhibition on protein expression and RNA transcription.