表达的siRNA对人乳腺癌SKBr-3细胞中survivin基因的干涉作用

The Interference Effect of Vector-based siRNA on Survivin Gene of Human Breast Cancer SKBr-3 Cells

目的:构建针对人survivin基因的siRNA真核表达载体,检测其对人乳腺癌SKBr-3细胞中survivin基因表达的干 涉作用。方法:将合成的寡核苷酸链退火形成双链,连接入经HindⅢ和BglⅡ双酶切后的pSUPER真核表达载体。对重组质 粒进行酶切分析和测序鉴定。通过脂质体介导,把重组质粒稳定转染入SKBr-3细胞,RT-PCR、Western blot印记杂交和细胞免 疫化学法检测其对mRNA和蛋白表达的干涉效果。结果:经酶切鉴定及基因测序证实,重组质粒中已插入目的基因片段。 RT-PCR显示两个干涉载体均抑制了目的基因的转录;Western blot印记杂交和细胞免疫化学检测结果显示pSUPER-S1对蛋 白表达的抑制作用更强。结论:成功地构建了针对人survivin基因的RNA干涉真核表达载体pSUPER-S1和pSUPER-S2,并在 人乳腺癌细胞株SKBR-3中有效发挥了对survivin基因的干涉作用。

Objective: To construct the eukaryotic expression vector for RNA interferencing human survivin gene and detect its interference effect in human breast cancer cell line(SKBr-3). Methods: Two target gene segments were synthesized and cloned into pSUPER vector respectively to construct two recombinant eukaryotic expression vectors: pSUPER-S1 and pSUPER-S2. The two recombinant vectors were identified by enzyme digestion analysis and DNA sequencing. Then SKBr-3 cells were transfected with pSUPER-S1 or pSUPER-S2, together with pCDNA3 plasmid, and subjected to G418 selection. In G418-resistant cells, the interference effect was detected by RT-PCR, Western blot and immunocytochemical staining. Results: Enzyme digestion analysis and DNA sequencing showed that the target segments were cloned into pSU PER vector respectively. The results of RT-PCR, Western blot and immunocytochemical staining indicated that both vectors could knock down the transcription and expression of survivin gene, and that pSUPER-S1 had better interference effect than pSUPER-S2. Conclusion: The transcription and expression of survivin gene were inhibited effectively by the constructed RNAi eukaryotic expression vectors in the breast cancer cells.