survivin启动子调控siRNA真核表达载体的构建及对HeLa细胞中stathmin基因的干涉作用

The interferencing effects of survivin promoter-directed siRNA on stathmin gene transcription human HeLa cells

目的:探讨survivin基因启动子调控的siRNA真核表达载体对HeLa细胞中stathmin基因表达的肿瘤特异性封闭作用。方法:合成针对人stathmin基因的siRNA cDNA寡核苷酸链退火形成双链,连接入经BamHI和HindIII双酶切后的pSilencer4.1-CMV neo真核表达载体。PCR扩增suvivin基因启动子并测序,用EcoRI和BamHI分别双酶切,连接至经相同内切酶消化的载体,获得survivin启动子调控的siRNA真核表达载体,酶切及测序鉴定。脂质体法转染重组质粒入HeLa细胞,G418筛选。RT-PCR扩增stathmin基因,检测其对stathmin基因表达的干涉效果;流式细胞仪分析转染后HeLa细胞增殖周期的改变。结果:经酶切及测序鉴定,成功构建重组质粒;RT-PCR结果显示所构建的干涉载体可有效封闭stathmin基因表达;流式细胞仪分析结果显示,Hale细胞在stathmin siRNA作用下G2/M期细胞的比例明显增加。结论:survivin基因启动子调控的siRNA真核表达载体可有效地封闭HeLa细胞中stathmin基因表达并使HeLa细胞阻断于G2/M期,为以stathmin基因为靶点的恶性肿瘤生物治疗奠定了理论基础。

Objective：To evaluate the interferencing effects of stathmin gene siRNA driven by survivin gene promoter in HeLa cell line. Methods： Double chain siRNA for stathmin gene were obtained through annealing of ohgonucleotide of siRNA, then cloned into pSilencer4.1 - CMV vector together with PCR derived survivin gene promoter to construct the recombinant eukaryotic expression vectors ： pSilencer4.1 - surp neo. The recombinant vectors were identified by enzyme digestion analysis and DNA sequencing. Then HeLa cells were transfected with pSilencer4.1 - surp neo and pSilencer4.1 -CMV negtive control ,then subjected to G418 selection. In G418 -resistant cells, the interferencing effect was detected by RT - PCR and cell cycle was detected by flow cytometry. Results ： Enzyme digestion analysis and DNA sequencing showed that survivin promoter - directed siRNA on stathmin gene were cloned into pSilencer4.1 - CMV neo vector. The result of RT - PCR indicated that the transcription of stathmin gene was suppressed by siRNA. Flow cytometry test found that cells of G2/M stage increased significantly. Conclusion： The transcription of stathmin gene is inhibited effectively by the constructed RNAi eukaryotic expression vectors in HeLa cells.