survivin启动子驱动的shRNA在宫颈癌SiHa细胞中的特异性表达

研究survivin启动子驱动的小发夹RNA(shRNA)在宫颈癌SiHa细胞中的特异性表达.PCR扩增survivin启动子,构建以survivin启动子驱动增强绿色荧光蛋白(EGFP)表达的真核表达载体pEGFP-C1/Surv,并检测survivin启动子的活性;构建针对EGFP的shRNA真核表达载体pGenesil-1-EGFP-shRNA/U6,并检测shRNA在U6启动子驱动下的干扰效果;用有活性的survivin启动子取代pGenesil-1-EGFP-shRNA/U6载体中的U6启动子,从而获得新的shRNA真核表达载体pGenesil-1-EGFP-shRNA/Surv;将pGenesil-1-EGFP-shRNA/Surv转染到宫颈癌SiHa细胞及正常人脐静脉内皮HuVEC细胞,观察EGFP在两种细胞中的表达变化.结果表明,成功扩增survivin启动子并验证该启动子具有活性;成功构建EGFP shRNA真核表达载体pGenesil-1-EGFP-shRNA/U6,并验证shRNA在U6启动子驱动下具有较好的干扰效果;成功构建pGenesil-1-EGFP-shRNA/Surv载体,分别转染SiHa及HuVEC细胞,在SiHa 细胞中观察到较少的绿色荧光蛋白表达,而在HuVEC细胞中观察到较多的绿色荧光蛋白表达.survivin启动子能驱动shRNA在宫颈癌SiHa细胞中特异性表达,而在正常HuVEC细胞中不表达.

Specific Expression of shRNA Drived by Survivin Gene Promoter in SiHa Cells

It was to investigate the specific expression of shRNA drived by survivin gene promoter in SiHa cells. The survivin gene promoter was amplified by PCR and pEGFP-C1/Surv plasmid was constructed in which the EGFP was drived by survivin gene promoter, and the activity of survivin gene promoter was detected. shRNA eukaryotic expression vector pGenesil-1-EGFP-shRNA/U6 targeting EGFP was constructed,and the RNA interferencing effect of shRNA drived by U6 promoter was detected. shRNA eukaryotic expression vector pGenesil-1-EGFP-shRNA/Surv in which the U6 promoter was replaced by surviving gene promoter. SiHa and HuVEC cells were transfected by pGenesil-1-EGFP-shRNA/Surv plasmid and the expression of EGFP in two cell lines was observed . Results showed that the survivin gene promoter was amplified successfully and its activity was verified. The pGenesil-1-EGFP-shRNA/U6 plasmid was constructed successfully, and the RNA interferencing effect of shRNA drived by U6promoter was verified as good as expected. The pGenesil-1-EGFP-shRNA/Surv vector was constructed successfully and transfected into SiHa and HuVEC cell lines. After 72 h, there was less green fluorescence in SiHa cells, whereas there was more green fluorescence in HuVEC cells. Therefore,it proved that the survivin gene promoter can drive the specific expression of shRNA in SiHa cell line,which can not happened in normal HuVEC cell line.