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目的构建针对人Survivin基因的特异性短发卡RNA(shRNA)真核表达载体,并观察其在人脑胶质母细胞瘤U251细胞中对Survivin基因表达的抑制作用。方法根据SurvivincDNA编码序列,设计并合成针对Survivin基因的特异性RNA干涉片断,将其克隆入pWH1质粒载体,构建Survivin基因shRNA真核表达载体pWH1SR。通过脂质体介导将pWH1SR载体和空载体pWH1分别转染入U251细胞,经G418筛选,建立稳定转染的细胞系U251SR和U251P。采用RTPCR、Westernblot和免疫组化方法检测SurvivinmRNA和蛋白在U251、U251P和U251SR细胞中的表达水平。结果成功构建了Survivin基因shRNA真核表达载体pWH1SR,经酶切鉴定证实克隆正确。获得了稳定转染pWH1SR载体和空载体pWH1的细胞系U251SR和U251P。U251SR细胞SurvivinmRNA和蛋白表达均受到明显抑制,抑制率分别达87%和91%;U251P细胞Survivin基因表达水平无明显变化。结论特异性shRNA能够明显抑制Survivin基因在U251细胞中的表达,这为进一步研究Survivin在U251细胞中的生物学功能和作用机制奠定了实验基础。
Objective To construct a specific short hairpin RNA (shRNA) eukaryotic expression vector targeting human Survivin gene and observe its inhibitory effect on Survivin gene expression in human glioma U251 cells. Methods According to the coding sequence of Survivinc DNA, a specific RNA interference fragment targeting Survivin gene was designed and synthesized. The fragment was cloned into pWH1 plasmid vector to construct shRNA eukaryotic expression vector pWH1SR of Survivin gene. The pWH1SR vector and empty vector pWH1 were transfected into U251 cells by lipofectamine respectively, and then were selected by G418 to establish stable transfected cell lines U251SR and U251P. The expression of Survivin mRNA and protein in U251, U251P and U251SR cells was detected by RTPCR, Western blot and immunohistochemistry. Results The shRNA eukaryotic expression vector pWH1SR of Survivin gene was successfully constructed and confirmed by restriction enzyme digestion. The cell lines U251SR and U251P stably transfected with pWH1SR vector and empty vector pWH1 were obtained. U251SR cells Survivin mRNA and protein expression were significantly inhibited, the inhibition rates were 87% and 91%; U251P cells Survivin gene expression levels did not change significantly. Conclusion The specific shRNA can significantly inhibit the expression of Survivin gene in U251 cells, which will lay the foundation for the further study on the biological function and mechanism of Survivin in U251 cells.