存活素siRNA表达质粒的构建及其对MCF-7细胞细胞周期和增殖的调控(英文)

Construction of Survivin siRNA Expression Vector and Its Regulation on Cell Cycle and Proliferation in MCF-7 Cells

背景与目的存活素特异地在肿瘤组织中过表达,许多报道表明抑制它的功能对肿瘤治疗有利。RNA干扰被证明是一项能有效而特异地抑制基因表达的新技术。本研究的目的在于构建存活素基因的小分子干扰RNA(siRNA)表达质粒,并检测该质粒在乳腺癌细胞中的生物学功能。方法应用含有U6启动子的mU6pro载体构建存活素siRNA质粒,RT-PCR和Westernblot法检测该质粒转染MCF-7细胞后存活素表达的变化,流式细胞仪和MTT法分别检测其对细胞周期和对细胞增殖的影响。结果存活素siRNA质粒明显下调MCF-7细胞中存活素的表达,阻断细胞周期在G1期,显著抑制细胞增殖,促进细胞凋亡。结论通过RNA干扰,存活素的表达在mRNA和蛋白质水平上被下调。

BACKGROUND & OBJECTIVE: Survivin is specifically overexpressed in tumor tissues. Many reports have shown that the abrogation of its functions is useful for tumor therapy. RNA interference is a new technique that proved to be effective for suppressing gene expression. The aim of this study was to construct a siRNA expression plasmid against gene survivin, and then to assess its functions on breast cancer cells MCF 7. METHODS: A survivin siRNA plasmid was constructed using a mU6pro vector contained U6 promotor, the expression change of survivin was examined by semi quantitative reverse transcription polymerase chain reaction (RT PCR) and Western blot analysis after the plasmid had been transfected into MCF 7 cells, and the effect of the plasmid on the cell cycle and cell proliferation was analyzed by flow cytometry and MTT method,respectively. RESULTS: Survivin siRNA plasmid knocked down survivin expression in MCF 7 cells obviously, arrested the cell cycle in G1 phase, inhibited the cell proliferation significantly, and promoted cell apoptosis in a tendency. CONCLUSION: Survivin expression was knocked down at mRNA and protein levels by RNAi.