prohibitin在乳腺癌MCF-7细胞表达抑制的鉴定

Effect of shRNA Expression Vectors for Prohibitin Transfected MCF-7 Cells in Vitro

目的 利用靶向prohibitin基因的shRNA表达质粒转染乳腺癌MCF-7细胞,进而观察prohibitin 在转染组细胞的表达情况.方法 将构建的prohibitin基因shRNA表达质粒用脂质体法转染人乳腺癌MCF-7细胞,通过荧光定量PCR和Western blot 检测其在mRNA及蛋白水平的干扰效应.结果 转染Ⅰ组、转染Ⅱ组、转染Ⅲ组细胞PHB mRNA表达量分别是阴性对照组的27.2%,45.6%和29.4%;空白对照组的23.7%,39.7%和25.6%;转染Ⅰ组、转染Ⅱ组、转染Ⅲ组细胞PHB/β-actin灰度值比值分别是阴性对照组的27.4%,39.4%和30.1%;空白对照组的26.9%,38.7%和29.5%.构建的shRNA表达载体可以使MCF-7细胞中prohibitin基因的mRNA及其蛋白含量降低.结论 构建的针对prohibitin基因的shRNA表达载体,转染MCF-7细胞后可有效抑制细胞中prohibitin的表达,证实构建有效.

Objective To observe the effect of shRNA expression vectors for prohibitin transfeeted MCF-7 cells in vitro. Methods After transfeetion with shRNA eukaryotie expression vectors,prohibitln mRNA and protein levels was analysed using Real-time PCR and Western blot. Results After transfected 48 h,the mRNA and protein level of PHB had no changes in MCF-7/pGenesil-non cells and MCF-7 cells, but significantly decreased in MCF-7/pGenesil- PHBI, MCF-7/pGenesil- PHB2 and MCF-7/pGenesil-PHB3,which indicated PHB was down-regulated in MCF-7/pGenesil-PHB], MCF-7/pGenesil- PHB2 and MCF-7/pGenesil- PHB3. Conclusion The shRNA expression vector can effectively inhibit the expression of prohibitin in the MCF-7 cells.