FANCF-shRNA质粒构建及其对卵巢癌细胞OVCAR3沉默效应的观察

Construction of shRNA expression plasmid vector targeting FANCF gene and silencing effect on the ovarian carcinoma cells OVCAR3

目的:构建针对人FANCF基因的特异性shRNA真核表达载体,体外观察siRNA对卵巢癌OVCAR3细胞系FANCF基因的沉默效应。方法:采用基因克隆技术,将设计合成的能表达短发夹RNA的寡核苷酸序列插入真核表达质粒载体pSilencerTM4.1-CMV-neo,构建能表达FANCF-shRNA的真核表达载体;转染人卵巢癌OVCAR3细胞,提取总RNA和蛋白,RT-PCR验证mRNA表达的变化,蛋白质印迹法验证蛋白表达的变化。结果:质粒酶切和测序证实成功构建了FANCF-shRNA的真核表达载体。与野生型OVCAR3相比,脂质体法转染卵巢癌OVCAR3细胞后,FANCFmRNA 24、48和72 h的表达水平与阴性控制组比较均下调,抑制率分别为(23.91±6.58)%(、51.23±5.86)%和(47.42±7.52)%;FANCF蛋白在244、8和72 h的表达水平与阴性控制组比较亦下调,抑制率分别为(16.28±5.46)%(、24.42±6.85)%和(36.05±8.26)%。证实FANCF-shRNA具有沉默OVCAR3细胞FANCF基因表达的效应。结论:siRNA-FANCF干扰可引起卵巢癌OVCAR3细胞系FANCF基因沉默,可能为卵巢肿瘤基础和临床研究提供一种有效的方法。

OBJECTIVE： To investigate the silencing effect of FANCF-specific siRNA on the human ovarian carcinoma cell lines OVCAR3, the short-hairpin RNA eukaryotic expression vector was constructed. METHODS： The FANCF gene-targeted hairpin shRNA was designed, synthesized,and inserted into psilencerTM4. 1-CMV-neo. The recombination vector was transfected into the human ovarian carcinoma cell lines OVCAR3 by Lipofectamine^TM 2000, mRNA and protein expression were detected by RT-PCR and western blotting. RESULTS： Restricted enzyme digesting and sequencing confirm the proper sequences of shRNA among the eukaryotic expression vector. Compard with untransfected and nonspecific groups, mRNA and protein expression level of FANCF gene on the ovarian carcinoma cells OVCAR3 decreased by transfection of Lipofectamine^TM 2000 which confirmed that The FANCF gene-targeted hairpin shRNA could reduce the expression level of FANCF-gene. Compared with nonspecific groups, mRNA expression level decreased （23.91±6.58）%,（51.23±5.86） %, （47.42±7.52）% 24,48, 72 h after transfection, and protein expression level reduced （16.28±5.46）%,（24.42±6.85）% ,（36.05±8.26）%. CONCLUSION： siRNA interfering can induce the silencing of FANCF gene on the human ovarian carcinoma cell lines OVCAR3, which provide a possible methed for foundational and clinical ovarian cancer research.