靶向ER-α36的vshRNA真核表达慢病毒载体的构建、鉴定及其对胃癌细胞增殖的影响

目的:针对雌激素受体(ER)-α36基因的特异性靶序列,构建并鉴定靶向ER-α36基因RNAi慢病毒载体,研究ER-α36沉默后对胃癌细胞增殖的影响。方法:筛选确定的ER-α36基因RNAi有效靶序列,合成靶序列的Oligo DNA,与慢病毒载体(GV307)连接,测序鉴定。转染293T细胞,包装产生慢病毒,感染胃癌SGC7901细胞株。荧光显微镜下观察SGC7901感染后荧光表达情况,real-time PCR和Western blotting方法检测ER-α36的表达变化。用1×10-10mol/L的17β-雌二醇处理沉默ER-α36的SGC7901细胞,用细胞计数法观察细胞增殖能力的变化及检测相关下游信号通路分子Src、ERK1/2、cyclin D1表达的变化。结果:阳性克隆PCR及测序证明成功构建慢病毒载体LV-ER-α36-RNAi,倒置显微镜下观察LV-ER-α36-RNAi慢病毒载体感染率达80%以上。Real-time PCR和Western blotting方法证实四环素(Te T)诱导下LV-ER-α36-RNAi明显抑制SGC7901细胞内ER-α36 mRNA和蛋白质的表达。与对照组相比,沉默ER-α36的SGC7901细胞增殖能力减弱,Src、ERK1/2、cyclin D1蛋白表达明显降低,Src蛋白活化能力减弱(P0.05)。结论:我们构建的Te T诱导靶向ER-α36的vshRNA慢病毒载体LVER-α36-RNAi,可明显沉默ER-α36的表达,为研究ER-α36蛋白的作用机理提供实验依据,ER-α36与胃癌细胞等肿瘤细胞的增殖有关。

Construction of lentiviral vector-mediated siRNA knockdown of ER-α36and its action on gastric cancer cell growth

AIM: To construct a lentiviral vector for stable delivery of  the ER-α36gene and to detect its effect on SGC7901 cell growth. METHODS: The efficient RNAi targeting sequences identified for the ER-α36gene were screened. The Oligo DNA was synthesized with target sequences and annealed to form double-stranded DNA. Then it was digested by XhoI and EcoR I and connected with GV307 vector to produce LV-ER-α36-RNAi lentiviral vector. PCR was used to screen the positive clones and sequence. The LV-ER-α36-RNAi, pHelper 1.0 and pHelper 2.0 plasmids were co-transfected into 293T cells for producing lentiviral vector and infecting SGC7901 cell line. Fluorescence microscopy, real-time PCR and Western blotting were used to detect the transfection efficiency and gene silencing effect. 17β-estrodial at concentration of 1×10-10 mol/L was used to stimulate the recombinant cell line, and the action on the growth of gastric cancer cells and the expression of Src, ERK1/2 and cyclin D1 were determined. RESULTS: DNA sequencing analysis confirmed the identity of recombinant shRNA expression vectors. Immunofluorescence assay demonstrated that transfection efficiency was above 80%. Transfection of LV-ER-α36-RNAi significantly knocked down the expression of ER-α36 at mRNA and protein levels with tetracycline (TeT) simulating as revealed by real-time PCR and Western blotting. Compared with control group, the growth of the recombinant cell line declined and the expression of Src, ERK1/2 and cyclin D1 and the activation of Src decreased (P<0.05).CONCLUSION: Lentiviral vectors that silenceER-α36expression are constructed successfully and can be used to study the role of ER-α36 in gastric cancer. The ER-α36is related with many kinds of cancer cell growth, including gastric cancer cells.