Osteopontin特异性siRNA真核表达载体的构建及其在GC9811胃癌细胞中沉默效应的鉴定

Construction of eukaryotic expression vector of siRNA specific for osteopontin and characterization of its efficiency in GC9811 gastric carcinoma cell line

目的:构建骨桥蛋白(OPN)特异性siRNA(smallin terfereRNA)真核表达载体,体外观察对OPN基因的沉默作 用.方法:采用基因克隆技术,将合成的特异性OPNRNA干 扰寡核苷酸序列插入真核表达载体mU6pro,构建OPNsiRNA 真核表达载体.以脂质体法将mU6pro空载体和2个 (mU6pro/OPN siRNA1和mU6pro/OPN siRNA2)重组质粒分 别导入具有高转移特性的GC9811胃癌细胞系.72h后用 RT PCR和Westernblotting技术检测各实验组胃癌细胞内 OPNmRNA及蛋白水平的表达情况.结果:成功构建OPN siRNA真核表达载体.转染OPN siRNA的胃癌细胞,72h后 OPNmRNA及蛋白表达下调,而以mU6pro/OPN siRNA1的抑 制效果更为明显.结论:构建的RNA干扰真核表达载体能明 显干扰OPNmRNA及蛋白的表达,为将其进一步应用于胃癌 的治疗研究奠定了基础.

AIM: To construct the small interfering RNA (siRNA) eukaryotic expression vector specific for human OPN gene and to observe its silencing effect on OPN gene. METHODS: The expression vectors of mU6pro/OPN-siRNA1 and mU6pro/OPN-siRNA2 were constructed by gene recombination and then were transfected by liposome-mediated transfection into the GC9811 gastric carcinoma cell line with high metastasis potential recently established in our laboratory. At 72 h after transfection, the expression of OPN in the levels of mRNA and protein was detected by RT-PCR and Western-blotting. RESULTS: The eukaryotic expression vectors of mU6pro/OPN-siRNA1 and mU6pro/OPN-siRNA2, which down-regulated mRNA and protein of OPN at 72 h after transfection, were successfully constructed. mU6pro/OPN-siRNA1 was more effective than the other one. CONCLUSION: Eukaryotic expression vector of siRNA specific for OPN is successfully constructed, which lays the basis for its application in the treatment of gastric carcinoma.