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

目的：构建骨桥蛋白(OPN)特异性siRNA(small interfere RNA)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OPN siRNA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Western blotting技术检测各实验组胃癌细胞内OPN mRNA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OPN mRNA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OPN mRNA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。

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

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.