糖皮质激素受体αsiRNA真核表达载体的构建与鉴定

Construction and Identif ica tion of GRαsiRNA Eukaryotic Expression Vectors

目的构建糖皮质激素受体α(GRα)小干扰RNA(siRNA)的真核表达载体,并检测其抑制结肠癌细胞株Lovo中糖皮质激素受体α表达的效果。方法根据基因文库中GRα的cDNA序列,设计并合成两段siRNA,分别插入到pGenesil-1质粒中,并对重组质粒(命名为pGenesil-1/GRα1和pGenesil-1/GRα2)进行测序鉴定。脂质体介导下转染Lovo细胞株,RT-PCR和Western Blot检测转染后GRα的表达。结果成功构建GRαsiRNA真核表达载体(pGenesil-1/GRα1和pGenesil-1/GRα2);pGenesul-1/GRα2转染(脂质体法)Lovo细胞后,RT-PCR和Western blot检测细胞内GRα的表达显著降低。结论GRαsiRNA真核表达载体的成功构建为进一步研究GR在结肠癌化疗中的作用奠定了重要的实验基础。

Objective To construct eukaryotic expression vectors of siRNA tageting glucocoriticoid receptor α （GRα） ,and measure its inhibition efficiency in GRα expression of the human colon cancer cell line Lovo. Methods Two pairs of siRNA were designed and synthesized according to the GRαcDNA sequence in Genebank,and they were then inserted into pGenesil-1 plasmids respectively. The recombinants （ named pGenesil-1/GRαl and pGenesil-1/GRα2 ） were sequenced and identified. The vectors were transfected into Lovo cell line. RT-PCR and Western blot were performed to examine the expression of GRα. Results GRα siRNA eukaryotic expression vectors were successfully constructed. RT-PCR and Western blot analysis demonstrated that siRNA against GRα reduced the expression of GRα mRNA and protein after pGenesil-1/GRα2 transference. Conclusion Construction of GRα siRNA eukaryotic expression vectors lays an experimental foundation for the role of GR during the treatment of colon cancer.