利用RNA干扰抑制细丝蛋白A基因的表达

Inhibition of Expression of Filamin A Gene by RNA Interference

目的:构建细丝蛋白A(FLNa)基因的小干扰RNA(siRNA)表达载体,并观察其对FLNa基因表达的抑制作用。方法:利用RNA干扰(RNAi)技术设计并合成1条针对FLNa的siRNA,将其克隆到siRNA表达载体pSilencer4.1-CMV-hygro中;将重组质粒pSilencer-FLNa、pSilencer-negative(阴性对照)转染293T人胚肾细胞,通过Western印迹检测FLNa的表达;通过潮霉素筛选建立干扰FLNa表达的前列腺癌细胞。结果:PCR鉴定证明构建了FLNa基因RNAi载体;Western印迹表明构建的FLNa基因干扰载体能够有效地抑制FLNa基因的表达;建立了稳定干扰FLNa表达的前列腺癌C4-2细胞。结论:构建了FLNa基因RNAi载体,该载体能够有效地抑制FLNa基因的表达。

Objective： To construct the short ingerfering RNA（siRNA） vector of filamin A（FLNa） gene and investigate the effect of it on the expression of FLNa. Methods： One FLNa gene siRNA was designed by RNA interference（RNAi） technique and was inserted into expression vector pSilencer4.1-CMV-hygro. The human embryo kidney 293T cells were transfected with the recombination plasmids pSilencer-FLNa and pSilencer-negative respectively. The effect of RNAi on the expression of FLNa gene were identified by Western blot. The prostate cancer cell lines that express the FLNa siRNA stably were selected through hygromycin-resistant selection. Results： The RNAi vector of FLNa was constructed and confirmed by PCR. Western-blot showed that RNAi vector could effectively inhibit the expression of FLNa. The C4-2 cells that interfere the FLNa expression stably were constructed. Conclusion： The RNAi vector of FLNa was constructed successfully. The RNAi vector could effectively inhibit the expression of FLNa gene.