应用荧光定量PCR和Western-blot检测RNA干扰肺腺癌A549细胞STAT3基因表达水平的实验研究

EXPERIMENTAL STUDY OF RNA IN TERFERENCE INHIBITING STAT3 GENE EXPRESSION IN LUNG ADENO CARCINOMA A549 CELLS BY FIUORESCENCE QUANTITATIVE PCR AND WESTERN-BLOT

目的:研究RNAi技术对人肺腺癌A549细胞系中STAT3基因的阻断效应,探讨STAT3在肺癌中的重要作用,为肺癌的基因治疗寻找新的靶点。方法:将化学合成siRNA用脂质体介导法转染肺腺癌A549细胞,应用荧光定量PCR法检测RNA干扰后STAT3 mRNA表达量,得出抑制率。应用Western-blot检测RNA干扰后对STAT3蛋白表达的影响。结果:成功将siRNA转染肺腺癌A549细胞,荧光定量PCR结果显示,在转染24h、48h、72h后,各干扰组与阴性组比较STAT3基因表达量明显下调(P<0.05),Western blot显示转染前后STAT3蛋白的表达水平明显受到抑制,干扰组与阴性组空白组相比具有显著差异(P<0.05),内参基因在各组的表达量无明显差异。结论:将合成的STAT3 siRNA能有效抑制STAT3蛋白的表达、降低STAT3 mRNA水平,对STAT3有高效和特异的沉默作用,以STAT3为靶点的RNA干扰技术可望成为肺癌基因治疗的新策略。

Objective：To study blocking effects of RNAi technology on STAT3 expression in lung adeno carcinoma A549 cells..Methods： After liposome-mediated siRNA transfection of A549 cells,the expression of STAT3mRNA was determined by fluorenscence quantitative PCR,STAT3 protein expression was detected by Western-blot Results：lung adenocarcinoma A549 cells ware Successfully transfected with siRNA.Fluorescence quantitative PCR results showed that after transfected 24h,48h,72h,the interference group STAT3 gene expression was significantly reduced（P〈0.05）,Western-blot resnlt showed that STAT3 protein expression was significantly inhibited in interference group（P〈0.05）.Conclusion： The synthetic STAT3siRNA can effectively inhibit the expression of STAT3 protein and lower the levels of STAT3.mRNA of STAT3.It has an efficient and specific silencing effect to STAT3.RNA interference technology with STAT3 as target is expected to become a new strategy for gene therapy of lung cancer.