Ku70基因RNA干扰载体的构建及干扰效果鉴定

Construction and assessment of effects of Ku70 RNA interference vector

目的构建靶向Ku70基因的RNA干扰(RNAi)表达载体并进行RNAi效果鉴定。方法设计3个针对Ku70基因的小干扰RNA(small interfering RNA,siRNA),转染Hela细胞系。在转染24、48、72 h后,通过免疫印迹分析检测Ku70蛋白的表达量。把RNAi效果最显著的siRNA设计成短发夹RNA(short-hairpin RNA,shRNA),克隆入pSi-lencerTM 4.1-CMV hygro载体,转染Hela细胞系并筛选稳转细胞株,在mRNA和蛋白水平,评估Ku70 siRNA的干扰效果。结果 siRNA转染Hela细胞24、48 h后,Ku70蛋白的表达没有显著变化,但在72 h后,蛋白的表达量显著下降。在具有稳定表达siRNA的稳转细胞株中,Ku70基因的表达在mRNA水平和蛋白水平都受到了非常明显的抑制。结论成功构建靶向Ku70基因RNAi载体,并筛选出效能最优序列,为进一步研究Ku70基因的表达与宫颈癌放射治疗敏感性的关系奠定了基础。

Objective To construct the RNA interference（RNAi）expression vector of Ku70 gene and evaluate the effect of RNAi.Methods Three small interfering RNA（siRNA） targeting Ku70 was designed for transfecting Hela cell line.Through immunoblotting assay,the expression of Ku70 was examined at 24,48 and 72 hours after transfection into Hela cell line.The siRNA which have the most effect of RNAi was designed into short-hairpin RNA and cloned into pSilencerTM 4.1-CMV-hygro vector,then transfection Hela cell line and the stable strain were selected.The interference effects of Ku70 siRNA was evaluated at mRNA and protein levels.Results The expression of Ku70 was not obviously changed at 24 and 48 hours after siRNA transfected Hela cells,but was significantly down-regulated at 72 hours.At the stable transfection strain which with stable expression of siRNA,the expression of Ku70 gene was inhibited apparentely at mRNA and protein levels.Conclusion The RNAi vector targeting Ku70 gene is successfully constructed,and the best efficacy series are selected,which lays solid foundation for further study of the relationship between Ku70 gene expression and the cervical cancer susceptibility with radiation therapy.