ATM基因siRNA真核表达载体的构建及其对转染宫颈癌细胞株ATM表达的抑制作用

目的:研究特异性siRNA对宫颈癌ATM基因的阻抑效果以及用RNAi效应对宫颈癌做基因治疗的可行性. 方法:构建可表达人ATM基因siRNA的重组真核表达质粒pSuppressorNeo-ATM,电穿孔法转染Siha宫颈癌细胞株. 应用RT-PCR,Western blot,流式细胞术、免疫荧光法等方法检测转染的宫颈癌细胞中ATM基因的表达水平. 结果:RT-PCR, Western-blot均表明瞬时转染pSuppressorNeo-ATM的宫颈癌细胞中ATM 基因的表达受到明显抑制,流式细胞术、免疫荧光法检测表明ATM蛋白含量明显下降. 结论:pSuppressorNeo-ATM质粒构建成功,瞬时转染宫颈癌细胞后可以明显抑制ATM基因的表达.

Construction of eukaryotic expression plasmid expressing siRNA targeting ATM gene and its inhibitory effect on ATM expression in transfected human cervical carcinoma cells

AIM: To study inhibitory effect of siRNA on ATM expression in human cervical carcinoma cells and to determine the feasibility of RNAi application in gene-therapy of cervical cancer. METHODS: Hairpin siRNA templates were designed based on ATM gene sequence and mRNA structure and was cloned into pSuppressor vector. Siha cells were transfected by pSuppressor-ATM with electroporation and the non-transfected cells and non-specific siRNA transfected cells were used as controls. The inhibitory effect of ATM mRNA was detected by semi-quantitative RT-PCR and Western-blot, and the inhibitory effect of ATM protein expression was detected by FCM and immunofluorescency. RESULTS: Semi-quantitative RT-PCR, Western-blot, FCM and immunofluorescency all revealed a potent inhibition of the expression of ATM in the transfected Siha cells. CONCLUSION: The plasmid vector was successfully constructed. Transient transfection can significantly decrease the ATM level in transfected cells.