人乳腺癌MCF-7细胞的TRF2 siRNA表达载体的构建和鉴定

Construction and identification TRF2 siRNA expression vector on the breast cancer MCF-7 cells

目的:构建表达端粒重复序列结合因子2(TRF2)基因小干扰RNA(siRNA-TRF2)的腺病毒表达载体(rAd-shRNA-TRF2),并检测该载体对人乳腺癌(MCF-7)细胞TRF2基因表达的沉默效应。方法:采用同源重组法构建重组腺病毒rAd-shRNA-TRF2表达载体,鉴定正确后转染MCF-7细胞,用FQ-RT-PCR和Western blot检测转染后48 h TRF2基因的表达,MTT法和克隆形成实验检测细胞生长状况。结果:重组腺病毒rAd-shRNA-TRF2包装成功,转染MCF-7细胞后,TRF2基因的表达明显被抑制,细胞生长显著抑制,细胞克隆形成能力显著下降。结论:腺病毒介导的TRF2 RNAi表达载体rAd-shRNA-TRF2构建成功,可有效抑制人乳腺癌MCF-7细胞TRF2基因的表达,抑制细胞生长。

Objective： To construction adenovirus - mediated RNA interference vector expressing TRF2 small interfering RN A （rAd- shRNA- TRF2） , and explore the specific inhibitory effect of this vector on TRF2 mRNA expression in MCF- 7 cell. Methods： The recombinant adenovirus rAd - shRNA - TRF2 was obtained, and the vector was transfected into MCF - 7 cells , then the expression of TRF2 mRNA and protein at 48 h after transfection was detected by FQ - RT - PCR and Western blot, the cell growth was detected by M＇FF colorimetry and clone forming test. Results： The recombinant adenovirns vectors were successfully constructed; The expression of TRF2 group was obviously decreased, cells proliferation were inhibited and cell colony forming ability were decreased obviously after transfected tad - shRNA - TRF2 into MCF - 7 cells. Conclusion： Adenovirus - mediated RNA interference vector has been constructed succeessfully, expression of TRF2 and cell growth is obviously decreased after transfection.