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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 construct an adenovirus expression vector (rAd-shRNA-TRF2) expressing the small interfering RNA (siRNA-TRF2) of telomeric repeat-binding factor 2 (TRF2) gene and to detect the effect of the vector on human breast cancer (MCF-7) Silencing effects of TRF2 gene expression. Methods: The recombinant adenovirus rAd-shRNA-TRF2 expression vector was constructed by homologous recombination and transfected into MCF-7 cells. The expression of TRF2 gene was detected by FQ-RT-PCR and Western blot 48 h after transfection. MTT Method and clone formation assay to detect cell growth status. Results: The recombinant adenovirus rAd-shRNA-TRF2 was successfully packaged. The expression of TRF2 gene was significantly inhibited after transfected with MCF-7 cells. The cell growth was significantly inhibited and the ability of cell cloning was significantly decreased. Conclusion: The successful construction of adenovirus-mediated TRF2 RNAi expression vector rTR-shRNA-TRF2 can effectively inhibit TRF2 gene expression and inhibit cell growth in human breast cancer MCF-7 cells.