Twist特异性小分子干扰RNA对乳腺癌MDA-MB-231细胞生长作用的影响

Effect of silencing Twist gene using small interfering RNA on growth of breast cancer cells

目的 观察Twist特异性小分子干扰RNA（siRNA）对乳腺癌细胞生长作用的影响.方法 设计、合成Twist特异性siRNA,转染至乳腺癌MDA-MB-231细胞.逆转录-聚合酶链反应（RT-PCR）及Western blot法检测细胞中Twist基因的mRNA和蛋白表达；Western blot法检测细胞浸润、凋亡相关蛋白表达；噻唑蓝（MTT）法及流式细胞术检测细胞生长及凋亡.结果 Twist-siRNA转染组与阴性对照组比较,mRNA和蛋白表达量明显降低（0.873±0.071比0.323±0.035、0.964±0.021比0.419±0.010,P＜0.05）；细胞浸润相关蛋白E-钙黏蛋白（E-cadherin）表达增加（1.102±0.051比1.583±0.031,P＜0.05）；凋亡相关蛋白半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-3表达增加（1.059±0.070比1.502±0.029,P＜0.05）；细胞生长速度明显降低；空白对照组、阴性对照组和转染组的细胞凋亡率分别为15.3％、16.1％和47.2％（P＜0.05）.结论 构建的Twist特异性siRNA能够显著下调人乳腺癌MDA-MB-231细胞株Twist基因表达,降低细胞体外增殖能力及肿瘤细胞浸润能力,提高细胞凋亡率.

Objective To observe the effect of the down-regulated expression of Twist gene on growth and infiltration of the breast cancer cells. Methods The small interfering RNA （siRNA） vectors targeting Twist gene were designed and synthesized, and transfeeted into MDA-MB-231 cells. The expression levels of Twist mRNA and protein were detected by using reverse transeriptase-polymerase chain reac tion （RT-PCR） and Western blotting. Western blotting was used to detect genes about infiltration and apoptosis. Methyl thiazol tetrazolium （MTF） assay and flow cytometry were used to detect the growth and apopto sis. Results The expression of Twist mRNA and protein which was silenced in the cell line MDA-MB-231 was lower than that of control group （0. 873 ± 0. 071 vs. 0. 323 ± 0. 035, 0. 964± 0. 021 vs. 0. 419 ± 0. 010, P 〈 0. 05 ） , that of E-cadherin for infiltration was higher than that of control group （ 1. 102 ± 0. 051 vs. 1. 583 ± 0. 031, P 〈 0. 05 ） , and that of Caspase-3 for apoptosis was higher too （ 1. 059 ± 0. 070 vs. 1. 502 ± 029,P 〈0. 05）. The growth of the cells was slowed down. The apoptosis rate was higher in ex perimental group than in blank group and control group （15.3% , 16. 1% and 47. 2% respectively, P 〈 0. 05 ）. Conclusion The siRNA vectors targeting Twist gene we designed and synthesized could down-reg-ulate the expression of Twist in MDA-MB-231 cells, slow down the invasion capability and increase apoptosis rate.