沉默叉头框C2基因对乳腺癌MDA-MB-231细胞的化疗增敏作用

Enhanced sensitivity of breast cancer MDA-MB-231 cells to chemotherapy by silencing forkhead box C2 gene with RNA interference technology

目的 观察沉默叉头框C2 （FOXC2）基因对乳腺癌MDA-MB-231细胞多西紫杉醇化疗敏感性的影响.方法 通过脂质体介导将FOXC2短发卡RNA干扰表达质粒FOXC2-小干扰RNA（siRNA）转染至MDA-MB-231细胞,并检测转染48 h后的FOXC2 mRNA及蛋白表达；荧光显微镜观察72 h后的细胞凋亡形态学；流式细胞术检测72 h后的细胞凋亡率及细胞周期.结果 与未转染组细胞及空载体组细胞比较,干扰组细胞出现典型凋亡形态学改变,且FOXC2 mRNA及蛋白表达明显降低（P＜0.05）；噻唑蓝（MTT）显示干扰组细胞对多西紫杉醇敏感性增加,半数抑制浓度（IC50）由（6.08±1.23） mg/L降至（0.65 ±0.01） mg/L；同时,干扰组细胞早期凋亡率为（20.01±0.19）％,明显高于其他3组（P＜0.05）,且S期及G0/G1期细胞比例减少,G2/M期细胞比例增多（P＜0.05）.结论 以siRNA下调FOXC2表达可有效诱导乳腺癌MDA-MB-231细胞凋亡,并增强其对多西紫杉醇的化疗敏感性.

Objective To observe the effects of forkhead box C2 gene （FOXC2） short hairpin RNA on docetaxel chemotherapy sensitivity of human breast cancer MDA-MB-231 cells.Methods MDA-MB-231 cells were transfected with recombinant expression plasmid FOXC2-small interfering RNA （siRNA） by lipofectamine 2000,and the expression of FOXC2 mRNA and protein was detected at 48 h after transfection.The morphological changes of apoptotic cells were observed under fluorescent microscope at 72 h after transfection.Methyl thiazol tetrazolium （MTT） assay was used to examine the inhibitory rate and the 50％ inhibitory concentration （IC50）.Flow cytometry was applied to examine apoptosis and cell cycle at 72nd h after transfection.Results As compared with negative control plasmid and FOXC2-siRNA non-transfection group,the typical apoptotic morphology of MDA-MB-231 cells was observed,and the mRNA and protein expression levels of FOXC2 were obviously reduced in FOXC2-siRNA transfection group （P ＜ 0.05）.Silencing FOXC2 increased sensitivity of MDA-MB-231 cells to docetaxel （P ＜ 0.05）,and IC50 was decreased from （6.08 ±1.23） mg/L to （0.65 ±0.01） mg/L.Meanwhile,the apoptosis rate of MDA-MB-231 cells transfected with pFOXC2-shRNA was （20.01 ± 0.19）％,which was significantly higher than other three groups,and the proportion of cells in S phase and G0/G1 phase was decreased and that in G2/M phase was increased （P ＜0.05）.Conclusion Down-regulation of FOXC2 expression by siRNA in breast cancer MDA-MB-231 cells could effectively induce apoptosis of MDA-MB-231 cells and increase their sensitivity to docetaxel.