联合下调Survivin和KSP表达对乳癌细胞MDA-MB-231凋亡影响

EFFECTS OF COMBINED DOWN REGULATION OF EXPRESSIONS OF SURVIVIN AND KSP ON APOPTOSIS OF BREAST CANCER CELL MDA-MB-231

目的探讨联合下调Survivin和纺锤体驱动蛋白（KSP）基因表达对乳癌细胞增殖和凋亡影响。方法分别化学合成针对Survivin和KSP基因mRNA的小干扰RNA（siRNAs）,分别或联合转染乳癌细胞MDA-MB-231,以空白对照组作为阴性对照。采用MTT法检测细胞增殖率,荧光定量PCR（qRT-PCR）检测Survivin、KSP、Bax和Bcl-2的mRNA表达,蛋白印迹法检测Survivin和KSP的蛋白表达,流式细胞仪检测细胞凋亡情况。结果Survivin、KSP和Survivin＋KSP转染组转染48h的细胞增殖率分别为0.66±0.01、0.67±0.03、0.54±0.01,转染72h的细胞凋亡率分别为（11.61±0.89）%、（16.33±0.94）%、（45.73±1.11）%,与空白对照组相比差异有统计学意义（F=1 307.81、1 450.76,P〈0.01）,其中以双干扰组细胞增殖率最低、凋亡率最高。在各转染组中,Survivin、KSP的mRNA和蛋白表达水平均显著下降（F=48.68~490.20,P〈0.01）,Bax mRNA的表达显著增加（F=65.73,P〈0.01）,Bcl-2mRNA的表达显著减少（F=27.41,P〈0.01）。结论利用siRNAs联合下调Survivin和KSP基因表达,能有效抑制乳癌细胞的增殖、促进其凋亡,效果优于下调单个基因的表达。

Objective To investigate the effects of combined down regulation of expressions of Survivin and kinesin spindle protein（KSP）on proliferation and apoptosis of human breast cancer cells（MDA-MB-231）. Methods Small interfering RNAs（siRNAs）targeting Survivin and KSP mRNA were chemically synthesized and jointly or separately transfected to breast cancer cells（MDA-MB-231）.A blank control group was served as negative control.Employing MTT method to measure the cell proliferation rate,qRT-PCR to detect the expressions of Survivin,KSP,Bax and Bcl-2mRNA,Western blotting to detect the expressions of Survivin and KSP proteins,and flow cytometry to detect the cell apoptosis. Results After 48 hours of transfection,the cell proliferation rates in Survivin,KSP and Survivin＋KSP groups were 0.66±0.01,0.67±0.03,and 0.54±0.01,respectively,and the apoptotic rates were（11.61±0.89）%,（16.33±0.94）%,and（45.73±1.11）%,respectively,the differences were statistically significant as compared with the blank control group（F=1 307.81,1 450.76;P〈0.01）,of which,the lowest rate of cell proliferation and the highest rate of apoptosis were noted in double-interference group.In each transfection group,the mRNA and protein expressions of Survivin and KSP were significantly declined（F=48.68-490.20,P〈0.01）,the expression of Bax mRNA increased（F=65.73,P〈 0.01）,and the mRNA expression of Bcl-2reduced（F=27.41,P 〈0.01）. Conclusion Applying siRNAs to jointly down regulation of both Survivin and KSP expressions can more effectively inhibit the proliferation and promote apoptosis of breast cancer cells than down-regulating the expression of individual gene.