沉默Smad4基因对乳腺癌MCF-7细胞增殖和凋亡的影响

目的：研究降低Smad4表达对乳腺癌MCF-7细胞增殖和凋亡的影响，并探讨其可能的作用机制。方法：体外培养人乳腺癌MCF-7和MDA-MB-231细胞，采用逆转录-聚合酶链反应（RT-PCR）法检测MCF-7细胞和MDA-MB-231细胞中Smad4 mRNA表达水平；Smad4-shRNA与Scramble-shRNA质粒分别稳定转染Smad4高表达MCF-7细胞，实验分为未转染MCF-7细胞组（正常对照组），采用Smad4基因沉默组和Scramble组（阴性对照组）；采用CCK-8法检测各组细胞增殖能力，流式细胞术检测各组细胞凋亡率，实时荧光定量PCR（Real-time PCR）法检测增殖相关基因CDKN1A、CDK1和CDK2以及凋亡相关基因Suvivin、bcl-2、caspase3和caspase9 mRNA表达水平。结果：各组细胞增殖能力比较差异无统计学意义（P > 0.05），各组细胞CDKN1A、CDK1和CDK2 mRNA表达水平比较差异无统计学意义（P > 0.05）；与正常对照组和阴性对照组比较，Smad4基因沉默组细胞凋亡率明显降低（P < 0.01），Smad4基因沉默组细胞Suvivin和bcl-2 mRNA表达水平明显升高（P < 0.01），caspase3和caspase9 mRNA表达水平明显降低（P < 0.05）。结论：Smad4可以通过下调Suvivin和bcl-2的表达、上调caspase3和caspase9表达引起细胞凋亡。

Influence of silencing Smad4 gene in proliferation and apoptosis of breast carcinoma MCF-7 cells

Objective:To study the effects of down-regulated Smad4 expression on the proliferation and apoptosis of breast carcinoma MCF-7 cells,and to explore their mechanisms.Methods:The human breast carcinoma MCF-7 cells and MDA-MB-231 cells were cultured in vitro.RT-PCR method was used to detect the expression levels of Smad4 mRNA in MCF-7 cells and MDA-MB-231 cells.The Smad4-shRNA plasmid and Scramble-shRNA plasmid were respectively stably transfected into the MCF-7 cells with high expression of Smad4.The experiment was divided into non-transfected MCF-7 cells (normal control) group,Smad4 gene silencing group,Scramble(negative control) group.The proliferation abilities of the cells in various groups were detected by CCK-8 method.The apoptotic rates of the cells in various groups were detected by flow cytometry.Real-time PCR method wasused to detect the mRNA expression levels of the proliferation-related genes CDKN1A,CDK1 and CDK2 and the apoptosisrelated genes Suvivin,bcl-2,caspase 3 and caspase 9.Results:The proliferation abilities of cells had no statistical significance between various groups (P＞0.05).The mRNA expression levels of CDKN1A,CDK1 and CDK2 in the cells had no statistical significance between various groups (P＞0.05).Compared with normal control group and negative control group,the apoptotic rate of the cells in Smad4 gene silencing group was significantly decreased (P＜0.01),the expression levels of Suvivin and bcl-2 mRNA in Smad4 gene silencing group were significantly increased (P＜0.01),and the mRNA expression levels of caspase 3 and caspase 9 in Smad4 gene silencing group were significantly decreased (P＜0.05).Conclusion:Smad4 could induce the apoptosis of MCF-7 cells by downregulating the expressions of Suvivin and bcl-2 and up-regulating the expressions of caspase 3 and caspase 9.