弓形虫ROP16蛋白对人乳腺癌MCF-7细胞增殖、周期和凋亡的影响

Effects of Toxoplasma gondii ROP16 protein on the proliferation,cell cycle and apoptosis of MCF-7 human breast cancer cells

目的探讨弓形虫Ⅰ型(RH)ROP16蛋白在人乳腺癌MCF-7细胞增殖、周期及凋亡方面的作用。方法以空载体(MCF-7-HBLV)和过表达ROP16蛋白的慢病毒(HBLV-RH ROP16)分别感染MCF-7细胞,嘌呤霉素筛选出稳定表达ROP16的细胞株,Real time PCR、Western blot法检测MCF-7细胞中ROP16 mRNA和蛋白表达水平。CCK-8和流式细胞术检测细胞增殖、周期和凋亡。Western blot法检测细胞周期及凋亡相关蛋白表达。结果相比MCF-7-HBLV(空载体组)和MCF-7细胞组(对照组),MCF-7-RH ROP16细胞组中ROP16 mRNA和蛋白表达升高;细胞增殖率降低(P<0.01);S期细胞比例、细胞凋亡率升高(P<0.01)。促凋亡因子Bax、P53、Caspase-9、Caspase-3及细胞周期蛋白依赖激酶抑制因子P21表达增高(P<0.01);抗凋亡蛋白Bcl-2及细胞周期蛋白A1(CyclinA1)、周期蛋白依赖性激酶2(CDK2)的表达均下降(P<0.01)。结论弓形虫Ⅰ型(RH)ROP16蛋白可通过调节与凋亡、周期相关的细胞因子的表达,诱导MCF-7细胞凋亡,抑制其增殖并引起细胞周期阻滞。

This study aimed to investigate the role of Toxoplasma gondii typeⅠ(RH)ROP16 protein in the proliferation,cell cycle and apoptosis of human breast cancer MCF-7 cells.We constructed a typeⅠprotein-specific lentiviral vector(MCF-7-RH ROP16)and a MCF-7-HBLV lentiviral vector,and transfected them into human breast cancer MCF-7 cells.The expression of ROP16 mRNA and protein in MCF-7 cells was detected by RT-PCR and Western blotting,respectively.The proliferation activity of MCF-7 cells was measured with the CCK-8 method.The cell cycle and apoptosis were detected by flow cytometry.The expression of proteins related to the cell cycle(p21,CDK2 and CyclinA1)and apoptosis(Bcl-2,Bax,P53,Caspase-3 and Caspase-9)was detected by Western blotting.The MCF-7 cell line was transduced to overexpress recombinant lentiviral ROP16,whose mRNA and protein levels significantly increased.In MCF-7-RH ROP16 cells,compared with MCF-7-HBLV and MCF-7 cells,the cell proliferation rate,apoptosis rate and S-phase cell percentage were significantly lower(P<0.01);the expression of the pro-apoptotic factors Bax,Caspase-3,Caspase-9,P53 and cyclin-dependent kinase inhibitor P21 protein was significantly higher(P<0.01);and the expression of Bcl-2,CDK2 and CyclinA1 protein was significantly lower.Thus,the overexpression of Toxoplasma gondii ROP16 protein in MCF-7 breast cancer cells induced apoptosis and cell cycle arrest,and inhibited proliferation.