沉默CTHRC1调控p53介导的线粒体凋亡诱导乳腺癌MCF-7细胞凋亡

Silencing CTHRC1 induces apoptosis of breast cancer MCF-7 cells by regulating p53-mediated mitochondrial apoptosis pathway

目的探究短发夹RNA(shRNA)干扰载体沉默胶原三螺旋重复蛋白1(CTHRC1)诱导人乳腺癌MCF-7细胞凋亡的分子机制。方法将shRNA-CTHRC1质粒和阴性对照质粒转染至乳腺癌MCF-7细胞,采用qPCR法和Westernblot法分别检测CTHRC1mRNA和蛋白表达水平;CCK-8法和流式细胞术法分别检测细胞增殖活力和凋亡率;线粒体膜电位试剂盒(JC-10染色法)检测细胞线粒体膜电位变化;Western blot法检测凋亡相关蛋白caspase3、细胞色素C(CytC)以及p53介导的线粒体凋亡途径相关蛋白p53、Apaf-1、Cleaved-caspase9、Bcl-2、Bax和PUMA等表达水平。将shRNA-CTHRC1质粒和si-p53干扰质粒分别或同时转染至MCF-7细胞中,Westernblot法检测p53和CytC蛋白表达水平;流式细胞术检测细胞凋亡率。结果沉默CTHRC1可降低MCF-7细胞中CTHRC1mRNA和蛋白表达水平,抑制细胞增殖活力,提高细胞凋亡率,降低线粒体膜电位,上调Cleaved-caspase3、p53、Apaf-1、Cleaved-caspase9、Bax、PUMA和胞质中CytC等蛋白表达水平,下调Bcl-2和线粒体中CytC蛋白表达水平。然而,干扰p53表达可逆转CTHRC1沉默对MCF-7细胞凋亡的促进作用。结论CTHRC1沉默通过激活p53介导的线粒体凋亡途径诱导人乳腺癌MCF-7细胞发生凋亡。

Objective To investigate the molecular mechanism by which short hairpin RNA(shRNA) interferes with silencing of collagen triple helix repeat containing 1(CTHRC1) to induce apoptosis in human breast cancer MCF-7 cells. Methods The shRNA-CTHRC1 plasmid and negative control plasmid were transfected into breast cancer MCF-7 cells. The mRNA and protein expression levels of CTHRC1 were determined by qPCR and Western blot respectively. The cell proliferation activity and apoptosis rate were detected by CCK-8 and flow cytometry respectively. The changes of mitochondrial membrane potential were detected by mitochondrial membrane potential kit(JC-10 staining). The expression of apoptosis-related proteins Caspase 3, Cyt C and p53-mediated mitochondrial apoptosis pathway related proteins p53, Apaf-1, Cleaved-caspase 9, Bcl-2, Bax and PUMA was detected by Western blot. The shRNA-CTHRC1 plasmid and si-p53 interfering plasmids were transfected into MCF-7 cells, respectively or simultaneously, and the expression levels of p53 and Cyt C protein were detected by Western blot, the apoptosis level was detected by flow cytometry. Results Silencing CTHRC1 reduced the mRNA and protein expression levels of CTHRC1, inhibited cell proliferation, promoted cell apoptosis, decreased mitochondrial membrane potential, and upregulated the protein expression levels of Cleaved-caspase 3, p53, Apaf-1, Cleaved-caspase 9, Bax, PUMA and the cytoplasm protein of Cyt C in MCF-7 cells, while down-regulated the protein expression levels of Bcl-2 and the mitochondrial Cyt C in MCF-7 cells. However, interference with p53 expression could reverse the pro-apoptotic effect of CTHRC1 silencing on MCF-7 cells. Conclusion CTHRC1 silencing is dependent on the p53-mediated mitochondrial apoptosis pathway to induce apoptosis in human breast cancer MCF-7 cells.