Mfn2过表达诱导乳腺癌细胞线粒体自噬促进细胞凋亡的机制研究  

Study on the mechanism of mitochondrial autophagy in breast cancer cells induced by Mfn2 overexpression

作　　者：茅芯慧 张建庆[1] 王珍[1] 朱成斌[1] MAO Xinhui;ZHANG Jianqing;WANG Zhen;ZHU Chengbin(Radiotherapy Center,Xinjiang Uygur Autonomous Region People's Hospital,Xinjiang Urumqi 830000,China.)

机构地区：[1]新疆维吾尔自治区人民医院放疗中心,新疆乌鲁木齐830000

出　　处：《现代肿瘤医学》2024年第19期3670-3676,共7页Journal of Modern Oncology

基　　金：新疆维吾尔自治区自然科学基金项目(编号:2022D01C642)。

摘　　要：目的:探究线粒体融合基因2(Mfn2)过表达诱导乳腺癌细胞凋亡的作用及机理。方法:收集乳腺癌患者肿瘤组织及癌旁组织标本,通过实时荧光定量聚合酶链式反应(qRT-PCR)、蛋白质免疫印迹(Western Blot)及免疫组织化学染色检测两种组织中Mfn2表达差异。将MCF-7细胞分为对照组、NC组、Mfn2组、Mfn2+3-MA组,按照分组进行对应处理后,收集4组细胞,CCK-8法测定细胞增殖活性,流式细胞术检测细胞凋亡率,DCFH-DA探针检测细胞内活性氧(ROS)水平,JC-1染色检测线粒体膜电位,Western Blot检测细胞中PTEN诱导激酶1(PINK1)、E3泛素连接酶(Parkin)、微管相关蛋白1轻链3(LC3)Ⅱ/LC3Ⅰ、p62蛋白表达。结果:与癌旁组织比较,乳腺癌组织中Mfn2 mRNA相对表达量和蛋白相对表达量显著下调(P<0.05),阳性细胞比例显著减少(P<0.05)。转染Mfn2重组过表达质粒的MCF-7细胞中Mfn2 mRNA相对表达量和蛋白相对表达量显著高于未转染的MCF-7细胞、转染阴性对照NC重组质粒的MCF-7细胞(P<0.05)。与对照组比较,Mfn2组MCF-7细胞增殖活性显著降低(P<0.05),细胞凋亡率显著增加(P<0.05),细胞内ROS水平显著升高(P<0.05),线粒体膜电位显著下降(P<0.05),细胞中PINK1、Parkin、LC3Ⅱ/LC3Ⅰ蛋白表达水平均显著上调(P<0.05),p62蛋白表达水平显著下调(P<0.05);与Mfn2组比较,Mfn2+3-MA组MCF-7细胞增殖活性显著升高(P<0.05),细胞凋亡率显著减少(P<0.05),细胞内ROS水平显著降低(P<0.05),线粒体膜电位显著升高(P<0.05),细胞中PINK1、Parkin、LC3Ⅱ/LC3Ⅰ蛋白表达水平均显著下调且p62蛋白表达水平显著上调(P<0.05)。结论:乳腺癌组织中Mfn2低表达,在乳腺癌细胞中过表达Mfn2能够通过诱导线粒体自噬促进细胞凋亡,起到肿瘤抑制作用。Objective:To explore the role and mechanism of overexpression of mitochondrial fusion gene 2(Mfn2)in inducing apoptosis of breast cancer cells.Methods:The tumor tissues and adjacent tissues of breast cancer patients were collected,and the difference of Mfn2 expression in the two tissues was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR),Western Blot and immunohistochemical staining.MCF-7 cells were divided into control group,NC group,Mfn2 group and Mfn2+3-MA group,after corresponding treatment according to the groups,the cells in 4 groups were collected.The cell proliferation activity was determined by CCK-8 method,the apoptosis rate was detected by flow cytometry,the level of intracellular reactive oxygen species(ROS)was detected by DCFH-DA probe,the mitochondrial membrane potential was detected by JC-1 staining,and the protein expressions of PTEN-induced kinase 1(PINK1),E3 ubiquitin ligase(Parkin),microtubule-associated protein 1 light chain 3(LC3)Ⅱ/LC3Ⅰand p62 were detected by Western Blot.Results:Compared with the adjacent tissues,the relative expression of Mfn2 mRNA and protein in breast cancer tissues was significantly down-regulated(P<0.05),and the proportion of positive cells was significantly reduced(P<0.05).The relative expression levels of Mfn2 mRNA and protein in MCF-7 cells transfected with Mfn2 recombinant overexpression plasmid were significantly higher than those in untransfected MCF-7 cells and those transfected with negative control NC recombinant plasmid(P<0.05).Compared with the control group,the proliferation activity of MCF-7 cells in Mfn2 group was significantly decreased(P<0.05),the apoptosis rate was significantly increased(P<0.05),the ROS level was significantly increased(P<0.05),and the mitochondrial membrane potential was significantly decreased(P<0.05),the protein expression levels of PINK1,Parkin,LC3Ⅱ/LC3Ⅰwere significantly up-regulated(P<0.05),while the protein expression level of p62 was significantly down-regulated(P<0.05).Compared with

关 键 词：乳腺癌 线粒体融合基因2 线粒体 自噬 细胞凋亡 

分 类 号：R737.9[医药卫生—肿瘤]