MicroRNA-431-5p在胃癌组织中低表达:基于线粒体和Bax/Bcl-2/caspase3信号通路

Overexpression of miR-431-5p impairs mitochondrial function and induces apoptosis in gastric cancer cells via the Bax/Bcl-2/caspase3 pathway

目的探究microRNA-431-5p(miR-431-5p)在胃癌细胞的表达特点及其对胃癌细胞凋亡及线粒体功能的影响。方法荧光定量PCR检测miR-431-5p在50例胃癌组织及癌旁组织中的表达,并分析其与患者临床病理特征的关系。采用脂质体转染技术构建miR-431-5p过表达组细胞(miRNAmimics)及对照组细胞(NCmimics),分别采用CCK-8、流式细胞术、荧光探针标记以及ATP检测试剂盒评估miR-431-5p对MKN-45细胞线粒体数量、膜电位完整性、通透性转换孔开放程度、活性氧生成水平以及ATP含量的影响,Westernblot检测MKN-45细胞内凋亡蛋白表达水平的变化。结果miR-431-5p在胃癌组织中的表达水平较癌旁组织明显下调(P<0.001),其表达水平与患者的肿瘤分化程度(P=0.0227)、T分期(P=0.0184)、N分期(P=0.0005)、TNM分期(P=0.0414)和血管侵犯(P=0.0107)密切相关。细胞功能实验显示:过表达miR-431-5p抑制MKN-45细胞增殖能力并诱导细胞凋亡,miR-431-5p过表达后MKN-45细胞内线粒体数量减少、膜电位完整性下降、通透性转换孔开放程度增加,ROS生成增多,ATP含量降低。Westernbot显示:miR-431-5p过表达后MKN-45细胞内抗凋亡蛋白Bcl-2表达下调,而促凋亡蛋白p53、Bax以及cleavedcaspase3表达上调。结论miR-431-5p低表达于胃癌组织,其可能通过介导Bax/Bcl-2/caspase3信号通路,继而抑制胃癌细胞线粒体功能并促进细胞凋亡,是胃癌潜在的治疗靶点。

Objective To investigate the expression of microRNA miR-431-5p in gastric cancer(GC)tissues and its effects on apoptosis and mitochondrial function in GC cells.Methods The expression level of miR-431-5p in 50 clinical samples of GC tissues and paired adjacent tissues was detected using real-time fluorescence quantitative PCR,and its correlation with the clinicopathological features of the patients was analyzed.Acultured human GC cell line(MKN-45 cells)were transfected with a miR-431-5p mimic or a negative control sequence,and the cell proliferation,apoptosis,mitochondrial number,mitochondrial potential,mitochondrial permeability transition pore(mPTP),reactive oxygen species(ROS)production and adenosine triphosphate(ATP)content were detected using CCK-8 assay,flow cytometry,fluorescent probe label,or ATP detection kit.The changes in the expression levels of the apoptotic proteins in the cells were detected with Western blotting.Results The expression level of miR-431-5p was significantly lower in GC tissues than in the adjacent tissues(P<0.001)and was significantly correlated with tumor differentiation(P=0.0227),T stage(P=0.0184),N stage(P=0.0005),TNM stage(P=0.0414)and vascular invasion(P=0.0107).In MKN-45 cells,overexpression of miR-431-5p obviously inhibited cell proliferation and induced cell apoptosis,causing also mitochondrial function impairment as shown by reduced mitochondrial number,lowered mitochondrial potential,increased mPTP opening,increased ROS production and reduced ATP content.Overexpression of miR-431-5p significantly downregulated the expression of Bcl-2 and increased the expressions of pro-apoptotic proteins p53,Bcl-2 and cleaved caspase-3 protein.Conclusion The expression of miR-431-5p is down-regulated in GC,which results in mitochondrial function impairment and promotes cell apoptosis by activating the Bax/Bcl-2/caspase3 signaling pathway,suggesting the potential role of miR-431-5p in targeted therapy for GC.