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. A cultured 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.
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(miRNA mimics)及对照组细胞(NC mimics)，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，Western blot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。Western bot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cleaved caspase 3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。
Keywords: cell apoptosis; gastric cancer; miRNA-431-5p; mitochondrial function.