【摘要】 目的:探讨miRNA-1287(miR-1287)通过靶向结合并负向调控谷胱甘肽过氧化物酶4(GPX4)表达在乳腺癌增殖中的作用及相关分子机制。方法:收集2018年01月至2019年01月期间来我院就诊的50位乳腺癌患者肿瘤标本及癌旁标本;运用实时定量聚合酶链式反应(qRT-PCR),检测乳腺癌患者肿瘤标本及癌旁标本miR-1287、GPX4 mRNA的表达水平;运用Western blot检测乳腺癌患者肿瘤标本及癌旁标本GPX4蛋白表达水平;运用Starbase 2.0软件预测miR-1287与GPX4 mRNA的结合位点;构建野生型位点(wt)和突变型位点(mut)GPX4荧光素酶报告基因质粒,探索miR-1287能否靶向结合并影响GPX4的表达;通过MTT实验检测miR-1287对乳腺癌细胞增殖能力的影响;通过谷胱甘肽(GSH)试剂盒检测乳腺癌细胞GSH水平,运用活性氧(ROS)试剂盒检测乳腺癌细胞活性氧水平。结果:乳腺癌患者肿瘤标本中miR-1287水平显著低于癌旁组织;而乳腺癌患者肿瘤标本中GPX4 mRNA及蛋白表达水平明显高于癌旁组织,其表达水平与miR-1287水平呈负相关关系;Starbase 2.0软件分析结果提示,miR-1287可以直接靶向结合GPX4 mRNA序列中的潜在位点;荧光素酶报告基因实验结果发现,miR-1287 mimic显著降低GPX4(wt)荧光素酶活性,对GPX4(mut)荧光素酶活性无明显影响;过表达miR-1287明显降低乳腺癌细胞GSH水平,增加ROS水平并显著抑制乳腺癌细胞增殖能力,而铁死亡抑制剂Fer-1预处理可以逆转miR-1287对乳腺癌细胞的上述作用,提示miR-1287通过促进铁死亡来执行其抑癌功能。结论:miR-1287通过抑制GPX4表达,进而促进肿瘤细胞铁死亡过程,最终抑制乳腺癌细胞增殖能力。更多还原

【Abstract】 Objective:To explore the effect of miR-1287 on the proliferation of breast cancer cells via regulating GPX4.Methods:Fifty breast cancer patients treated in our hospital were enrolled from January 2018 to January 2019 in our study.qRT-PCR was used to explore the expression of miR-1287 and the GPX4 mRNA in the breast cancer tissues and corresponding adjacent tissues.Western blot was utilized to detect the expression of GPX4 protein in the breast cancer tissues and corresponding adjacent tissues.Using Starbase 2.0 software,GPX4 mRNA sequence was found to harbor a potential binding site for miR-1287.The luciferase assay was used toverify the relation between miR-1287 and GPX4.MTT assay was conducted to observe the effect of miR-1287 on the proliferation ability of breast cancer cells.GSH and ROS test kit was used to examine the level of GSH and ROS in breast cancer cells.Results:The expression of miR-1287 was down-regulated in breast cancer tissues compared with corresponding adjacent tissues.The GPX4 mRNA and protein were increased in breast cancer tissues compared with corresponding adjacent tissues,which was negatively correlated with the expression of miR-1287.The Starbase2.0 result indicated that there was apotential binding site for miR-1287 in the mRNA sequence of GPX4.The luciferase assay result showed that transfection of miR-1287 mimic decreased the luciferase activity of wild type(wt) GPX4 luciferase vector while had little effect on the mutant(mut) GPX4 luciferase vector.Transfection of miR-1287 mimic reduced GSH production,increased the level of ROS and repressed the proliferation ability of breast cancer cells,hinting that miR-1287 down-regulated the proliferation ability of breast cancer cells via inducing the ferroptosis.Conclusion:miR-1287 inhibited the proliferation of breast cancer cells via down-regulating the expression of GPX4 and inducing ferrotosis.更多还原