| 路 华,石红光,谢 震,李庆超,李 佳.LncRNA KCNQ1OT1靶向调控miR-124-3p/HMGB1轴对高糖诱导肾小球系膜细胞增殖、凋亡及纤维化的影响[J].,2023,(14):2625-2631 |
| LncRNA KCNQ1OT1靶向调控miR-124-3p/HMGB1轴对高糖诱导肾小球系膜细胞增殖、凋亡及纤维化的影响 |
| Effects of LncRNA KCNQ1OT1 Targeting Regulation on miR-124-3p/HMGB1 Axis on Proliferation, Apoptosis and Fibrosis of Human Mesangial Cells Induced by High Glucose |
| 投稿时间：2023-02-07 修订日期：2023-02-28 |
| DOI：10.13241/j.cnki.pmb.2023.14.005 |
| 中文关键词: 长链非编码RNA KCNQ1OT1 miR-124-3p HMGB1 高糖 肾小球系膜细胞 细胞增殖 细胞凋亡 细胞纤维化 |
| 英文关键词: Long chain noncoding RNA KCNQ1OT1 miR-124-3p HMGB1 High glucose Human mesangial cells Cell proliferation Cell apoptosis Cell fibrosis |
| 基金项目:山东省医药卫生科技发展计划项目（202103050754）；青岛市医药科技指导计划项目（2020WJZD186） |
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| 中文摘要: |
| 摘要 目的：探讨长链非编码核糖核酸（LncRNA）KCNQ1OT1靶向调控miR-124-3p/高迁移率族蛋白B1（HMGB1）轴对高糖诱导肾小球系膜细胞（HMC）增殖、凋亡及纤维化的影响。方法：将人HMC分为对照组（NC组）、高糖组（30 mmol/L葡萄糖）、阴性序列（si-NC）组、KCNQ1OT1小干扰核糖核酸（RNA）（si-KCNQ1OT1）组、si-KCNQ1OT1+模拟对照序列（miR-NC）组、si-KCNQ1OT1+miR-124-3p抑制剂（miR-124-3p inhibitor）组，各组在转染后进行高糖处理。实时荧光定量聚合酶链式反应（RT-qPCR）检测LncRNA KCNQ1OT1信使核糖核酸（mRNA）、miR-124-3p mRNA、HMGB1 mRNA表达；四甲基偶氮唑盐比色法（MTT）检测细胞增殖活性；流式细胞术检测细胞凋亡率；蛋白印迹法（Western blot）检测HMGB1蛋白、增殖相关蛋白[细胞周期蛋白1（CyclinD1）]、细胞凋亡蛋白[半胱氨酸蛋白酶-3（caspase-3）、半胱氨酸蛋白酶蛋白9（caspase-9）]、细胞纤维化蛋白[纤维连接蛋白（FN）、细胞黏附分子-1（ICAM-1）]表达；双荧光素酶报告基因实验验证LncRNA KCNQ1OT1与miR-124-3p与HMGB1之间的靶向关系。结果：与NC组比较，高糖组KCNQ1OT1 mRNA、HMGB1 mRNA及HMGB1蛋白、CyclinD1蛋白、FN蛋白、ICAM-1蛋白表达、HMC活性（24 h、48 h和72 h）明显上升，miR-124-3p mRNA、caspase-3蛋白及caspase-9蛋白表达、HMC凋亡率明显下降（P<0.05）；与高糖组、si-NC组比较，si-KCNQ1OT1组KCNQ1OT1 mRNA、HMGB1 mRNA及HMGB1蛋白、CyclinD1蛋白、FN蛋白、ICAM-1蛋白表达、HMC活性（24 h、48 h和72 h）明显下降，miR-124-3p mRNA、caspase-3蛋白及caspase-9蛋白表达、HMC凋亡率明显上升（P<0.05）；与si-KCNQ1OT1组、si-KCNQ1OT1+miR-NC组比较，si-KCNQ1OT1+miR-124-3p inhibitor组HMGB1 mRNA及HMGB1蛋白、CyclinD1蛋白、FN蛋白、ICAM-1蛋白表达、HMC活性（24 h、48 h和72 h）明显上升，miR-124-3p mRNA、caspase-3蛋白及caspase-9蛋白表达、HMC凋亡率明显下降（P<0.05）。较miR-NC组与KCNQ1OT1-WT共转染组而言，miR-124-3p mimic组与KCNQ1OT1-WT共转染组细胞荧光素酶活性明显降低（P<0.05）；较miR-NC组与HMGB1-WT共转染组而言，miR-124-3p mimic组与HMGB1-WT共转染组细胞荧光素酶活性明显降低（P<0.05）。结论：LncRNA KCNQ1OT1可以靶向下调miR-124-3p mRNA表达，上调HMGB1 mRNA及HMGB1蛋白表达，促进高糖诱导HMC增殖，抑制凋亡，促进细胞纤维化发展。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the effects of long chain noncoding ribonucleic acid (LncRNA) KCNQ1OT1 targeting regulation on miR-124-3p/ high mobility group protein B1 (HMGB1) axis on proliferation, apoptosis and fibrosis of human mesangial cells (HMC) induced by high glucose. Methods: Human HMC were divided into control group (NC group), high glucose group (30 mmol/L glucose), negative sequence (si-NC) group, KCNQ1OT1 small interfering RNA (si-KCNQ1OT1) group, si-KCNQ1OT1+simulated control sequence (miR-NC) group, si-KCNQ1OT1+miR-124-3p inhibitor (miR-124-3p inhibitor) group, each group was treated with high glucose after transfection. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the LncRNA KCNQ1OT1 messenger ribonucleic acid (mRNA), miR-124-3p mRNA and HMGB1 mRNA expression. Methyl thiazolyl tetrazolium assay (MTT) was used to detect cell proliferation activity. The cell apoptosis rate was detected by flow cytometry. HMGB1 protein, Proliferation related protein (CyclinD1) and the cells of apoptotic proteins [cysteine proteinase 3 (caspase-3), cysteine proteinase 9 (caspase-9)] and the cells of fibrotic proteins [fibronectin (FN), intercelluar adhesion molecule-1 (ICAM-1)] expressions were detected by Western blot. Dual luciferase reporter assay was applied to verify the targeting relationship between LncRNA KCNQ1OT1 and miR-124-3p and HMGB1. Results: Compared with the NC group, the expression of KCNQ1OT1 mRNA, HMGB1 mRNA and HMGB1 protein, CyclinD1 protein, FN protein, ICAM-1 protein, HMC activity (24 h, 48 h and 72 h) in the high glucose group were significantly increased, the expression of miR-124-3p mRNA, caspase-3 protein and caspase-9 protein, HMC apoptosis rate were significantly decreased (P<0.05). Compared with the high glucose group and the si-NC group, the expression of KCNQ1OT1 mRNA, HMGB1 mRNA and HMGB1 protein, CyclinD1 protein, FN protein and ICAM-1 protein, HMC activity (24 h, 48 h and 72 h) in the si-KCNQ1OT1 group were significantly decreased, the expression of miR-124-3p mRNA, caspase-3 protein and caspase-9 protein, HMC apoptosis rate were significantly increased (P<0.05). Compared with the si-KCNQ1OT1 group and the si-KCNQ1OT1+miR-NC group, the expression of HMGB1 mRNA and HMGB1 protein, CyclinD1 protein, FN protein and ICAM-1 protein, HMC activity (24 h, 48 h and 72 h) in the si-KCNQ1OT1+miR-124-3p inhibitor group were significantly increased, the expression of miR-124-3p mRNA, caspase-3 protein and caspase-9 protein, HMC apoptosis rate were significantly decreased (P<0.05). Compared with the miR-NC group and the KCNQ1OT1-WT co transfection group, the luciferase activity of cells in the miR-124-3p mimic group and the KCNQ1OT1-WT co transfection group were significantly decreased (P<0.05). Compared with the miR-NC group and the HMGB1-WT co-transfection group, the luciferase activity of cells in the miR-124-3p mimic group and the HMGB1-WT co transfection group were significantly decreased(P<0.05). Conclusion: LncRNA KCNQ1OT1 can targetingly down-regulate the expression of miR-124-3p mRNA, up-regulate the expression of HMGB1 mRNA and HMGB1 protein, promote the proliferation of HMC induced by high glucose, inhibit apoptosis, and promote the development of cell fibrosis. |
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