miR-192-5p通过靶向Notch3减轻顺铂所致的肾小管上皮细胞凋亡

miR-192-5p attenuates cisplatin-induced apoptosis of tubular epithelial cells by inhibiting expression of Notch3

目的:探讨Notch3在顺铂诱导的人肾小管上皮细胞(HK-2)损伤中的表达变化及其潜在的相关上游微小RNA(microRNA,miRNA)调控机制。方法:体外培养HK-2细胞,Western blot及实时定量PCR检测Notch3在顺铂(20μmol/L,24 h)诱导HK-2细胞凋亡模型及正常细胞中的表达;Targetscan预测靶向Notch3的miRNA,实时定量PCR测定相关miRNA在顺铂诱导HK-2细胞凋亡模型及正常细胞中的表达;然后,将HK-2细胞分别转染阴性对照模拟物(NC mimic)及miR-192-5p模拟物(miR-192-5p mimic),并依据给予或不给予顺铂处理(20μmol/L,24 h),分为NC mimic组、NC mimic+Cisplatin组、miR-192-5p mimic组、miR-192-5p mimic+Cisplatin组。Western blot及实时定量PCR检测Notch3的表达水平;流式细胞术检测细胞凋亡率,研究miR-192-5p对顺铂所致肾小管上皮细胞凋亡及Notch3表达的影响;荧光素酶报告实验证实miR-192-5p与靶基因Notch3的靶向关系。结果:Notch3在顺铂诱导肾小管上皮细胞凋亡中的表达上调,同时miR-192-5p在顺铂诱导肾小管上皮细胞凋亡中的表达下调;实时定量PCR证实转染miR-192-5p模拟物后,miR-192-5p的表达量升高;过表达miR-192-5p能够减轻顺铂诱导的肾小管上皮细胞凋亡,同时负向调控Notch3的表达;荧光素酶报告实验结果表明miR-192-5p直接靶向作用于Notch3的3′-UTR,抑制Notch3的表达,证实Notch3是miR-192-5p的直接靶标。结论:miR-192-5p可通过直接抑制Notch3的表达而减轻顺铂所致的肾小管上皮细胞凋亡。

Objective:To investigate the expression changes of Notch3 in cisplatin-induced renal tubular epithelial cells(HK-2)injury and the potential regulation mechanism related to upstream microRNA.Methods:Based on HK-2 cells apoptosis model induced by cisplatin(20μmol/L,24 h),Western blot and RT-qPCR were used to analyze the expression of Notch3 in HK-2 cells.Targetscan was used to search potential miRNAs that bind the 3’-UTR of Notch3.RT-qPCR was used to detect the expression of miR-192-5p in HK-2 cells after cisplatin treatment.Then,HK-2 cells transfected with negative control mimic(NC mimic)and miR-192-5p mimic respectively,and treated with or without cisplatin(20μmol/L,24 h),were divided into four groups:NC mimic group(NC);NC mimic+cisplatin group(NC+Cis);miR-192-5p mimic group(OE);miR-192-5p mimic+cisplatin group(OE+Cis).Western blot and RT-qPCR were used to demonstrate the effect of miR-192-5p on the expression of Notch3 after cisplatin treatment,while Western blot and flow cytometry were used to demonstrate the effect of miR-192-5p on apoptosis of HK-2 cells.Dual luciferase reporter assay was used to verified whether miR-192-5p targets Notch3.Results:Notch3 was up-regulated while miR-192-5p was down-regulated in HK-2 cells after cisplatin treatment compared with that in the normal controls.RT-qPCR showed that the expression of miR-192-5p in HK-2 cells was significantly increased after miR-192-5p mimic transfection.The overexpression of miR-192-5p alleviated cisplatin-induced apoptosis of HK-2 cells and negatively regulated the expression of Notch3.The dual luciferase reporter assay results verified that miR-192-5p directly binds with 3’-UTR of Notch3.Conclusion:miR-192-5p can attenuate the apoptosis of renal tubular epithelial cells induced by cisplatin through directly inhibiting the expression of Notch3.