摘要
目的 探讨微小RNA(miR)-34a-5p/跨膜融合蛋白1(Notch1)信号轴介导内质网应激对缺氧/复氧(H/R)人心肌细胞的改善作用.方法 人心肌细胞随机分为对照组(Con-trol组)、缺氧复氧组(H/R组)、模拟物阴性对照组(mimic NC组)、模拟物组(mimic组)、抑制物阴性对照组(inhibitor NC组)、抑制物组(inhibitor组).除Control组外,其余组细胞建立H/R损伤模型.定量反转录聚合酶链式反应(qRT-PCR)法检测miR-34a-5p和Notch1表达量,噻唑蓝(MTT)法检测细胞存活率,流式细胞仪检测细胞凋亡率,双荧光素酶基因报告法检测miR-34a-5p和Notch1的靶向关系,蛋白印迹法检测转录激活因子6(ATF6)、肌醇需求酶1(IRE1)、蛋白激酶样内质网激酶(PERK)以及葡萄糖调节蛋白78(GRP78)表达量.结果 与Control组比较,H/R组miR-34a-5p表达量、细胞凋亡率以及ATF6、IRE1、PERK和GRP78蛋白表达量均升高,细胞存活率以及Notch1 mRNA和蛋白表达量均降低(P<0.05);与H/R组和mimic NC组比较,mimic组miR-34a-5p表达量、细胞凋亡率以及ATF6、IRE1、PERK和GRP78蛋白表达量均升高,细胞存活率以及Notch1 mRNA和蛋白表达量均降低(P<0.05);与H/R组和inhibitor NC组比较,inhibitor组miR-34a-5p表达量、细胞凋亡率以及ATF6、IRE1、PERK和GRP78蛋白表达量均降低,细胞存活率以及Notch1 mRNA和蛋白表达量均升高(P<0.05).结论 下调miR-34a-5p可抑制H/R人心肌细胞凋亡,其可能是通过靶向激活Notch1介导内质网应激发挥作用.
Abstract
Objective To investigate the improvement of endoplasmic reticulum stress mediated by microRNA (miR)-34a-5p/transmembrane fusion protein 1(Notch1) signaling axis on hypoxia/reoxygenation (H/R) human cardiomyocytes.Methods Human cardiomyocytes were randomly divided into Control group, H/R group, mimic NC group, mimic group, inhibitor NC group and inhibitor group.Except the Control group, H/R injury model was established in other groups.The expression levels of miR-34a-5p and Notch1 were detected by quantitative real-time polymerase chain reaction (qRT-PCR) , cell survival rate was detected by thiazolyl blue (MTT) , cell apopto-sis rate was detected by flow cytometry, and the targeting relationship between miR-34a-5p and Notch1 was detec-ted by dual luciferase gene reporting method.The expressions of transcriptional activator 6 (ATF6), inositol demand enzyme 1 (IRE 1), protein kinase - like endoplasmic reticulum kinase (PERK) and glucose regulatory protein 78 (GRP78) were detected by Western blot.Results miR-34a-5p targeted Notch1 (P<0.05); compared with Con-trol group, the expression level of miR-34a-5p, apoptosis rate and protein expressions of ATF6, IRE1, PERK and GRP78 in H/R group increased, while the cell survival rate and Notch1 mRNA and protein expressions decreased (P<0.05).Compared with H/R group and mimic NC group, miR-34a-5p expression, apoptosis rate, and protein expressions of ATF6, IRE1, PERK and GRP78 in mimic group increased, while cell survival rate and Notch1 mR-NA and protein expressions decreased (P<0.05).Compared with H/R group and inhibitor NC group, the expres-sion of miR-34a-5p, apoptosis rate and protein expressions of ATF6, IRE1, PERK and GRP78 decreased in inhibi-tor group, while cell survival rate and Notch1 mRNA and protein expressions increased (P<0.05).Conclusion miR-34a-5p can inhibit the apoptosis of H/R human cardiomyocytes, possibly through the targeted inhibition of Notch1-mediated endoplasmic reticulum stress.
基金项目
安徽高校自然科学研究项目(KJ2021ZD0027)
安徽医科大学第一附属医院与合肥中科离子医学技术装备有限公司合作项目(CIM-HT-2018-327)
国家科技期刊平台
NSTL
万方数据