摘要
目的:探讨环状RNA(circRNA)-6874/miR-30a/基质相互作用分子2(STIM2)通路在缺氧/复氧(H/R)诱导的心肌细胞损伤中的作用及机制。方法:采用RNA结合蛋白免疫沉淀(RIP)和双荧光素酶报告基因实验检测circRNA-6874和miR-30a的结合情况、miR-30a是否靶向调控STIM2。心肌细胞转染或共转染后进行H/R处理,荧光定量聚合酶链式反应(RT-qPCR)检测circRNA-6874、miR-30a和STIM2的表达,细胞计数试剂盒(CCK-8)检测细胞存活力,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL)检测细胞凋亡,蛋白免疫印迹法(Western Blot)检测STIM2蛋白水平、生化分析仪检测乳酸脱氢酶(LDH)的释放、荧光分光光度计检测活性氧(ROS)的生成和荧光酶标仪检测Caspase-3活性。结果:circRNA-6874靶向结合miR-30a。H/R诱导心肌细胞表达circRNA-6874,其与miR-30a结合后抑制miR-30a的表达,进而促进下游靶基因STIM2的表达(P<0.05)。H/R处理明显抑制心肌细胞的存活力,促进LDH释放、ROS生成、凋亡增加和Caspase-3活性上调(P<0.05);分别转染circRNA-6874 siRNA、miR-30a mimics或STIM2 siRNA后,H/R的上述作用明显减弱(P>0.05);分别共转染circRNA-6874 siRNA和miR-30a inhibitors、miR-30a mimics和STIM2过表达载体后,H/R对心肌细胞的影响作用仍然明显(P<0.05)。结论:circRNA-6874/miR-30a/STIM2通路通过促进氧化应激和凋亡促进H/R诱导的心肌细胞损伤。
Objective:To explore the effect of circRNA-6874/miR-30a/stromal interaction molecule 2 pathway on cardiomyocyte injury induced by hypoxia/reoxygenation(H/R).Methods:RNA binding protein immunoprecipitation(RIP)and dual luciferase reporter gene assay were used to detect the binding of circRNA-6874 and miR-30a,and whether miR-30a,targeted STIM2 regulation.After transfection or co-transfection,cardiocytes were treated with H/R,and the expressions of circRNA-6874,miR-30a and STIM2 were detected by fluorescence quantitative polymerase chain reaction(RT-qPCR).Cell viability was detected by cell counting kit(CCK-8).Apoptosis was detected by dUTP notch end labeling(TUNEL)assay mediated by terminal deoxynucleotide transferase.The protein level of STIM2 was detected by Western Blot.The release of lactate dehydrogenase(LDH)was detected by biochemical analyzer.The production of reactive oxygen species(ROS)was detected by fluorescence spectrophotometer,and the activity of Caspase-3 was detected by fluorescence enzyme labelling instrument.Results:circRNA-6874 targeted miR-30a.H/R induced the expression of circRNA-6874 in cardiomyocytes,which inhibited the expression of miR-30a after binding with miR-30a,and then promoted the expression of downstream target gene STIM2(P<0.05).H/R treatment significantly inhibited the viability of cardiomyocytes,promoted LDH release,ROS production,increased apoptosis and up-regulation of Caspase-3 activity(P<0.05).After transfection with circRNA-6874 siRNA,miR-30a mimics or STIM2 siRNA,the above effects of H/R treatment were significantly weakened(P>0.05).After co-transfection of circRNA-6874 siRNA and miR-30a inhibitors,miR-30a mimics and STIM2 overexpression vectors,the effect of H/R on cardiomyocytes was still significant(P<0.05).Conclusion:The circRNA-6874/miR-30a/STIM2 pathway promotes H/R-induced cardiomyocyte injury by promoting oxidative stress and apoptosis.
作者
别自东
郭影
BIE Zidong;GUO Ying(Fei County People′s Hospital Affiliated of Shandong Medical College,Linyi 273400,Shandong,China)
出处
《中西医结合心脑血管病杂志》
2023年第11期1964-1970,共7页
Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease
基金
宁夏自然科学基金项目(No.2020AAC03366)。
