摘要
目的探讨miR-124-5p在心肌细胞缺氧/复氧(hypoxia/reoxygenation,H/R)损伤中的作用及其机制。方法通过将H9c2细胞置于缺氧环境(1%O^(2)、94%N2和5%CO^(2))中培养不同时间后再复氧24 h,制作心肌细胞H/R损伤模型;转染miR-124-5p mimic及miR-124-5p靶向基因质粒改变miR-124-5p及其靶向基因表达水平;qRT-PCR检测miR-124-5p表达水平,Western blot检测相应靶基因蛋白表达水平;CCK-8法检测细胞活力,TUNEL染色法和Annexin V-FITC/PI双染法流式细胞术检测细胞凋亡,通过检测细胞中肌酸激酶(creatine kinase,CK)和乳酸脱氢酶(lactic dehydrogenase,LDH)活性评估心机受损情况;生物信息学分析miR-124-5p的作用靶点并利用双荧光素酶报告实验进行验证。结果H9c2细胞活力及miR-124-5p表达水平随缺氧时间延长逐渐降低。与H/R+miR-NC组相比,H/R+miR-124-5p mimic组细胞活力增加,而CK及LDH活性、TUNEL阳性细胞比例和凋亡水平均降低。miR-124-5p靶向抑制TRAF6表达,过表达TRAF6抑制miR-124-5p对H/R损伤的心肌细胞的保护作用。结论miR-124-5p可通过对TRAF6的靶向负调控而抑制心肌细胞的H/R损伤。
Objective To investigate the role and mechanism of miR-124-5p in myocardial cell injury induced by hypoxia/reoxygenation(H/R).Methods The H9c2 cells were cultured under hypoxic conditions(1%O2,94%N2,and 5%CO2)for varying durations,followed by reoxygenation for 24 hours to establish an H/R injury model.The expression levels of miR-124-5p and its target genes were altered by transfecting miR-124-5p mimic and target gene plasmids.The expression level of miR-124-5p was assessed by qRT-PCR,and the protein expression of corresponding target genes was analyzed by Western blot.Cell viability was measured using CCK-8 assay,apoptosis was detected by TUNEL staining and Annexin V-FITC/PI double staining flow cytometry,and myocardial injury was assessed by measuring the activity of creatine kinase(CK)and lactic dehydrogenase(LDH).Bioinformatics analysis was used to predict the targets of miR-124-5p,and a dual-luciferase reporter assay was performed for validation.Results The viability of H9c2 cells and miR-124-5p expression gradually decreased with prolonged hypoxia.Compared to the H/R+miR-NC group,the H/R+miR-124-5p mimic group showed increased cell viability,while CK and LDH activity,the proportion of TUNEL-positive cells,and apoptosis levels were reduced.miR-124-5p targeted and inhibited TRAF6 expression,while overexpression of TRAF6 suppressed the protective effect of miR-124-5p on H/R-injured cardiomyocytes.Conclusion miR-124-5p could inhibits H/R-induced myocardial injury by negatively targeting TRAF6 expression.
作者
王雪
付建平
牛媛媛
牟丽娜
李勇
李敬
Wang Xue;Fu Jianping;Niu Yuanyuan;Mou Lina;Li Yong;Li Jing(Drug Preparation Room,Hengshui People’s Hospital,Hengshui 053000,China;Department of Cardiology,Hengshui People’s Hospital,Hengshui 053000,China;Department of Emergency,Hengshui People’s Hospital,Hengshui 053000,China)
出处
《中国组织化学与细胞化学杂志》
CSCD
2024年第6期547-554,共8页
Chinese Journal of Histochemistry and Cytochemistry
基金
衡水市科技计划项目(2021014084Z)
河北省卫健委医学科学研究课题计划项目(20232173)。
作者简介
王雪,男(1987年),汉族,药师;通讯作者:付建平,fujianpingzzss22@yeah.net。
