miR-878靶向调控Pim1促进线粒体分裂导致心肌细胞缺氧/复氧损伤  

MiR-878 Aggravates Hypoxia/Reoxygenation Injury in H9c2 Cardiomyocytes by Inducing Pim1-mediated Mitochondrial Fission

作　　者：胡淑文 张晶晶 白明[4,5,6] 牛小伟 HU Shu-Wen;ZHANG Jing-Jing;BAI Ming;NIU Xiao-Wei(The First Clinical College of Lanzhou University,Lanzhou 730099,China;Maternal and Child Health Care Hospital of Gansu Province/Medical Genetic Center of Gansu Central Hospital,Lanzhou 730079,China;Clinical Medical Research Center for Birth Defects and Rare Diseases of Gansu Province,Lanzhou 730210,China;Heart Center,The First Hospital of Lanzhou University,Lanzhou 730099,China;Gansu Clinical Medical Research Center for Cardiovascular Diseases,Lanzhou 730099,China;Gansu Provincial Key Laboratory of Cardiovascular Diseases,Lanzhou 730099,China)

机构地区：[1]兰州大学第一临床医学院,兰州730099 [2]甘肃省妇幼保健院/甘肃省中心医院,医学遗传中心,兰州730079 [3]甘肃省出生缺陷与罕见病临床医学研究中心,兰州730210 [4]兰州大学第一医院心脏中心,兰州730099 [5]甘肃省心血管病临床医学研究中心,兰州730099 [6]甘肃省心血管疾病重点实验室,兰州730099

出　　处：《生物化学与生物物理进展》2024年第4期912-923,共12页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(82000277,82060807);甘肃省科技计划(21JR1RA100);甘肃卫生行业科研计划(GSWSKY2020-64);兰州市科技计划(2020-ZD-72);甘肃省教育厅青年博士基金(2022QB-011);甘肃省军民融合发展专项(2060303);兰州大学第一医院院内基金(ldyyyn2021-115)资助项目。

摘　　要：目的心肌缺血/再灌注(MI/R)损伤是导致急性心肌梗死患者不良心血管结局的重要原因。然而,目前对MI/R损伤的分子机制仍不明确。本文旨在确定微小RNA-878(miR-878)对MI/R损伤的影响及其分子机制。方法在H9c2细胞中建立缺氧/复氧(H/R)模型。采用CCK-8法检测细胞活力。采用生化试剂盒检测乳酸脱氢酶(LDH)含量。流式细胞术分析细胞凋亡水平。采用免疫荧光法及激光共聚焦显微镜分析线粒体形态。采用免疫荧光法检测线粒体活性氧(mtROS)水平。使用双荧光素酶报告基因实验研究miR-878与Pim1的结合位点。RNA免疫沉淀(RIP)实验验证miR-878与Pim1的结合关系。实时荧光定量PCR(RT-qPCR)和蛋白质印迹法(Western blot)检测基因的表达水平。结果与对照组相比,miR-878在H/R处理的H9c2细胞中表达显著升高((1.00±0.25)vs(9.70±2.63),P<0.01)。在H/R诱导的细胞中,转染miR-878抑制剂能够显著增加细胞活力((46.67±3.00)vs(74.62±4.08),P<0.0001),并降低LDH释放量((358.58±41.71)vs(179.09±15.59),P<0.0001)及细胞凋亡率((43.41±0.72)vs(27.42±4.48),P<0.01)。同时,下调miR-878表达能够显著抑制DRP1介导的线粒体过度分裂及mtROS产生((6.60±0.57)vs(4.32±0.91),P<0.0001)。机制研究显示,miR-878能够靶向结合Pim1 mRNA的3'-UTR区域并抑制Pim1的表达水平。挽救实验证明,下调Pim1表达能够显著逆转miR-878抑制剂抗H9c2细胞损伤的作用(均P<0.01),并出现线粒体过度分裂及mtROS产生增加(均P<0.05)。结论在H/R条件下,miR-878通过靶向抑制Pim1表达而促进DRP1介导的线粒体过度分裂,最终导致心肌细胞损伤。Objective Acute myocardial infarction(AMI)is a highly prevalent and deadly disease globally,with its incidence continuing to rise in recent years.Timely reperfusion therapy is crucial for improving the prognosis of AMI patients.However,myocardial reperfusion can lead to irreversible myocardial ischemia/reperfusion(MI/R)injury,which is associated with adverse cardiovascular outcomes following AMI.Studies have shown that microRNAs(miRNAs)are abnormally expressed during MI/R injury and play an important role in the fate of cardiomyocytes.Effective preventive and therapeutic strategies against MI/R injury remain lacking in clinical practice,necessitating elucidation of the molecular mechanisms underlying MI/R onset and progression.This study investigated the role of microRNA-878(miR-878)in the regulation of mitochondria-mediated apoptosis in MI/R injury.Methods The H9c2 cells were flushed with a gas mixture containing 1%O2,5%CO2 and 94%N2 for 3 h.Then the cells were incubated in complete culture medium under 5%CO2 and 95%air for 6 h to mimic in vivo hypoxia/reoxygenation(H/R)injury.Cell viability were detected by CCK-8 assay.The concentrations of lactate dehydrogenase(LDH)were then measured.The level of apoptosis was analyzed by flow cytometry.The morphology of mitochondria was analyzed by immunofluorescence and laser confocal microscopy.The levels of mitochondrial reactive oxygen species(mtROS)were detected by immunofluorescence.Dual luciferase reporter gene assay was used to study the binding site of miR-878 and Pim1.RNA immunoprecipitation(RIP)assay was used to verify the binding relationship between miR-878 and Pim1.The gene expression levels were detected by real-time fluorescent quantitative PCR(RT-qPCR)and Western blot.Results The study found that compared with the control group,the expression of miR-878 in H/R-treated H9c2 cells was significantly increased((1.00±0.25)vs(9.70±2.63),P<0.01).In H/R-induced cells,transfection of miR-878 inhibitor significantly increased cell viability((46.67±3.00)vs(74.62±4.0

关 键 词：miR-878 Pim1 发动蛋白相关蛋白1 心肌缺血/再灌注损伤 

分 类 号：R542.4[医药卫生—心血管疾病]