摘要
目的探索在心肌缺血再灌注损伤(myocardial ischemia reperfusion injury,MIRI)中组蛋白3赖氨酸4三甲基化(trimethylation of lysine 4 on histone H3,H3K4me3)促进长链非编码RNA核富集转录本1(nuclear enriched abundant transcript 1,NEAT1)表达介导铁死亡的作用及机制。方法16只C57BL/6小鼠随机分为假手术组(sham)组和缺血/再灌注(I/R)组,采用氯化三苯基四氮唑(triphenyltetrazolium chloride,TTC)及苏木精-伊红(hematoxylin-eosin,HE)染色观察小鼠心肌梗死和病理结构变化;用H/R条件培养HL-1心肌细胞的方法复制体外MIRI模型,选择缺氧4 h(H4)及复氧1 h(H4/R1)、2 h(H4/R2)、4 h(H4/R4)时间点进行观察,用cell counting kit-8(CCK-8)法检测细胞活力,DCFH-DA探针检测细胞内活性氧(reactive oxygen species,ROS),比色法分别检测丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)和铁离子水平,用蛋白免疫印迹法(Western blot)检测心肌组织和细胞谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、长链酰基辅酶A合成酶4(acyl-CoA synthetase long-chain family member 4,ACSL4)、转铁受体蛋白1(transferrin receptor 1,TfR1)、赖氨酸甲基转移酶2A(lysine methyltransferase 2A,KMT2A)、赖氨酸特异性组蛋白去甲基酶1A(lysine-specific demethylase 1A,KDM1A)等蛋白质表达水平及H3K4me3水平,qRT-PCR法检测心肌组织和细胞NEAT1表达水平;用慢病毒载体稳定转染细胞敲低NEAT1后进行H4/R4处理,观察细胞铁死亡各项指标变化;再用特异性KMT2A抑制剂MM-102(50μmol/L)预处理细胞2 h,或用慢病毒载体稳定转染细胞敲低KMT2A或过表达KDM1A后进行H4/R4处理,观察H3K4me3和NEAT1表达水平变化。结果与sham组相比,I/R组小鼠心肌梗死面积约41%,心肌组织GPX4蛋白水平降低,ACSL4和TfR1蛋白水平增加,NEAT1表达水平升高,H3K4me3和KMT2A蛋白水平增加,KDM1A蛋白水平降低(P<0.05);与Control组相比,缺氧后随着复氧时间延长,细胞活力下降,ROS、MDA、Fe3+和Fe2+水平均升高,GSH含量降低,GPX4蛋白水平降低,ACSL4和TfR1蛋白水平增加,NEAT1表达水平增加,H3K4me3和KMT2A蛋白水平增加,KDM1A蛋白水平降低(P<0.05);与敲低NEAT1 NC组相比,敲低NEAT1+H4/R4组ROS、MDA、Fe3+和Fe2+均降低,GSH升高,GPX4蛋白表达升高,ACSL4和TfR1蛋白表达下降(P<0.05);与H4/R4组相比,MM-102+H4/R4组H3K4me3水平和NEAT1表达水平均降低(P<0.05);与敲低KMT2A Vector+H4/R4组相比,敲低KMT2A+H4/R4组H3K4me3和NEAT1表达水平降低(P<0.05);与过表达KDM1A Vector+H4/R4组相比,过表达KDM1A+H4/R4组H3K4me3和NEAT1表达水平降低(P<0.05)。结论MIRI诱导NEAT1表达上调可介导心肌细胞铁死亡,NEAT1表达上调可能是KMT2A表达增多和KDM1A表达减少导致NEAT1基因H3K4me3修饰水平增高引起的。
Objective To explore the role and mechanism of histone H3K4me3 in promoting long non-coding RNA nuclear enriched abundant transcript 1(NEAT1)expression to mediate ferroptosis in myocardial ischemia-reperfusion injury(MIRI).Methods Sixteen C57BL/6 mice were randomly divided into sham group and ischemia/reperfusion(I/R)group.Myocardial infarction and pathological changes were assessed using triphenyltetrazolium chloride(TTC)and hematoxylin-eosin(HE)staining.An in vitro MIRI model was established by subjecting HL-1 cardiomyocytes to hypoxia/reoxygenation(H/R).Cells were observed at hypoxia for 4 h(H4)followed by reoxygenation for 1 h(H4/R1),2 h(H4/R2),or 4 h(H4/R4).Cell viability was measured using the CCK-8 assay.Intracellular reactive oxygen species(ROS)levels were detected using the DCFH-DA probe.The levels of malondialdehyde(MDA),glutathione(GSH),and iron levels via colorimetric assays.The protein expression levels of glutathione peroxidase 4(GPX4),acyl-CoA synthetase long-chain family member 4(ACSL4),transferrin receptor protein 1(TfR1),lysine methyltransferase 2A(KMT2A),lysine-specific histone demethylase 1A(KDM1A),and the level of trimethylation of lysine 4 on histone H3(H3K4me3)in myocardial tissue and cells were detected by Western blot.NEAT1 expression levels in myocardial tissues and cells were detected by qRT-PCR method.NEAT1-knockdown cells(via lentiviral transduction)underwent H4/R4 treatment to assess ferroptosis markers.Cells pretreated with KMT2A-specific inhibitor MM-102(50μmol/L,2 h),or subjected to KMT2A-knockdown via lentivirus,or KDM1A-overexpression via lentivirus followed by H4/R4 treatment to evaluate H3K4me3 and NEAT1 expression.Results Compared with the sham group,the I/R group exhibited 41%myocardial infarction area,and ecreased GPX4,d increased ACSL4/TfR1 protein levels,and elevated NEAT1 expression,higher H3K4me3 and KMT2A,but lower KDM1A protein levels(P<0.05).Compared with the Control group,prolonged reoxygenation after hypoxia,cell viability decreased,the levels of ROS,MDA,Fe 3+and Fe 2+all increased,GSH content decreased,GPX4 protein level decreased,ACSL4 and TfR1 protein levels increased,NEAT1 expression level increased,H3K4me3 and KMT2A protein levels increased,and KDM1A protein level decreased(P<0.05).Compared with the NEAT1 knockdown NC group,the NEAT1 knockdown+H4/R4 group exhibited decreased ROS,MDA,Fe 3+and Fe 2+levels,increased GSH,increased GPX4 protein expression,and decreased ACSL4 and TfR1 protein expressions(P<0.05).Compared with the H4/R4 group,the H3K4me3 level and NEAT1 expression level both decreased in the MM-102+H4/R4 group(P<0.05).Compared with the KMT2A Vector knockdown+H4/R4 group,the H3K4me3 and NEAT1 expression levels decreased in the KMT2A knockdown+H4/R4 group(P<0.05).Compared with the KDM1A Vector overexpression+H4/R4 group,the H3K4me3 and NEAT1 expression levels decreased in the KDM1A overexpression+H4/R4 group(P<0.05).Conclusion MIRI-induced upregulation of NEAT1 mediates cardiomyocyte ferroptosis,likely through increased H3K4me3 modification at the NEAT1 gene locus via elevated KMT2A and reduced KDM1A expression.
作者
张荣瑞
丁菁
梁政伟
谢莹
吕莎
张倩
柴鑫
陆德琴
ZHANG Rongrui;DING Jing;LIANG Zhengwei;XIE Ying;LYU Sha;ZHANG Qian;CHAI Xin;LU Deqin(Guizhou Provincial Key Laboratory of Pathogenesis and Drug Research on Common Chronic Diseases,Guiyang 550025,Guizhou,China;Department of Pathophysiology,School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China;Department of Radiology,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China)
出处
《贵州医科大学学报》
2025年第8期1120-1131,1143,共13页
Journal of Guizhou Medical University
基金
国家自然科学基金项目(32160206)
贵州省科技厅基础研究计划项目(黔科合基础-ZK-2024-249)。
