摘要
目的探讨LncRNA Mirt2在心肌细胞H9C2缺氧复氧损伤过程中的作用及其机制。方法检测LncRNA Mirt2在急性心肌损伤患者血液中的水平。建立H9C2心肌细胞缺氧复氧模型,检测LncRNA Mirt2在H9C2缺氧复氧模型中的表达。采用脂质体转染法将LncRNA Mirt2 mimics和阴性对照转染入H9C2细胞中,qRT-PCR验证转染效率。实验分为对照组、缺氧复氧组、过表达对照组和Mirt2过表达组。Western blot分析IL-1β和IL-6的蛋白表达;EILSA分析IL-1β和IL-6浓度变化;CCK-8分析细胞活力,Caspase-3相对活性检测,TUNEL染色分析细胞凋亡;Western blot分析核糖聚合酶(PARP)、剪切的核糖聚合酶(Cleaved PARP)、Caspase-3前体(Pro-Caspase-3)、Caspase-3剪切体(Cleaved Caspase-3)、丝裂原活化蛋白激酶磷酸酶-1(MKP-1)、c-Jun氨基末端激酶(t-JNK)、磷酸化c-Jun氨基末端激酶(p-JNK),t-c-Jun,磷酸化c-Jun蛋白(p-c-Jun)的蛋白表达。结果Mirt2在急性心肌损伤患者血液中表达下调,同时在H9C2缺氧2 h复氧12 h细胞中表达下调。在H9C2细胞缺氧复氧模型中:与对照组相比,Mirt2过表达组炎性因子IL-1β和IL-6的表达显著降低;CCK-8实验和凋亡实验检测结果表明,与过表达对照组相比,Mirt2过表达组细胞活力增强,凋亡数减少;Western blot结果表明,与过表达对照组相比,过表达Mirt2减少Cleaved PARP/Cleaved Caspase-3的表达,p-JNK、p-c-Jun的表达并增加了MKP-1的蛋白表达。结论Mirt2在缺氧复氧诱导的H9C2心肌细胞损伤过程中发挥保护作用,其作用机制可能是通过激活MKP-1/JNK通路。
Objective To investigate the role of LncRNA Mirt2 in the process of hypoxic reoxygenation injury in cardiomyocytes H9 C2 and its mechanism.Methods The expression of lncRNA Mirt2 in acute cardiac myocardial injury cells was analyzed.A hypoxia-reoxygenation model of H9 C2 cardiomyocytes was established to detect the expression of LncRNA Mirt2 in the H9 C2 hypoxia-reoxygenation model.LncRNA Mirt2 mimics and negative control(NC)were transfected into H9 C2 cells by liposome transfection method.qRT-PCR was performed to verify the transfection efficiency.The cells were divided into control,hypoxic reoxygenation,overexpression control and Mirt2 overexpression groups.Western blotting analyzed IL-1βand IL-6 protein expression.EILSA analyzed IL-1βand IL-6 concentration changes.Cell viability was assessed by CCK-8,Caspase-3 relative activity was analyzed,and cell apoptosis evaluated by TUNEL staining.Western blot analysis was performed to analyze PARP,cleaved PARP,Pro-Caspase-3,Cleave Caspase-3,MKP-1,and t-JNK,p-JNK,t-c-Jun,p-c-Jun protein expression.Results Mirt2 was down-regulated in the blood of patients with acute myocardial injury and concurrently in H9 C2 cells with hypoxia 2 h/reoxygenation 12 h.In the hypoxia/reoxygenation model of H9 C2 cells,the expression of inflammatory factors IL-1βand IL-6 was significantly reduced in the Mirt2 overexpression group compared with the control group.CCK-8 assay and apoptosis assay results showed that the cell viability was enhanced and the number of apoptosis was reduced in the Mirrt2 overexpression group compared with the control group.Western blotting results indicated that,compared with the control group,overexpression of Mirt2 decreased the expression of Cleaved PARP/Cleaved Caspase-3,p-JNK,p-c-Jun and increased the protein expression of MKP-1 compared with the control group.Conclusion Mirt2 plays a protective role in hypoxia-reoxygenation-induced H9 C2 cardiomyocyte injury,and its mechanism of action may be related with activation of the MKP-1/JNK pathway.
作者
申健
李梦豪
邓焕堂
李志明
何丽珍
张宇
罗艳芳
谢雄伟
刘锦光
Shen Jian;Li Menghao;Deng Huantang(Department of Cardiovascular Medicine,Huizhou Municipal Central Hospital of Guangdong Province,Huizhou 516001,China)
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2021年第1期50-55,共6页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
广东省医学科研基金资助项目(No.A2020163)。
