The Role And Mechanism Of MiR-92a-3p In Apoptosis Of H9c2 Cardiomyocytes Induced By Ethanol

Background:The incidence of alcoholic cardiomyopathy is high year by year and the therapeutic effect is not effective.It is a general trend to find a new generation of molecular targeted therapy.miRNAs play an important regulatory role in the cardiovascular system and are expected to become a new generation of therapeutic targets.Exosomes show great advantages in drug delivery of miRNAs,and finding methods and basis for targeted delivery of miRNAs via exosomes will have great application prospects in the future.Objective:To find the differentially expressed miRNAs in the exosomes of H9c2 cardiomyocytes stimulated by ethanol,study the function of miRNAs in ethanol-induced apoptosis of H9c2 cardiomyocytes,explore the direct targets of their apoptotic regulation.Based on this,provides a new idea for diagnosis and treatment of alcoholic cardiomyopathy.Methods:Exosomes were collected in exosome-free medium,separated by differential centrifugation,and then detected by electron microscopy,particle size and exosome marker proteins.The uptake process of exosomes by H9c2 cardiomyocytes was observed by fluorescent dye tracer.The miRNAs in exosomes were sequenced and analyzed by Illumina high-throughput sequencing technology.Among the differentially expressed miRNAs,miR92a-3p related to the purpose of the study was selected,and the differentially expressed miR92a-3p was verified in H9c2 cell exosomes and cells by qRT-PCR.Annexin V-FITC/PI apoptosis detection kit and apoptosis-related antibody were used for apoptosis-related detection by flow cytometry and Western blotting,respectively.TargetScan was used to predict the direct targets of miR-92a-3p related to apoptosis.The targets were verified by qRT-PCR,Western blotting and double luciferase reporter gene assay.Results:Exosomes of H9c2 cardiomyocytes meeting the research criteria could be collected by differential centrifugation,and the process of exosomes uptake by H9c2 cardiomyocytes could also be observed.There were 123 differentially expressed miRNAs in ethanol-induced H9c2 cardiomyocyte exosomes,including 12 up-regulated miRNAs and 111 down-regulated miRNAs.miR-92a-3p was significantly overexpressed in both exosomes and cells of ethanol-stimulated H9c2 cardiomyocytes(P<0.001).After ethanol stimulation,H9c2 myocardial cells showed obvious apoptosis,and the apoptosis rate of miR-92a-3p mimics group was higher than the control group,the apoptosis rate of miR-92a-3p inhibitor group was lower than the control group,suggesting that miR-92a-3p plays a promoting role in the ethanolinduced appptosis of H9c2 myocardial cells.The apoptosis-related targets of miR-92a-3p include MSK2 and CREB3L2,suggesting that miR-92a-3p can inhibit the MSK2/CREB/Bcl-2 pathway through the targeted inhibition of MSK2 and CREB3L2,thus promoting the apoptosis of H9c2 cardiomyocytes.Conclusion:1.After ethanol stimulation,the expression level of miRNAs in the exosomes of H9c2 cardiomyocytes changed significantly,and miR-92a-3p was significantly overexpressed in both H9c2 cells and exosomes.2.Apoptosis of H9c2 cardiomyocytes was increased after ethanol stimulation,and miR92a-3p was involved in ethanol-induced apoptosis of H9c2 cardiomyocytes.3.miR-92a-3p can directly target MSK2 and CREB3L2 and inhibit their expression,miR92a-3p may be involved in regulating ethanol-induced apoptosis of H9c2 cardiomyocytes by targeting MSK2 and CREB3L2.