Overexpression Of Exosomal Cardioprotective MiRNAs Mitigates Hypoxia-Induced H9c2 Cells Apoptosis

Oxygen is indispensable substance to mammalian cells in maintaining cell normal metabolism,a variety of biochemical reactions in living cells can run smoothly under in sufficient oxygen concentrations.In recent years,the incidence of ischemic/hypoxic cardiovascular disease is on the rise,such as acute myocardial infarction,angina pectoris,the low oxygen environment inside the heart caused by ischemic/hypoxic cardiovascular disease can induce myocardial cell apoptosis.miRNA is a class of endogenous non-coding RNA in eukaryotic cells,and regulate target gene expression mainly through mediating mRNA degradation or inhibiting translation.Exosome is a vesicle structure with a diameter of approximate 100 nm secreted by living cells and play an important role in intercellular communication by transporting cytosolic components including miRNAs,mRNAs,and proteins.Recent studies have found that different types of cardiac cells can secrete miRNA-enriched exosome and involve in a variety of physiological or pathological processes.In this study,H9c2 cells were used as experimental materials to establish hypoxia model of cardiomyocytes in vitro.Small RNA-seq was used to identify the discrepant transcripts of hypoxia-induced H9c2 cells and corresponding exosome and to excavate the hypoxia-related miRNAs in cardiomyocytes,and to elucidate the potential function of candidate miRNAs in hypoxic adaptability of cardiomyocytes by overexpression and inhibition of miRNAs,in order to provide basic data for the detection and treatment of ischemic/hypoxic cardiovascular disease and the animal molecular breeding of anti-plateau hypoxia.The main results of this study are as follows:(1)Hypoxia stress can inhibit H9c2 cell activity,destroy cell membrane integrity,and induce apoptosis.(2)Using small RNA-seq,338,331 mature miRNAs were identified from normoxic and hypoxic H9c2 cells respectively.Among them,there were 316 overlappingmiRNA.A total of 92 differentially-expressed miRNAs were screened in H9c2 cells after hypoxia according to the standard(miRNA expression:fold-change<0.5 or>2).By comparing with previous reports,31 hypoxamiRs were found to besignificantly enriched in hypoxic H9c2 cells differentially expressed miRNA.(3)Exosome was isolated and purified from normoxic and hypoxic H9c2 cells culture medium by ultracentrifuge.NTA analysis found that the particle size distribution was in 50-200nm,which was similar to the typical distribution characteristics of exosomal particle size.In addition,exosome concentration significantly increased after hypoxia,which indicated that environmental stress(such as hypoxia)can promote cell exosome production.A total of 74,144 mature miRNAs were identified from the exosome produced by normoxic and hypoxic H9c2 cells using small RNA-seq,which indicated that hypoxia significantly altered the miRNA species in the exosome.(4)A total of 62 differentially expressed miRNAs were screened in the exosome after hypoxia according to the standard(miRNA expression:fold-change<0.5 or>2),all of which were up-regulated in the hypoxic exosome and there were 25 known hypoxamiR.The expression of top 12 miRNA showed that hypoxia hadlittle effect on thetypes of high abundance miRNA but significantly changed the expression levels of these miRNAs.Functional enrichment analysis by DIANA revealed that the differential miRNAs in exosome were mainly involved in the HIF-1 signalling pathway and apoptosis-related pathways including the TNF,MAPK,and mTOR pathways.(5)The results of overexpression and inhibition of known anti-apoptotic miRNA(miR-21-5p,miR-378-3p)were consistent with previous studies,which proved the feasibility of hypoxic model in this study and the accuracy of small RNA-seq.The results of overexpression and inhibition of novel potential anti-apoptotic miRNA(miR-152-3p、let-7i-5p)showed that miR-152-3p and let-7i-5p could inhibit hypoxia-induced H9c2 cells damage and mitigate hypoxia-induced apoptosis.(6)Dual luciferase reporter assay confirmed that Atg12 and Faslg are apoptosis-related targets of miR-152-3p and let-7i-5p,respectively.Overexpression and inhibition of miRNA showed that miR-152-3p and let-7i-5p could mitigate the hypoxia-induced apoptosis by inhibiting the apoptosis-related target genes Atg12 and Faslg,and then affected the mitochondrial apoptotic pathway and death receptor apoptosis pathway.In conclusion,this study revealed that exosomes derived from H9c2 cells in response to hypoxia loaded with large amounts of cardioprotective miRNA,which can mitigate hypoxia-induced H9c2 cells apoptosis and improve cell hypoxia adaptability.Moreover,this study demonstrated for the first time that miR-152-3p and let-7i-5p,which were enriched in exosomes derived from H9c2 cells under hypoxia,mitigated hypoxia-induced H9c2 cells apoptosis via the mitochondrial and the death receptor pathways,respectively.