Effect Of Endothelial Cell-derived Extracellular Vesicle MiRNA On Cardiomyocyte Apoptosis In The Context Of Hypoxia Reoxygenation

Background: Extracellular vesicles(EVs)are a type of phospholipid bilayer membrane structure released by cells through membrane budding or endosomal exocytosis,and can be found in almost all body fluids.As a multifunctional signaling complex,EVs transmit information between cells and play various biological functions.In patients with coronary heart disease,endothelial cells release a large number of EVs during the process of ischemia-reperfusion,and these EVs and their contents,such as mi RNA,may participate in the process of myocardial cell reperfusion apoptosis.Objective : In this study,endothelial cells were used as an in vitro source of EVs to construct a hypoxia-reoxygenation model.The impact of endothelial cell-derived EVs on myocardial cell apoptosis during hypoxia-reoxygenation was investigated,and the expression changes of four mi RNA in EVs,namely let-7c-5p,mi R-30d-5p,mi R-181a-5p,and mi R-423-3p,were analyzed to predict their potential modes of action in this process.Method: First,plasma samples were collected from coronary heart disease patients undergoing percutaneous coronary intervention(PCI)at the First Affiliated Hospital of Henan University.Real-time fluorescent quantitative PCR technology was used to quantify the expression of EV-associated mi RNA including let-7c-5p,mi R-30d-5p,mi R-181a-5p,and mi R-423-3p.Subsequently,a human umbilical vein endothelial cell model was constructed to analyze the expression of these mi RNA in EVs under hypoxia/reoxygenation conditions.Finally,EVs derived from the endothelial cell hypoxia/reoxygenation model were used to treat hypoxic cardiomyocytes,followed by reoxygenation culture,and the effects of EVs on cardiomyocyte apoptosis and viability were studied using flow cytometry and CCK-8 assays.The meaningful mi RNAs were then subjected to bioinformatics analysis using software such as R version 4.0.2,Fun Rich,and Cytoscape to predict relevant target genes.Results: The real-time PCR results showed that the expression of let-7c-5p,mi R-30d-5p and mi R-181a-5p in plasma extracellular vesicles in patients with coronary heart disease were significantly reduced after PCI surgery,and the expression of extracellular vesicles let-7c-5p,mi R-30d-5p and mi R-181a-5p in the supernatant of endothelial cells after reoxygenation also decreased after reoxygenation.Co-culture of endothelial cell-derived extracellular vesicles with cardiomyocytes showed that post-oxygenated endothelial extracellular vesicles reduced apoptosis in cardiomyocytes,and this effect was significantly better than hypoxic endothelial-derived extracellular vesicles.We hypothesize that three mi RNA,let-7c-5p,mi R-30d-5p,and mi R-181a-5p,may play an important role in this process,so we predict the relevant target genes.Through prediction,we found that these three mi RNAs can target and regulate a variety of apoptosis-related genes such as BCL2,MCL1,TNFRSF10 B,MYC,ATG5,ATM,XIAP,CDC25 A,etc.,and may be involved in apoptosis-related signaling pathways such as TRAIL signaling pathway,S1 P pathway,AP-1 transcription factor network,apoptosis,ATM pathway,etc.Conclusion: During reoxygenation,endothelial cell-derived extracellular vesicles can protect myocardial cells from reperfusion injury-induced apoptosis.During the process of reoxygenation,the expression of let-7c-5p,mi R-30d-5p,and mi R-181a-5p in endothelial cell-derived extracellular vesicles decreases,which may participate in the protective effect against myocardial cell apoptosis by targeting apoptotic genes such as BCL2,MCL1,TNFRSF10 B,MYC,ATG5,ATM,XIAP,and CDC25 A,and may be involved in apoptotic signaling pathways such as the TRAIL pathway,S1 P pathway,AP-1 transcription factor network,apoptosis,and ATM pathway.