Abstract
Recent evidence suggests that hypoxia caused by acute myocardial infarction can induce cardiomyocyte apoptosis. Exosomes are signalling mediators that contribute to intercellular communication by transporting cytosolic components including miRNAs, mRNAs, and proteins. However, the systemic regulation and function of exosomal miRNAs in hypoxic cardiomyocytes are currently not well understood. Here, we used small RNA sequencing to investigate the effects of hypoxia stress on miRNAome of rat cardiomyoblast cells (H9c2) and corresponding exosomes. We identified 92 and 62 miRNAs in cells and exosomes, respectively, that were differentially expressed between hypoxia and normoxia. Hypoxia strongly modulated expression of hypoxia-associated miRNAs in H9c2 cells, and altered the miRNAome of H9c2 cells-derived exosomes. Functional enrichment analysis revealed extensive roles of differentially expressed exosomal miRNAs in the HIF-1 signalling pathway and in apoptosis-related pathways including the TNF, MAPK, and mTOR pathways. Furthermore, gain- and loss-of-function analysis demonstrated potential anti-apoptotic effects of the hypoxia-induced exosomal miRNAs, including miR-21-5p, miR-378-3p, miR-152-3p, and let-7i-5p; luciferase reporter assay confirmed that Atg12 and Faslg are targets of miR-152-3p and let-7i-5p, respectively. To summarize, this study revealed that hypoxia-induced exosomes derived from H9c2 cells loaded cardioprotective miRNAs, which mitigate hypoxia-induced H9c2 cells apoptosis.
Highlights
Acute myocardial infarction (AMI), a common consequence of coronary artery occlusion and myocardial ischemia, is one of the leading causes of death worldwide [1,2]
A previous study demonstrated that hypoxia induced cardiomyocyte apoptosis, which was involved in the pathogenesis of AMI [15]
Cotransfection of pmirGLO-Faslg 3 -UTR and let-7i-5p mimic into HeLa cells demonstrated that let-7i-5p was capable of targeting the 3 -UTR of Faslg mRNA (0.79-fold change in luciferase activity, p = 0.016; Figure 5E). These results indicate that Atg12 and Faslg are the respective targets of miR-152-3p and let-7i-5p. miR-152-3p directly targeted Atg12 and activated the anti-apoptotic gene Bcl-2, attenuating hypoxia-induced H9c2 cells apoptosis [41]; simultaneously, let-7i-5p played an anti-apoptotic role by inhibiting Faslg and, attenuating the cell death-inducing signalling cascade [42,43]
Summary
Acute myocardial infarction (AMI), a common consequence of coronary artery occlusion and myocardial ischemia, is one of the leading causes of death worldwide [1,2]. Hypoxia caused by AMI leads to cardiomyocyte apoptosis [3]. Apoptosis is a process of programmed cell death that is actively controlled by a series of genes to maintain homeostasis [4]. HIF-α (HIF-1α, HIF-2α and HIF-3α) is activated and translocates from the cytoplasm to the nucleus, where it dimerizes with HIF-β and binds to hypoxia response elements (HRE) in the promoters of HIF-controlled genes [6]. Continuous hypoxic stress will eventually lead to apoptosis, or even necrosis [8]
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