miR-4463 Regulates Hypoxia-Induced Autophagy and Apoptosis by Targeting ULK1 in Endothelial Cells

Background: Our previous study revealed aberrant miR-4463 expression in the vascular tissues of patients with arteriosclerosis obliterans of the lower extremities (ASO), but the role of miR-4463 was largely ambiguous. In the current study, we aimed to explore the function of miR-4463 in hypoxia-induced endothelial cells and determine its molecular mechanisms. Methods: CCK-8 assay and flow cytometry were performed to evaluate cell viability and apoptosis. Adenovirus carrying mRFP-GFP-LC3 was employed to monitor cellular autophagy, and mitochondrial membrane potential was determined by JC-1 staining. Moreover, dual-luciferase reporter gene assay, qPCR, western blot and siRNA analysis were carried out to explore the potential molecular mechanisms. Results: Hypoxia significantly elevated the miR-4463 expression in primary human umbilical vein endothelial cells (HUVEC). Overexpression of miR-4463 inhibited hypoxia-induced autophagy by suppressing the formation of autophagosomes and autolysosomes, resulting in reduced cell viability and increased apoptosis, and these effects were reversed by miR-4463 inhibitor. Furthermore, activation of autophagy induced by miR-4463 inhibitor attenuated HUVECs apoptosis in hypoxic conditions. Mechanically, the results of the dual-luciferase reporter gene assay discovered that miR-4463 directly targeted Unc-51 like kinase 1 (ULK1). The silence of ULK1 blocked miR-4463 inhibitor-activated autophagy and further facilitated apoptosis under hypoxic conditions. Conclusions: Our findings indicate that miR-4463 is an essential regulator of hypoxia-induced autophagy and apoptosis in endothelial cells via directly targeting ULK1. Inhibition of miR-4463 might be a potential strategy to protect endothelial cells and maintain vascular function in patients with lower limb ischemia and its complications.

Abstract licence: CC BY

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