Abstract 15321: MicroRNA Mir-378-3p is a Novel Regulator of Endothelial Autophagy and Function

Abstract

Introduction: Autophagy maintains cellular homeostasis by degradation of unnecessary proteins for their recycling and reuse. MicroRNAs (miRNAs) regulate various biological processes including autophagy. MiRNA and autophagy both regulate endothelial function, however, role of miRNAs in endothelial autophagy and function is unknown. Methods and Results: To understand the role of miRNAs in endothelial autophagy, autophagy was genetically inhibited via ATG7 (autophagy-related gene 7) silencing in Human Umbilical Vein Endothelial Cells (HUVECs) and then a miRNA array was performed, which showed miR-378-3p as the most up-regulated miRNA. Pharmacological (via chloroquine treatment) inhibition of autophagy in HUVECs resulted in significant upregulation of miR-378a-3p. Autophagy activation via starvation in HUVECs significantly reduced miR-378a-3p expression. Next, we over-expressed miR-378a-3p (via mimic) in HUVECs and measured autophagy and endothelial function. MiR-378a-3p overexpression was associated with impaired autophagy (reduced LC3-II/LC3-I ratio), reduced proliferation, and increased migration in HUVECs. At the molecular level, miR-378a-3p overexpression was associated with reduced eNOS expression at both transcript and protein levels in HUVECs. MiR-378a-3p inhibition (via antagomir) led to reduced cell migration and upregulation of eNOS in HUVECs. In-silico study identified PDIA4 as a potential target of miR-378-3p, and our qPCR data and immunoblotting data confirmed increased and reduced PDIA4 expression in mimics and antagomir-transfected HUVECs, respectively. We then inhibited autophagy using chloroquine (50uM) and observed significantly reduced PDIA4 expression. Inhibition of PDIA4 expression via silencing inhibited autophagic flux indicated by reduced LC3-II/LC3-I in HUVECs. Conclusions: Our findings, for the first time, show an inverse relationship between miR-378a-3p expression and endothelial autophagy and endothelial function. PDIA4 was identified as a molecular target for miR-378a-3p in endothelial cells, which revealed PDIA4’s potential role in endothelial autophagy. Our data demonstrate that miR-378-3p regulates endothelial autophagy and function via modulating PDIA4 expression.