miR-486-5p Inhibits eNOS and Angiogenesis in Cultured Endothelial Cells by Targeting MAML3

Kidney ischemia–reperfusion (I/R) is associated with endothelial injury. Administration of miRNA (miR)-486-5p protects against rat kidney I/R injury, with localisation to capillary endothelial cells, although it inhibits I/R-induced endothelial nitric oxide synthase (eNOS) protein expression. Here, we studied the effect of miR-486-5p on eNOS and endothelial cell function and determined its mRNA targets. Human umbilical vein endothelial cells (HUVECs) were transfected with the miR-486-5p mimic and assayed for proliferation, migration and network formation. Biotinylated miR-486-5p was transfected for pulldown of bound mRNA, followed by RNA sequencing. miR-486-5p markedly decreased eNOS mRNA and protein in HUVECs (p < 0.001) and decreased eNOS protein in human pulmonary microvascular endothelial cells (p < 0.05), although eNOS was not a direct target of miR-486-5p. miR-486-5p inhibited angiogenesis, which was rescued with eNOS plasmid transfection. RNA sequencing of biotinylated miR-486-5p pulldown RNA revealed highly significant enrichment in predicted targets FOXO1, FOXP1, TNFSF4, MAML3 and CELSR3, and in the non-predicted target SPCS2. RT-qPCR validated these transcripts as inhibited by miR-486-5p. While silencing of FOXO1 had no impact on eNOS protein, MAML3 silencing inhibited eNOS levels. miR-486-5p inhibits angiogenesis in endothelial cells via eNOS down-regulation, which involves selective targeting of MAML3. These data support a novel pathway regulating endothelial cell function.