Fig. 3

NOS1 is a downstream direct target of miR-4640-5p. A A Venn diagram showed the numbers of potential target genes of miR-4640-5p predicted by TargetScan, miRDB, and miRWalk. B PASMCs were transfected with miR-4640-5p mimic or NC. The relative mRNA expression levels of commonly predicted genes were analyzed by RT-qPCR 48 h later. C Diagrams showed the putative binding sites between miR-4640-5p and corresponding wild type (WT) or mutant (MUT) sites of NOS1. PASMCs were co-transfected with reporter vector containing WT or Mut 3’-UTR of NOS1, together with NC or miR-4640-5p mimic. The dual luciferase activity was analyzed in PASMCs at 48 h post transfection. D The mRNA and (E) protein expression levels of NOS1 in PASMCs cultured under normoxia or hypoxia, transfected with miR-4640-5p mimic or NC. F Pearson analysis of the correlation between NOS1 mRNA expression and miR-4640-5p expression in lung tissue from COPD-PH patients (n = 14). G, H The mRNA (G) and protein (H) expression levels of NOS1 in PASMCs cultured under normoxia or hypoxia condition were analyzed by RT-qPCR and western blot. All experiments were independently repeated for at least three times. *p < 0.05, **p < 0.01, ***p < 0.001