DDAH1-V3 transcript might act as miR-21 sponge to maintain balance of DDAH1-V1 in cultured HUVECs

To investigate whether microRNA (miRNA) miR-21 regulates dimethylarginine dimethylaminohydrolase 1 (DDAH1) expression through binding 3′-UTR region directly in human umbilical venous endothelial cells (HUVECs) and to explore whether DDAH1-V2/V3 transcripts can function as microRNA sponge, thereby modulating DDAH1-V1 expression. The DDAH1 3′-UTR containing miR-21 recognizing sequence was cloned into PmirGLO dual-luciferase miRNA target expression plasmid to construct PmirGLO-miR-21. The plasmid and miR-21 (at concentrations of 25, 50, 100 nM, respectively) or negative control (100 nM) were co-transfected into HUVECs, luciferase activity was detected at 24 h. HUVECs were incubated with 2 μg/ml Actinomycin D for the indicated time after miR-21 (25 nM) transfection, half-lives of DDAH1 mRNA were determined. HUVECs were transfected with PmirGLO-miR-21 alone or co-transfected with miR-21 for 24 h, DDAH1 transcripts mRNA, eNOS activity and DDAH1 protein expression were determined. MiR-21 decreased luciferase activity of PmirGLO-miR-21 in a dose-dependent manner (P