Journal: Nitric oxide : biology and chemistry

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

PMID/MRN: 27663503

Publication Date: 2016-Nov-30

Abstract:

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, 100ÊnM, respectively) or negative control (100ÊnM) were co-transfected into HUVECs, luciferase activity was detected at 24Êh. HUVECs were incubated with 2Ê?g/ml Actinomycin D for the indicated time after miR-21 (25ÊnM) transfection, half-lives of DDAH1 mRNA were determined. HUVECs were transfected with PmirGLO-miR-21 alone or co-transfected with miR-21 for 24Êh, 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 (PÊ<Ê0.05 for 25ÊnM miR-21, PÊ<Ê0.01 for 50ÊnM and 100ÊnM miR-21), and miR-21 inhibitor increased reporter activity of PmirGLO-miR-21 and mRNA expression of all three DDAH1 transcript variants significantly (PÊ<Ê0.05, respectively). The degree of increase in endogenous DDAH1 mRNA expression by miR-21 inhibitor was more obvious for DDAH1-V3. Overexpression of miR-21 decreased mRNA expression and mRNA half-life time of all DDAH1 transcripts significantly (PÊ<Ê0.05), and DDAH1-V2 displayed significantly decreased half-life time than DDAH1-V1 and -V3 with or without miR-21 transfection (PÊ<Ê0.05, respectively). MiR-21 (100ÊnM) decreased DDAH1 protein expression and eNOS activity significantly (PÊ<Ê0.05), which was reversed by PmirGLO-miR-21 transfection (PÊ<Ê0.05). Transfection of PmirGLO-miR-21 alone increased intracellular miR-21 expression by approximately 5.6-fold, but only showed a trend of increase in DDAH1 protein expression. Our results confirmed DDAH1 3'-UTR as a target for miR-21, and endogenous miR-21 showed increased inhibitory effect on DDAH1-V3 transcript. DDAH1 3'-UTR, especially for DDAH1-V3, may function as miR-21 sponge to regulate DDAH1 protein expression. Modulation of miR-21-DDAH1 interaction may provide a new approach for tackling cardiovascular diseases. Copyright © 2016 Elsevier Inc. All rights reserved.