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 < 0.05 for 25 nM miR-21, P < 0.01 for 50 nM and 100 nM miR-21), and miR-21 inhibitor increased reporter activity of PmirGLO-miR-21 and mRNA expression of all three DDAH1 transcript variants significantly (P < 0.05, 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 (P < 0.05), and DDAH1-V2 displayed significantly decreased half-life time than DDAH1-V1 and -V3 with or without miR-21 transfection (P < 0.05, respectively). MiR-21 (100 nM) decreased DDAH1 protein expression and eNOS activity significantly (P < 0.05), which was reversed by PmirGLO-miR-21 transfection (P < 0.05). 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.
Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha 410008, Hunan, PR China; Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha 410078, Hunan, PR China; Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang 421001, Hunan, PR China.
Da-Bin
DB
Kuang
Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha 410008, Hunan, PR China; Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha 410078, Hunan, PR China; Department of Cardiovascular Medicine, Xiangya Hospital, Central South University, Changsha 410008, Hunan, PR China.
Ji-Peng
JP
Zhou
Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha 410008, Hunan, PR China; Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha 410078, Hunan, PR China.
Lin-Yu
LY
Yu
Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha 410008, Hunan, PR China; Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha 410078, Hunan, PR China; Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang 421001, Hunan, PR China.
Wen-Jing
WJ
Zeng
Department of Pharmacy, Xiangya Hospital, Central South University, Changsha 410008, Hunan, PR China.
Jian
J
Xiao
Department of Pharmacy, Haikou People's Hospital and Affiliated Haikou Hospital of Xiangya Medical School, Central South University, Haikou 570311, PR China.
Gang-Zhi
GZ
Zhu
Department of Pharmacy, Xiangya Hospital, Central South University, Changsha 410008, Hunan, PR China. Electronic address: zanlingzhang2015@126.com.
Zan-Lin
ZL
Zhang
Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha 410008, Hunan, PR China; Institute of Clinical Pharmacology, Central South University, Hunan Key Laboratory of Pharmacogenetics, Changsha 410078, Hunan, PR China; Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang 421001, Hunan, PR China. Electronic address: chenxp74@hotmail.com.
Xiao-Ping
XP
Chen
D000581:Amidohydrolases / Q000235:genetics*; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*
C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D000581:Amidohydrolases; C059999:dimethylargininase
DDAH1; miR-21; microRNA sponge