OBJECTIVE To investigate whether micro RNA(mi RNA)mi R-21 regulates dimethylarginine dimethylaminohydrolase 1(DDAH1)expression through binding 3′-UTR regiondirectly in human umbilical venous endothelial cells(HUVECs)...OBJECTIVE To investigate whether micro RNA(mi RNA)mi R-21 regulates dimethylarginine dimethylaminohydrolase 1(DDAH1)expression through binding 3′-UTR regiondirectly in human umbilical venous endothelial cells(HUVECs)and to explore whether DDAH1-V2/V3 transcripts can function as micro RNA sponge,thereby modulating DDAH1-V1 expression.METHODS The DDAH13′-UTR containing mi R-21 recognizing sequence was cloned into Pmir GLO dual-luciferase mi RNA target expression plasmid to construct PmirGLO-mi R-21.The plasmid and mi R-21(at concentrations of 25,50,100 nmol·L-1,respectively)or negative control(100 nmol·L-1)were co-transfected into HUVECs,luciferase activity was detected at 24 h.HUVECs were incubated with 2μg·m L-1actinomycin D for the indicated time after mi R-21(25 nmol.L-1)transfection,half-lives of DDAH1 m RNA were determined.HUVECs were transfected with Pmir GLO-mi R-21 alone or co-transfected with mi R-21 for 24 h,DDAH1 transcripts m RNA and DDAH1protein expression were determined.RESULTS Mi R-21decreased luciferase activity of Pmir GLO-mi R-21 in a dose-dependent manner(P<0.05 for 25 nmol·L-1mi R-21,P<0.01 for 50 nmol·L-1and 100 nmol·L-1mi R-21),and mi R-21 inhibitor increased reporter activity of PmirGLO-mi R-21 and m RNA expression of all DDAH1 three transcript variants significantly(P<0.05,respectively).The degree of increase in endogenous DDAH1 m RNA expression by mi R-21 inhibitor was more obvious for DDAH1-V3.Overexpression of mi R-21 decreased m RNA expression and m RNA half-life time of all DDAH1 transcripts significantly(P<0.05),and DDAH1-V2 displayed significantly decreased half-life time than DDAH1-V1and-V3 with or without mi R-21 transfection(P<0.05,respectively).Mi R-21(100 nmol·L-1)decreased DDAH1protein expression significantly(P<0.05),which was reversed by Pmir GLO-mi R-21 transfection(P<0.05).Transfection of Pmir GLO-mi R-21 alone increased intracellular mi R-21 expression by approximately 5.6-fold,but only showed a trend of increase in DDAH1 protein expression.CONCLUSION Our resu展开更多
基金The project supported by National Natural Science Foundation of China(81170091,81373489,81422052)Special Topic of the Major Subject of National Science and Technology(2013ZX09509-107)+1 种基金Provincial Natural Science Foundation of Hunan(13JJ1010)Funds for Hunan Education Department Program(12K006)
文摘OBJECTIVE To investigate whether micro RNA(mi RNA)mi R-21 regulates dimethylarginine dimethylaminohydrolase 1(DDAH1)expression through binding 3′-UTR regiondirectly in human umbilical venous endothelial cells(HUVECs)and to explore whether DDAH1-V2/V3 transcripts can function as micro RNA sponge,thereby modulating DDAH1-V1 expression.METHODS The DDAH13′-UTR containing mi R-21 recognizing sequence was cloned into Pmir GLO dual-luciferase mi RNA target expression plasmid to construct PmirGLO-mi R-21.The plasmid and mi R-21(at concentrations of 25,50,100 nmol·L-1,respectively)or negative control(100 nmol·L-1)were co-transfected into HUVECs,luciferase activity was detected at 24 h.HUVECs were incubated with 2μg·m L-1actinomycin D for the indicated time after mi R-21(25 nmol.L-1)transfection,half-lives of DDAH1 m RNA were determined.HUVECs were transfected with Pmir GLO-mi R-21 alone or co-transfected with mi R-21 for 24 h,DDAH1 transcripts m RNA and DDAH1protein expression were determined.RESULTS Mi R-21decreased luciferase activity of Pmir GLO-mi R-21 in a dose-dependent manner(P<0.05 for 25 nmol·L-1mi R-21,P<0.01 for 50 nmol·L-1and 100 nmol·L-1mi R-21),and mi R-21 inhibitor increased reporter activity of PmirGLO-mi R-21 and m RNA expression of all DDAH1 three transcript variants significantly(P<0.05,respectively).The degree of increase in endogenous DDAH1 m RNA expression by mi R-21 inhibitor was more obvious for DDAH1-V3.Overexpression of mi R-21 decreased m RNA expression and m RNA half-life time of all DDAH1 transcripts significantly(P<0.05),and DDAH1-V2 displayed significantly decreased half-life time than DDAH1-V1and-V3 with or without mi R-21 transfection(P<0.05,respectively).Mi R-21(100 nmol·L-1)decreased DDAH1protein expression significantly(P<0.05),which was reversed by Pmir GLO-mi R-21 transfection(P<0.05).Transfection of Pmir GLO-mi R-21 alone increased intracellular mi R-21 expression by approximately 5.6-fold,but only showed a trend of increase in DDAH1 protein expression.CONCLUSION Our resu
