Regulation Of DDAH1/ADMA System By Genetic Polymorphisms And MiRNA-mediated Epigenetics Mechanisms

Chapter1MicroRNA mediate the regulation of epgenetics and genetic polymorphism in DDAH1expressionBackgroundCardiovascular disease is a severel threaten to human health. CVD is the most important cause of death in Chinese, and keep increasing. In order to develop new therapeutic strategies and decrease mortality of CVD, further researches on the mechanism of CVD pathogenesis are needed.Endothelium dysfunction is the common pathophysiological basis for cardiovascular diseases such as hypertension, coronary artery disease and stroke. Asymmetric dimethylarginine (ADMA) is an endogenous NOS inhibitor of which can competitively inhibit NO production and results in endothelial dysfunction. Increased plasma ADMA level has been proved to be a new risk factor for cardiovascular events and a direct predictor of the prognosis for cardiovascular diseases.About80%of ADMA in plasma is metabolized and inactivated by dimethylarginine dimethylaminohydrolases (DDAH1and DDAH2). DDAH1is the key enzyme that metabolizes ADMA in plasma. Variation in DDAH activity may influence NO level through affecting plasma ADMA level and NOS activity, and consequently results in CVD. Genetic polymorphisms in DDAH1are factors contribute to variation in DDAH expression and activity. Genetic polymorphisms in DDAH1are also reported to be associated with the plasma level of ADMA and risk for cardiovascular diseases.MicroRNAs (miRNAs) are a class of short (21～25nt) non-coding RNA that can specially bind to3’-untranslated region of target mRNA and control the expression of target gene at post-transcriptional level. Genetic polymorphisms can alter the target gene expression by influence the binding between miRNA and target gene. Therefore, we hypothesis that sequence specific and post-transcriptional of miRNA is possible mechanism in DDAHl polymorphisms regulate the DDAH1expression and activity. This study was designed to evaluate whether DDAH1polymorphisms functions through affecting miRNA-mediated regulation of DDAH1experssion and ADMA metabolizing activity.METHODS1. Functional analysis of DDAH1SNPs by luciferase reporter gene assay systemThe encompassing of positively associated SNPs in3’-untranslated region (3’-UTR) of DDAH1were cloned to downstream of pGL3-Control vectors. And then, The vectors were transfected into human umbilical vein endothelial cells (HUVEC-C) and HepG2cells by using pRL-SV40vector as a control. Dual-luciferase reporter assay system was used to determine the luciferase activity.2. Functional analysis of DDAH13’-UTR SNPs after transfect miRNAs mimic or inhibitor by luciferase reporter gene assay systemThe sequences encompassing SNPs in3’-untranslated region (3’-UTR) of DDAH1were cloned to downstream of pGL3-Control vectors. The vectors were then transfected with miR-660, miR-218, or miR-455-5p mimic (40nM) or inhibitor (80nM), respectly, into human umbilical vein endothelial cells (HUVEC-C) and HepG2cells by using pRL-SV40vector as a control. Dual-luciferase reporter assay system was used to determine the luciferase activity at24h after transfection.3. Influence of DDAH1protein expression, ADMA metabolic activity and intracellular ADMA concentration after transfection of miRNA mimic or inhibitor in HUVEC-C.Transfect miR-660、miR-218、miR-455-5p mimic (5,10,20,40nM) or inhibitor (80nM) respectly into HUVEC-C for24h. DDAH1protein expression was determined by western-blot. ADMA concentrations in cell culture and in cell lysates were determined by ELISA.4. Verifying the function of SNPs in HUVECsHUVECs were isolated from27human umbilical cords, DDAH1 SNPs were genotyped, and the cells were cultured in EGM-2medium. Cells of the fourth passage were cultured for24hours, and then DDAH1mRNA expression were determined by real-time quatitative PCR. DDAH1protein expression was determined by western-blot.ADMA in cell culture and in cell lysates were determined by ELISA.5.Verifying the function of SNPs in PBMCsIsolate the PBMCs from health volunteers.DDAH1SNPs were genotyped,and then DDAH1mRNA expression were determined by real-time quatitative PCR.RESULTS(1)By transfect into HUVEC-C and HepG2, pGL3vectors bearing the rs233113T allele showed significantly higher luciferase activity than those bearing the rs233113A allele (p<0.01); pGL3vectors bearing the rs233112G allele showed significantly lower luciferase activity than those bearing the rs233112A allele (p<0.01).(2) miR-660inhibitor(80nM) can significantly increase pGL3-233113A vectors luciferase activity in both HUVEC-C and HepG2(p<0.05); miR-660inhibitor can significantly increase DDAH1protein expression in HUVEC-C (p<0.05);(3) By transfect into HUVEC-C and HepG2, miR-218mimic(40nM) can significantly decrease pGL3-233112A vectors luciferase activity (p<0.01); Transfect miR-218mimic(40nM) can significantly decrease pGL3-233112G vectors luciferase activity in HepG2(p<0.01); Transfect miR-218inhibitor(80nM) can significantly increase pGL3-233112G vectors luciferase activity in HepG2(p<0.05);Transfect miR-218mimic (20nM,40nM) can significantly decrease DDAH1protein expression and DDAH activity in HUVEC-C (p<0.05); miR-218mimic (20,40nM) can significantly increase intracellular ADMA concentration in HUVEC-C (p<0.05)(4) By transfect into HUVEC-C and HepG2, either miR-455-5p mimic(40nM) or miR-455-5p inhibitor (80nM) can significantly decrease pGL3-233112A vectors luciferase activity (p<0.01); miR-455-5p mimic (10nM,20nM,40nM) can significantly decrease DDAH1protein expression and DDAH activity in HUVEC-C (p<0.05); miR-455-5p mimic(10nM,20nM,40nM)can significantly increase intracellular ADMA concentration in HUVEC-C (p<0.05)(5) In all isolated27HUVECs from different individuals, carrying the rs233113Tallele (AT=10, TT=5) showed significantly higher DDAH1mRNA expression DDAH protein expression DDAH activity and lower intracellular ADMA levels as compared those with the rs233113AA genotype (AA=12)(p<0.05).(6) In all isolated27HUVECs from different individuals, carrying the rs233112GG genotype (n=4) showed significantly higher DDAH1mRNA expression DDAH protein expression DDAH activity and lower intracellular ADMA levels as compared those with the rs233112AA genotype (AA=12)(p<0.05).(7)In isolated PBMCs from health volunteers, carrying the rs233113T allele (AT=11, TT=4) showed significantly higher DDAH1mRNA expression as compared those with the rs233113AA genotype (AA=17, p=0.032)CONCLUSION1. DDAHlis target gene of miR-660、miR-218and miR-455-5p.2. By alter miR-218and miR-455-5p regulate DDAH1expression and DDAH activity, DDAH1rs233112polymorphism can affect ADMA metabolism.3. By alter miR-660regulate DDAH1expression and DDAH activity, DDAH1rs233113polymorphism can affect ADMA metabolism. Chapter24-HNE increases intracellular ADMA levels in cultured HUVECs: evidence for miR-21-dependent mechanismsBackgroundAsymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NOS which can not only regulate NO production,but also induce oxidative stress,accelerate inflammation,induce apoptosis of endothial cell. Increase of plasma ADMA level has been proved to new risk factor of cardiovascular events and direct predictor of Cardiovascular disease’s Prognosis. About80%of ADMA in plasma is metabolized and inactivated by dimethylarginine dimethylaminohydrolases (DDAH1and DDAH2). DDAH1is the key enzyme of metabolize the ADMA in plasma.MicroRNAs (miRNAs) are a class of short (21～25nt) non-coding RNA which play an important role in regulation of cell proliferation, cell differentiation and cell apoptosis. MicroRNA can specially bind to3’-untranslated region of target mRNA and control the expression of target gene at post-transcriptional level. miR-21is a kind of microRNA which is associate with Cardiovascular disease. By using target scan,DDAH1is predicted to be a potential miR-21target.4-Hydroxynonenal (4-HNE) is a major active product formed following lipid peroxidation and is considered as a new atherogenic factor.4-HNE can inhibit DDAH1activity and decrease NO generation in a dose-dependent manner through formation of Michael adducts on His173in DDAH1.In our study, we investigate whether4-HNE regulates asymmetric dimethylarginine (ADMA) metabolism through directly decrease ADMA metabolism enzyme expression and the involvement of microRNA (miRNA) miR-21in human umbilical venous endothelial cells (HUVECs).METHODS1. Obersve the influence of miR-21expression、DDAH1/2expression.. ADMA metabolic activity and concentration after miR-21transfect ion in HUVEC-C.HUVEC-C were transfected with miR-21(at concentrations of50nM and100nM) and cultured for12,24, and48h, respectively. DDAH1/2mRNA and miR-21expression in the HUVEC-C were determined by semi-quantitative real time PCR. DDAH1and DDAH2protein expression in the HUVEC-C were analyzed by Western blot. ADMA in cell medium and cell lysates were analyzed by ELISA. ADMA metabolic activity of the cell lysates was also determined.2. Obersve the influence of miR-21expression.. DDAH1expression ADMA metabolic activity and concentration after4-HNE treatment in HUVEC-C.Cultured HUVEC-C were treated with4-HNE (at concentrations of1,5, and10μM) for24h. MiR-21inhibitor (final concentration of100nM) was transfected at1h before4-HNE (10μM) treatment. DDAH1mRNA and miR-21expression in the HUVEC-C were determined by semi-quantitative real time PCR. DDAH1protein expression in the HUVEC-C were analyzed by Western blot. ADMA in cell medium and cell lysates were analyzed by ELISA. ADMA metabolic activity of the cell lysates was also determined. RESULTS(1) Transfection of miR-21at concentrations of both50nmol/L and100nmol/L increased miR-21expression in HUVEC-C significantly at12h、24h and48h(p<0.01, respectively). Both50and100nmol/L miR-21decreased DDAH1and DDAH2mRNA expression significantly at all three time points after transfection (p<0.05, respectively), both50and100nmol/L concentrations of miR-21decreased DDAH1protein exprssion at24h and48h after transfection(p<0.05, respectively). DDAH2protein expression was decreased at all three time points after miR-21transfection (p<0.05, respectively). ADMA metabolic activity of cell lysates was decreased significantly at all three time points after100nmol/L miR-21transfection (p<0.05for24h, and p<0.01for12and48h, respectively). Intracellular ADMA concentration was increased significantly at24and48h (p<0.05, respectively) after100nmol/L miR-21treatment(2) Treat with various concentrations of4-HNE(1,5,10μmol/L) for24hours decreased DDAH1/2mRNA expression significantly (p<0.05for1μmol/L; p<0.01for5μmol/L and10μmol/L, respectively);Significant decrease in DDAH1protein was observed in cells treated with10μmol/L (p<0.01) Intracellular ADMA metabolic activity was decreased while ADMA concentration was increased significantly by4-HNE treatment at all three concentrations (p<0.05). ADMA concentration in cell culture was also increased by5μmol/L and10μmol/L4-HNE treatment (p<0.05, respectively), treat with10μmol/L4-HNE increased both intracellular pri-miR-21/miR-21expression significantly by about2-fold (p<0.05). MiR-21inhibitor (100nmol/L) significantly decreased expression of miR-21in cells treated with or without10μmol/L4-HNE (p<0.01, respectively). The4-HNE (10μmol/L)+miR-21inhibitor group showed significantly increased DDAHl mRNA and protein expression (p<0.05, respectively), significantly increased intracellular ADMA metabolic activity (p<0.05), and significantly decreased intracellular ADMA concentration (p<0.05,) than the10μmol/L4-HNE treatment group. Transfection of miR-21inhibitor alone did not affect the intracellular DDAH1expression and ADMA metabolic activity significantly in HUVECs.CONCLUSION1.miR-21down-regulates both expression and activity of DDAH1/2.2.4-HNE down-regulates the DDAHl expression and increases intracellular ADMA accumulation in HUVEC-C through a miR-21-dependent mechanism.