Molecular Characteriation Of Coronary Artery Disease Susceptibility Gene ADTRP

Coronary artery disease,or coronary heart disease(CAD),is caused by the formation of coronary artery wall plaques(Atherosclerosis,AS),which causes vascular stenosis,resulting in insufficient supply of blood,oxygen and nutrients to the heart,chest pain(angina),shortness of breath,or other CAD signs and symptoms.A complete blockage of coronary arteries can cause a heart attack,referred to as myocardial infarction(MI).CAD is the world's leading cause of mortality and morbidity.In our country more than 700,000 people die of coronary heart disease each year.CAD has also become the world's “top ten killer”.CAD is a complex disease caused by genetic factors,environmental factors or their interactions.In 2011,our laboratory and collaborators used a Genome-Wide Association Study(GWAS)to identify a susceptible variant rs6903956 on chromosome 6p24.1 associated with coronary heart disease in the Chinese Han population.The rs6903956 variant is located within the gene C6orf105,and rs6903956 risk allele A was associated with decreased C6orf105 mRNA expression.These results suggest that the down-regulation of C6orf105 may be involved in the pathogenesis of CAD,but the specific molecular mechanism is still unclear and needs to be further studied.Subsequently,the Lupu team in august 2011 reported in the Blood journal that gene C6orf105 co-localized with another coronary artery disease susceptibility gene caveolin-1 and tissue factor pathway inhibitor TFPI in the cell,and that C6orf105 can regulate the expression of TFPI under the stimulation of androgen.Therefore,C6orf105 was named as Androgen-Dependent TFPI-Regulating Protein(ADTRP).However,the molecular mechanism by which the ADTRP gene mediates the development and progression of coronary heart disease is still unclear.In this thesis research project,ADTRP was analyzed by whole-genome expression profiling microarray to identify downstream genes regulated by ADTRP after it was knockdown.The results showed up-regulation of seven histone genes,down-regulation of four cell cycle regulatory genes and up-regulation of two apoptosis genes.These genes are involved in the regulation of cell cycle progression,cell proliferation,and endothelial cell apoptosis(Sub-project 1).Moreover,we identified another GWAS-proven coronary heart disease susceptibility gene MIA3 as a downstream gene regulated by ADTRP.We focused on the regulation of MIA3 by ADTRP and identified a novel biological pathway for the pathogenesis of CAD,which consists of ADTRP-PIK3R3-AKT activation-MIA3(Sub-project 2).In addition,we investigated the promoter of ADTRP,and found that androgen regulates the expression of ADTRP gene by androgen receptor(AR)in a dose-dependent manner(Sub-project 3).The details are as follows:Section1: Coronary artery disease susceptibility gene ADTRP regulates cell cycle progression,proliferation and apoptosis by global gene expression regulation.The ADTRP gene encodes the Androgen-Dependent TFPI-Regulating Protein and is a susceptibility gene for CAD.To identify other downstream targets of ADTRP,we performed global gene expression profiling in cells with knockdown of ADTRP expression.We performed global gene expression profiling for ADTRP knockdown using microarrays in human HepG2 cells.A total of 49,293 expression probes with intensities distinguishable from background noise(detection P value<0.05)in all RNA samples were filtered out for downstream differential analysis.Compared with NC siRNA group,probes or genes showing differential expression levels in the ADTRP siRNA group were identified by Student's t tests.Top genes with P<0.01,fold change>2,and average expression levels of greater than 7(usually ranging from 2 to 14)were selected for validation analysis.The selection resulted in a total of 120 probes/genes for validation analysis.To explore potential functional implications of top differentially expressed genes,top 120 expression probes were mapped to 94 unique genes based on the Affymetrix annotation database.For all 94 unique,downstream target genes of ADTRP identified by microarray analysis,we designed primers for qRT-PCR analysis.The primers were successful for qRT-PCR analysis of 71 genes,38 down-regulated genes and 33up-regulated genes with ADTRP knockdown.These 71 genes were then selected for follow-up validation.Follow-up real-time RT-PCR analysis demonstrated that ADTRP regulates a diverse set of genes,including up-regulation of 7 histone genes(HIST1H2AB,HIST1H2 AC,HIST2H2AA3/ HIST2H2AA4,HIST1H2 BD,HIST1H2BJ,HIST1H4 A,HIST1H1C),down-regulation of multiple cell cycle genes(CCND1,CDK4 and CDKN1A),and up-regulation of apoptosis genes(CASP7 and PDCD2)in HepG2 cells and endothelial cells.Because ADTRP downstream genes include those involved in cell cycle regulation and apoptosis as well as multiple histone genes,we carried out cellular studies on cell cycle,cell proliferation and apoptosis to further characterize the function of ADTRP.Consistently,ADTRP increases the number of S phase cells during cell cycle,promotes cell proliferation and inhibits apoptosis.Our study provides novel insights into the function of ADTRP and biological pathways involving ADTRP,which may be involved in the pathogenesis of CAD.Section 2: Identification of a molecular signaling gene-gene regulatory network between GWAS susceptibility genes ADTRP and MIA3 for coronary artery disease.GWAS have identified >50 genomic loci for CAD,including ADTRP(regulator of TFPI and coagulation)and MIA3(involved in ER trafficking of collagens).However,it is important to determine whether the GWAS genes form a molecular network in determining the genetic architecture of CAD.In this study,we have uncovered a novel molecular signaling pathway involving ADTRP and MIA3 for the pathogenesis of CAD.Global microarray gene expression and real-time RT-PCR analyses showed that knockdown of ADTRP expression markedly down-regulated expression of MIA3.Mechanistically,ADTRP positively regulates expression of PIK3R3 encoding the regulatory subunit 3 of PI3 K,which leads to activation of AKT,resulting in up-regulation of MIA3.Both ADTRP and MIA3 are involved in endothelial cell(EC)functions relevant to atherosclerosis.Knockdown of ADTRP expression by siRNA promoted oxidized-LDLmediated monocyte adhesion to ECs and transendothelial migration of monocytes,inhibited EC proliferation and migration,and increased apoptosis,which was reversed by expression of constitutively active AKT1,while the over-expression of ADTRP in ECs blunted these processes.Knockdown of MIA3 expression also promoted monocyte adhesion to ECs and transendothelial migration of monocytes.In conclusion,we have uncovered a novel molecular signaling pathway for the pathogenesis of CAD,which involves a novel gene-gene regulatory network.We show that ADTRP positively regulates PIK3R3 expression,which leads to activation of AKT and up-regulation of MIA3,thereby regulating endothelial cell functions directly relevant to atherosclerosis.Section 3: Study on the expression regulation of ADTRP by androgen.Low androgen levels are associated with an increased risk of cardiovascular disease and thrombosis,suggesting that androgen has a protective role.However,little is known about the underlying molecular mechanism.Our genome-wide association study identified the ADTRP gene encoding the androgen-dependent TFPI regulating protein as a susceptibility gene for CAD and MI.The expression level of ADTRP was regulated by androgen,but the molecular mechanism is unknown.In this study we identified the molecular mechanism by which androgen regulates ADTRP expression and tested the hypothesis that androgen plays a protective role in cardiovascular disease by activating ADTRP expression.Luciferase assays with an ADTRP promoter luciferase reporter revealed that androgen regulates ADTRP transcription in dose-dependent and timedependent manners,and the effects were abolished by three different androgen inhibitors pyrvinium pamoate,bicalutamide,and cyproterone acetate.Chromatin-immunoprecipitation showed that the androgen receptor binds to a half androgen response element(ARE,TGTTCT)located +324 bp from the ADTRP transcription start site,which is required for concentration-dependent transcription activation of ADTRP.HL-60 monocyte adhesion to EAhy926 endothelial cells(ECs)and migration across the EC layer,the two processes critical to development of CAD and MI,were inhibited by androgen,but the effect was rescued by ADTRP siRNA and exacerbated by overexpression of ADTRP.These data suggest that one molecular mechanism by which androgen protects one from CAD is stimulation of ADTRP expression.In summary,first,we have found that ADTRP increases the number of S phase cells during cell cycle,promotes cell proliferation and inhibits apoptosis.Second,we identified a novel biological pathway in which coronary heart disease susceptibility genes ADTRP and MIA3 regulate the pathogenesis of coronary artery disease.Third,we determined that androgen reduces monocyte adhesion to and transmigration across the endothelial layer,key processes involved in the pathogenesis of CAD,by regulating expression of ADTRP.Altogether,these studies start to provide important mechanistic insights into the molecularme chanisms by which ADTRP mediates the pathogenesis of CAD and MI.Further studies may establish ADTRP and the components of its signaling pathways as important targets for developing prevention and treatment strategies for coronary artery disease.