Functional GATA4 Gene Promoter Variants And Acute Myocardial Infarction

Background:Acute myocardial infarction(AMI)is a specific type of coronary artery disease(CAD),mainly caused by rupture of atherosclerotic plaques.At present,the pathogenesis of atherosclerosis has been involved in the processes of metabolism,inflammation,autophagy and aging.The occurrence of CAD is closely related to genetic and environmental factors.GATA4 is a zinc finger-containing transcriptional factor highly expressed in the heart and plays an important role in cardiovascular development,autophay,senescence and metabolism.The promoter regulates the level of gene expression.Therefore,we speculated that the genetic variation of GATA4 gene promoter may play an important role in the pathogensis of AMI.Objective:We aimed to find the variations of GATA4 gene promoter sequence in patients with AMI and healthy control group.Reporter gene expression vectors were constructed based on these genetic variants.We then tried to analyze the functional changes of GATA4 gene promoter by determining activity of the dual-luciferase assay,explore the effect of genetic variation on gene expression level.Methods:1.This study included two groups,395 AMI patients and 397 healthy control subjects.Peripheral blood mononuclear cells were collected and genomic DNAs were extracted.2.The PCR primer to analyze GATA4 gene promoter sequences were designed based on NCBI database.The GATA4 gene promoter was generated by PCR,which were sequenced and compared with the wild type GATA4 gene promoter.3.Wild type and variant GATA4 gene promoters were subcloned into pGL3-basic vector to construct reporter gene expression vectors.The GATA4-pGL3-basic vector and pRL-TK vector were transfected into H9c2 cells and HEK293 cells.Dual-luciferase activities were assayed by dual-luciferase reporter assay system.The effects of the variants of GATA4 gene promoter on the transcriptional activity were investigated.4.GATA4 gene sequences containing variations were designed and labeled by biotin as probes.Electrophoretic mobility shift assay(EMSA)was conducted with probes and HEK293 cells and H9c2 nuclear extracts.After separation and transferred to membrane.The biotin-labeled probes were detected by chemiluminescence assay to explore the effect of GATA4 gene promoter sequence variation on transcription factor binding sites.Results:1.A total of 14 DSVs were identified in this study,including one insertion DSV(g.31979-31980InsG)and two SNPs(g.31360T>C,rs372004083;g.31437C>A,rs769262495).Three novel heterozygous DSVs(g.31806C>T,g.31900G>C,g.32241C>T)were only identified in three AMI patients.Eight novel heterozygous DSVs(g.31403G>T,g.31492T>A,g.31566G>C,g.31567A>G,g.31715C>A,g.31730A>G,g.32171A>G,g.32190C>T)were only found in healthy controls.2.pGL3-basic reporter gene vectors containing GATA4 gene promoter wild type and sequence variation sites were constructed,including empty pGL3-basic(negative control),pGL3-WT(wild type),PGL3-31403T,PGL3-31492A,PGL3-31566C,PGL3-31715A,PGL3-31730G,PGL3-31806T,PGL3-31900C and PGL3-32241T.3.In HEK-293 cells and H9c2 cells,the DSVs(g.31806C>T,g.31900G>C and g.32241C>T)identified in AMI patients increased activity of the GATA4 gene promoter significantly(P<0.01).Others DSVs did not significantly alter the activity of the GATA4 gene promoter(P>0.05).4.In EMSA,g.31806C>T and g.31900G>C did not change significant specific binding sites.DSV g.32241C>T alter significant specific binding site of transcription factor.Conclusion:The DSVs of GATA4 gene promoter found in AMI patients may affect the transcriptional activity and transcription factor binding site of GATA4 gene and further affect its expression level.It plays an important role in the development of AMI and provides new ideas for the pathological mechanism,prevention,diagnosis and treatment of AMI.