Functional Genetic Variants Within The TBX5 Gene Promoter In Acute Myocardial Infarction

Background:Acute myocardial infarction(AMI)is an important classification of coronary heart disease.With the acceleration of social development and aging,the mortality and prevalence of cardiovascular disease(CVD)in China is still growing rapidly.Among them,coronary heart disease(CAD)has a high incidence in cardiovascular diseases,and most of them are caused by coronary arteriosclerosis.According to epidemiological studies,important risk factors associated with coronary arteriosclerosis are dyslipidemia,hypertension,diabetes,smoking,obesity,increased blood homocysteine,less physical activity,older age,and males.The occurrence of coronary heart disease is closely related to the surrounding environment and genetic factors.It is currently confirmed that the Genome-wide Association Study(GWAS)found that there are more than 200 CAD-related chromosomal loci,but only explained that 11%of CAD is genetically related.T-box gene coding has identified more than 20 genes in humans,of which the TBX5 gene has been confirmed to be involved in heart development and has an effect on the development of coronary arteries.Gene promoters regulate the level of transcription,further affecting the translation and expression of proteins.In this study,we speculated that the epigenetic gene TBX5 plays an important role in the pathogenesis of AMI.By studying the promoter of TBX5 gene,we investigated the relationship between TBX5 gene transcription and AMI.Objectiove:The analysis found that the occurrence of coronary heart disease is closely related to genetic inheritance.Bioinformatics methods were used to predict the analysis of the TBX5 gene promoter region and the TBX5 gene promoter was identified by designing a primer clone.Then the genetic variation sites were found by the sequencing.Based on this,the dual-luciferase repoter gene plasmid was constructed to investigate the effect of genetic variation on transcriptional activity.Then use EMSA experimental methods to further explore the changes in transcriptional regulation of genetic variation with transcriptional levels.This study provides an important experimental basis for the genetic and functional variation of the TBX5 gene promoter.Methods:1?In this experiment,432 newly diagnosed AMI patients and 448 healthy physical examination patients at the same time were recruited in the study.The clinical data of the two groups were entered,and peripheral blood was collected from all the patients and whole genomes were extracted.2?Bioinformatics was used to predict the promoter of TBX5 gene and primers were designed according to the sequences provided by the NCBI gene database.PCR were used to amplify gene promoters,and were sent to the company for sequencing and statistical analysis of the results of mutation sequence sites.3?The pGL3-basic luciferase reporter gene plasmid was constructed by counting the mutated sequences of the promoter of the TBX5 gene and the wild-type target gene fragment.After sequencing,the pGL3-basic luciferase reporter plasmid and the internal reference pRL plasmid was transiently transfected into HEK293 cells and H9c2 cardiomyocytes using liposomes.To test the activity of the dual-luciferase reporter gene expression product to identify the effect of the TBX5 gene promoter variant sequence on transcriptional activity.4?To further explore the site of variants of gene promoters that have transcriptional activity,use the electrophoretic mobility shift assay(EMSA)to,design the probes by designing the wild-type and target gene with sequence variation sites to verify transcription.The factors change to discover the effect on transcriptional regulatory elements.Result:1?The age of patients in the AMI group was higher than that of the normal control group,and the prevalence of men was higher.By comparison,there were statistical differences between triglyceride(TG),total cholesterol(TC),high-density lipoprotein(HDL-C)and low-density lipoprotein(LDL-C)values in the AMI group and the normal control group,P<0.001.According to statistics,the proportion of patients with smoking history,diabetes history,and hypertension history in the AMI group was higher than that in the normal control group,and the difference was statistically significant at P<0.01.2.After sequencing by PCR,14 DSVs were found,including 6 single nucleotide polymorphisms(SNPs).Six heterozygous DSVs were found in the AMI group,and two were SNPs:g.114408439G>T(rsl86960328),g.114408862A>G,g.114409193T>C,g.114409076C>A,g.114409249G>A(rs564965932)and g.114409343A>G.The above mutation sites were not found in the normal control group.Four new DSVs were found in the normal control group,including 2 heterozygous DSVs:g.114408539T>C,g.114409294A>G,one heterozygous deletion DSV:g.114408344delC,and one heterozygous insertion DSV:g.114408458insTAATAA.Has not yet appeared in patients with AMI.In the AMI group and the normal control group,four SNPs were found simultaneously:g.114408635(rs57820630)G>A,g.114409208(rsl83776658)G>A,g.114409331(rs7957609)C>T and g.114409337(rs79795050)G>C.The frequencies of the two groups were compared and the frequency of the two groups was similar.There was no statistical significance(P>0.05).3.Construction of TBX5 gene promoter pGL3-basic dual fluorescent reporter vectors,pGL3-WT(wild type),pGL3-114408439T,pGL3-114408862G,pGL3-114409193C,pGL3-114409076A,pGL3-114409249A,pGL3-114409343G,pGL3-114409294C,pGL3-114408344delC,pGL3-114408458insTAATAA,pGL3-114409208A(rsl83776658).After sequencing,it was confirmed that the insertion sequence was correct and the recombinant plasmid was successfully constructed.4.Liposomes were transiently transferred to human embryonic kidney cell line(HEK293)and rat cardiomyocyte cell line(H9c2).Transfection of HEK293 cells revealed that three DSVs in the AMI group significantly affected the transcriptional activity of the TBX5 gene promoter.DSVs g.114409193T>C(P<0.01)significantly reduced the transcriptional activity of TBX5 gene promoter;DSVs g.114408862A>G(P<0.01),g.114409076C>A(P<0.01)increased TBX5 the transcriptional activity of a gene promoter.The DSVs found in the control group:g.114408334delC,g.114408458insTAATAA,g.114409294A>G,did not change their transcriptional activity of the TBX5 gene promoter(P>0.05).In the AMI and control groups,DSVs g.114409208G>A(rs 183776658)did not find that it changed the transcriptional activity of the promoter.The relative transcriptional activity of DSVs was detected by transfecting H9c2 cells.DSVs were found in AMI:g.114409193T>C(P<0.01),g.114408862A>G(P<0.01),g.114409076C>A(P<0.01),g.114409249G>A(rs564965932)(P<0.01)and g.114409343A>G(P<0.05)all significantly reduced the transcriptional activity of TBX5 gene promoter.There was also no statistically significant difference in DSVs found in the normal control group(P>0.05).The results of EMSA experiments showed that a specific band formed by protein binding appeared in the wild type probe of DSVs g.114408862,but the specific band disappeared in the labeled mutant probe,and the remaining DSVs were not detected.We hypothesized that the TBX5 gene promoter DSVs g.114408862 altered its original transcriptional site.Conclusion:Through the study of the genetic and functional variants of the TBX5 gene promoter,it has been found in AMI patients that sequence variant sites may have an effect on the transcriptional activity of TBX5 and the binding of transcriptional sites,which may lead to changes in expression levels.Further research on the occurrence of myocardial infarction provides a new direction and provides a potential target for the treatment of myocardial infarction.