Functional Genetic Variants Of ATG5 Promoter Gene In Acute Myocardial Infarction

Background:Coronary atherosclerotic disease(CAD)can lead to cardiac dysfunction and heart failure.CAD is a vascular disease caused by the interaction of genetic and environmental factors.Myocardial infarction(MI)is the most serious type of CAD that can cause serious damage to the heart muscle tissue and the main reason for the increase of CAD mortality annually[1].Immediate family members and peers of the same risk factors for AMI patients compared to those who did not suffer from MI,the chance of heart damage increased by 50%to 80%[2,3],indicating that genetic variation has become one of the risk factors for CAD disease.Autophagy,as a normal physiological process widely existing in eukaryotic cells,is involved in inflammation,heart disease,immune disease,neurological disease and so on.Autophagy,as an internal regulatory mechanism of the body,maintains cell homeostasis by degrading essential intracellular proteins and damaged organelles.Autophagy activity is involved in the pathological process of cardiovascular diseases,such as cardiac hypertrophy,heart failure alu ischemic heart disease[4]·Increase myocardial autophagy in the heart decreases the infarct area[5,6].Autophagy-associated protein 5(ATG5)is a key molecule involved in autophagosome formation during autophagy.Studies have confirmed that ATG5 is involved in cardiac development,differentiation of cardiac progenitor cells,and the development of CAD[6].Therefore,we speculate that the variants of ATG5 gene promoter sequence play a very important role in acute myocardial infarction.Objective:In this study,a case-control study was used to identify the variants of ATG5 gene promoter sequence in patients with acute myocardial infarction and healthy controls,to identify mutation sites,to construct a pGL3-vector for the mutation site,and to detect the dual luciferase activity.Through statistical analysis,to investigate changes in the transcription level of the ATG5 gene promoter caused by mutation;finally,we will use JARSPER software to predict ATG5 gene promoter mutation related transcription factor changes,and through electrophoretic mobility shift assay(EMSA)test the protein-DNA interaction,further validation ATG5 mutation in AMi effect in the process of development.Methods:1.The study divided the eligible subj ects into the AMI group and the healthy control group.The final included subj ects were 378 cases in the AMI group and 3 86 cases in the healthy control group.The clinical data and fasting peripheral blood samples were collected from the two groups,and the whole genome DNA was extracted from the blood samples;2.Through NCBI gene database,human ATG5 gene promoter sequence was retrieved and primers were designed.The promoter fragment of ATG5 gene was amplified by PCR in vitro.After gene sequencing,the results were statistically analyzed to determine the relevant DNA sequence variants(DSVs)and single nucleotide polymorphisms(SNPs)of ATG5 gene promoter;3.The target fragment of DSVs and SNPs and the wild type sequence of the ATG5 gene promoter are ligated into the cloning vector(PMD19-T Vector),and the plasmid is extracted,and after amplification and cleavage,it is ligated to the pGL3-basic reporter vector;4.The internal reference plasmids pRL-TK and pGL3-basic reporter vectors were transiently transfected into HEK293 cells and H9c2 cardiomyocytes cultured in vitro by liposome co-transfection,and the dual luciferase activity of two cells were detected by means of a dual fluorescence reporter gene analyzer.By recording and counting fluorescence values,preliminary analysis of the effect of ATG5 gene promoter sequence mutation sites on gene transcription level;5.The JASPAR software predicts changes in the transcriptional factors of the ATG5 gene promoter sequence variants,and designs the DNA variant site sequence as a biotin-labeled probe for EMSA with nuclear extracts of HEK293 cells and H9 c2 cells.The chemiluminescence method was used to detect the binding of biotin-labeled probe to nuclear protein,and the effect of ATG5 gene promoter mutation on transcription factor binding site was further explored.Results:of1.By sequencing PCR products,a total 15 mutations of ATG5 gene promoter sequence were found,in group AMI:one deletion mutation:g.106326169 delAGA and the other is a gene mutation:g.106325751 C>G,the latter is located in the first exon of the ATG5 gene promoter sequence.In control group,we found three DSVs:g.106326292 C>A,g.106325849 G>A and g.106325912 A>G.Furthermore,in both AMI group and healthy control group,there are four DSVs:g.106326739 T>C?g.106326486C>T?g.106326330 G>A and g.106325874 C>T,one insert variation:g.106326426 insT,five SNPs were found:g.106326588(rs506027)C>T,g.106326583(rsl 87678668)T>A,g.106326547(rsl 17781908)C>A,g.106326542(rsl 82877945)C>A and g.106326154(rs510432)A>G,there were no statistical significance after statistical analysis.2.In the above sequencing results,DSVs and SNPs with statistical significance and ATG5 gene promoter sequence wild-type will be constructed with the vector plasmid of pGL3-basic:pGL3-Wild Type(WT),pGL3-g.106326169delAGA.pGL3-g.106326292 A,pGL3-g.106325849 A,pGL3-g.106325912 G and pGL3-g.106326154G.3.The constructed pGL3 reporter gene vector plasmid was transfected into HEK293 cells and H9c2 cardiomyocytes by the method of liposome transient transfection,to detect its dual luciferase activity.The results showed that DSV in the AMI group only:g.106326169 del AGA could change the transcriptional activity of ATG5 gene promoter(P<0.01).The other healthy control groups DSVs and SNPs had no significant effect on the transcriptional activity of ATG5 gene promoter(P>0.05).4.After the EMSA experiment of DSV of AMI group,it was found that there was a specific binding band between g.106326169 delAGA mutation and protein by chemiluminescence method,and the binding between WT and DSV and protein is obvious difference.Conclusion:This research mainly analyzed the ATG5 gene promoter sequence of genetic variation and functional variation,finding that ATG5 gene promoter by sequencing of variation,low-frequency loci in AMI patients may affect ATG5 gene transcription activity,causing ATG5 changes in the level of gene expression,and further development of AMI play an important role.Enrich the study of autophagy in the area of myocardial infarction.