Genetic And Functional The ATG12 Gene Promoter Variants In Acute Myocardial Infarction

Background:Acute myocardial infarction(AMI)is seriously endangering the lives of people all over the world.At present,the prevalence and mortality of AMI in China are on the rise.AMI is a very common complex disease,which is affected by environmental pollution,heredity and other factors.The main mechanism of AMI is to destroy or erode coronary atherosclerotic plaques,resulting in thrombus in circulating platelets and blockage of coronary artery lumen,thus causing myocardial ischemia and necrosis factors.So far,although people have more and more knowledge about AMI,the overall mortality rate of AMI patients is still very high.Therefore,the genetic factors and molecular mechanisms of AMI still need to be further studied and clarified.In recent years,autophagy has attracted extensive attention in the cardiovascular field.Autophagy is a major regulator of cardiac homeostasis and function,maintaining cardiac function and reducing myocardial injury by providing a substrate for regeneration of ATP during ischemia or starvation.During heart failure and aging,cardiac autophagy is inhibited;In the acute phase of ischemia/reperfusion,autophagy is overactivated.Mitochondrial autophagy is the basis of maintaining cardiac function.With the increase of age,the level of cardiac mitochondrial autophagy is down-regulated,while the mitochondrial function is decreased.Autophagy is involved in many physiological processes,including lipid metabolism and inflammation.ATG12 plays an important role in autophagy.In addition,ATG12 co-regulates mitochondrial homeostasis and cell death with ATG3.Animal experiments showed that ATG12 regulated autophagy and apoptosis of cardiomyocytes in vivo and in vitro.ATG12 levels were significantly reduced in arteriosclerosis mice.Therefore,mutations in the promoter region of ATG12 gene may cause changes in the expression level of ATG12 and affect the regulation of autophagy and mitochondrial autophagy,which may further affect the occurrence and development of AMI.AIM:The molecular genetic study on the genetic and functional variation of ATG12 gene promoter in AMI population may provide a more effective genetic basis for the prevention,treatment and disease mechanism of AMI.Methods:1.A case-control study was used to include 331 AMI patients and 349 healthy control population who underwent physical examination at the same time.Collected the clinical data of two groups,the extraction of peripheral blood on an empty stomach,extracting genome DNA.2.ATG12 primers were designed and amplified by PCR.Mutation sites were found by sequencing.3.Hardy-Weinberg test was performed on DSVs conforming to the standard using Halo View software.The difference of each DSVs between the control group and the AMI group was calculated using cross table analysis.SNP-Stats was used to analyze the association between AMI and five genetic models of SNPS that met the standard.4.The recombinant plasmid vector of ATG12 gene promoter was constructed to detect the mutation sites of ATG12 promoter in HEK-293 cells and NRCMs.5.TRANSFAC program was used to predict transcription factor binding sites that may be affected by ATG12 promoter site variation.6.EMSA further determined the binding mode of transcription factors affected by DSVs to ATG12 gene promoter.Results:1.Comparing the sequencing results,a total of 12 DSVs appeared in 680 samples.Two DSVs(g.115842475G>A and g.115842135C>G)and three SNPs[g.11584269 0C>G(rs1169604262);g.115842024G>C(rs1050864)and g.115841945T>G(rs117079134)]appeared in AMI population.Four SNPs[g.115841928 G>C(rsl 194285846);g.115841903 A>G(rs573530420);g.115842670 C>G(rs144189306);g.115841962 C>G(rs1362490071)]and two DSVs(g.115842132 G>C;g.115842090 A>G)appeared in healthy controls.In addition,The SNP[g.115842 698c>t(rs26538)]appeared in both groups at the same time,and its frequency was not statistically significant.2.SNP[g.115842698C>T(rs26538)]conforms to Hardy-Weinberg test(AMI group:0.538,control group:1.0),indicating that the sample conforms to population heritability.The correlation between rs26538 and AMI was analyzed by SNPStats online analysis software after gender,age and other confounding factors were corrected.The results showed P>0.05,indicating that there was no correlation between rs26538 and AMI,after all confounding factors were corrected.3.Using TRANSFAC(https://portal.genexplain.com/)online prediction program is used to predict the AMI patients in the group of five different mutation loci,and preliminarily determine whether the occurrence of each mutation site produces,eliminates or changes the binding of transcription factors to the corresponding binding sites of the ATG12 promoter sequence.speculated that the mutation point affect assumes that transcription factor binding sites.It was preliminarily determined whether the occurrence of each mutation site produced,eliminated or changed the binding of transcription factor to the corresponding binding site of ATG12 promoter sequence.4.Transfection data of HEK-293 cells and NRCMS showed that two DSVs(g.115842475G>A and g.115842135C>G)and three SNPs were found in AMI patients[g.115842690C>G(rs116904262),g.115842024G>C(rs1050864)and g.115841945T>G(rs1170791134)]strikingly down-regulation the transcriptional activity of ATG12 gene promoter(P<0.01).Only other DSVs found in the control group and the two groups did not affect the transcriptional activity of atg12(P>0.05).5.The results of EMSA showed that SNP[g.115842024G>C(rs1050864)]eliminated the binding of transcription factors in HEK-293 cells and created a binding site for new transcription factors,which may be due to tissue-cell specificity or sensitivity of EMSA experiment.NRCMs is not affected.DSV(g.115842135C>G)significantly enhanced the binding of unknown transcription factors in HEK-293 and NRCMs.DSV(g.115842475G>A)almost eliminated the binding of unknown transcription factors.Other SNPS[g.115842690C>G(rs1169604262)and g115841945T>G(rs1170791134)]did not affect the binding of transcription factors.Conclusion:A total of 12 DSVs were identified in 680 case samples.Initial findings from TRANSFAC predicted binding sites,transfection,and EMSA experiments revealed that the five DSVs present only in the AMI group altered the transcriptional activity of the ATG12 gene in HEK-293 cells and NRCMS,possibly by interfering with the binding of some transcription factors to the binding sites of the ATG12 promoter.These 5 DSVs may affect the binding of transcription factors and the promoter of ATG12,thereby affecting the normal expression of the ATG12 gene and may also affect the susceptibility to AMI.