| Background:The main pathological process of patients with coronary heart disease is coronary artery lumen stenosis caused by atherosclerosis.In severe cases,complete lumen occlusion often causes acute coronary syndrome,arrhythmia and heart failure,sometimes it even leads to sudden cardiac death.It is the main cause of clinical mortality worldwide.Inflammation is associated to the occurrence and development of atherosclerosis and its complications?including coronary heart disease and acute myocardial infarction?.Inflammatory response may protect ischemic myocardium by stimulating myo-cardial cells to undergo autophagy.In addition,abnormal lipid metabolism is also important for the occurrence of these diseases.The transcription factor E3?TFE3?belongs to the MiTF family.Overexpression of TFE3 can amend metabolic abnormalities caused by ingestion of large amounts of fat.TFE3 also regulate lysosomal signal transduction with TFEB and MITF?both belonging to the MiTF family?through various pathways,and promote autophagy and lysosomal catabolism.In recent years,methodologies of genotyping and gene sequencing have continued to make progress.Some researchers has focused on the population of comm-on mutations and found polymorphisms of new loci associated with CHD,but these can only explain a few cases.However,the low-frequency variation of genes may have great signific-ance.Therefore,human genetics is a very useful and valuable tool that can help us explore the role of low-frequency genetic variation of the TFE3 gene promoter in the pathogenesis of acute myocardial infarction further.Objective:Find out the low-frequency genetic mutations and single-nucleotide polymorphisms of the TFE3 gene promoter both in the experimental group?patients with acute myocardial infarction?and the control group?healthy population?by gene sequencing.Construct pGL3-basic reporter gene vector including the mutation site of the TFE3 gene promoter to study the effects of genetic and functional variations of the TFE3 gene promoter on the development of acute myocardial infarction.Methods:1.Choose 352 patients with acute myocardial infarction and collect anticoagulant peripheral blood of these patients,then extract the whole genomic DNA of the blood as the samples of the experimental group.The clinical data of the patients are queried and recorded in the hospital electronic system.Similarly,collect the anticoagulated peripheral blood of 343 healthy individuals from physical examination center to prepare the samples of control group.2.Use the Primer 6.0 software to design primers and amplify the promoter region of the TFE3 gene.Compare the target fragment sequences and the nomal TFE3 gene promoter sequences and find the low-frequency genetic mutations in the samples.The target fragments containing these mutation sites are connected to the PGL3-basic reporter vector and transformed into E.coli,culture the special bacteria to extract the corresponding plasmids,and transfect the target fragment plasmids?add internal reference pRL-TK?into human embryonic kidney cells and rat cardiomyocytes,detected and compared the firefly-renilla luciferase activity of two kinds of target fragments in the same kind of cells as well as the same kind of target fragments in different cells,and speculate the connection of low-frequency mutation sites and promoter transcriptional activity.3.Use the TRANSFAC program to search low-frequency mutations in the TFE3 gene promoter that may affect sequence binding,synthesize biotin-labeled oligonucleotide probes of normal and mutant TFE3 promoters and perform electrophoretic mobility experiments to analyze the changes of transcription factors bound with mutated promoter sequence.Results:1.Search the normal TFE3 gene promoter sequences in the gene bank and compare it with the target sequences,find that a total of 5 DNA sequence variation sites in the two types of samples,the AMI group includes 1 DSV[g.49043430 G>A?rs 1265786010?],the control group includes 2 DSVs[g.49043390 G>T,g.49043757 T>A?rs 1019053326?]and one deletion[g.49043737/8/9 A],and another DSV[g.49044029 G>T?rs 782026714?]presents in the AMI group as well as the control group.2.Construct the PGL3-basic reporter vector with the target fragment containing wild type or mutation site,and transiently transfect with pRL-TK internal plasmid into HEK-293 and H9C2 cells in vitro and detect the luciferase activity.?1?Detection and comparison of luciferase activity in rat myocardial cell lysate reveal that only one DSV in the AMI group could reduce the transcriptional activity of the TFE3 gene promoter?P<0.05?significantly.Neither the two DSVs and one deletion in the control group nor the one DSV in the two groups could change the transcriptional activity of the TFE3 gene promoter?P>0.05?.?2?Detection and comparison of luciferase activity of human embryonic kidney cell lysate,the results are consistent with H9C2 cells.3.The results of the TRANSFAC program suggests that the low-frequency variation of TFE3 gene promoter may eliminates the binding sites of transcription factors RREB1,EGR2 and SP2,and it modifies the binding sites of transcription factors MZF15-13 and EGR1.The result of EMSA shows that DSV[g.49043430 G>A?rs 126578 6010?]can enhance the binding of transcription factors in HEK-293 cells and produce a new binding band in H9C2 cells.Conclusion:Among the study we will know that the transcription activity of the promoter and the binding efficiency of transcription factors can be changed by the low-frequency genetic variation of the TFE3 gene promoter.Therefore,DSVs may cause various reaction mechanisms in the human body which are disturbed and unable to exert normal physiological functions.Ultimately,they promote the occurrence and development of acute myocardial infarction in many aspects. |
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