| Background:Coronary atherosclerotic heart disease is referred to as coronary heart disease(CHD),which is mainly caused by lesion of atherosclerotic plaque in coronary artery,induced myocardial ischemia,hypoxia or necrosis.Abnormal lipid metabolism and inflammation play critical roles in the initiation and progression of atherosclerosis and its complications,including CAD and AMI[1,2]CAD and AMI are caused by interactions of genetic and environmental factors.To date,genome-wide association studies have identified a great number of genetic loci for CAD and AMI.However,the collective genetic locicould explain only<10%of cases[3-5].Autophagy is a highly conserved mechanism of lysosomal-mediated degradation of proteins and cell organs that plays a key role in maintaining cellular homeostasis[6].Autophagy can remove damaged proteins and organelles,which is essential for ensuring the homeostasis of the cells,but excessive autophagy or defects may degrade the important proteins or organelles required for cell survival.The transcription factor TFEB is a helix-loop-helix-leucine zipper bHLH-LZ transcription factor,and belongs to the MiTF/TFE family with MITF,TFEB,TFE3,and TFEC[7,8].TFEB plays a key role in the regulation of basic cellular processes,such as lysosomal biogenesis and autophagy[9].Overexpression of TFEB significaatly increased autophagosome production and expression of lysosomal-associated proteins[10].Therefore,we provide genetic support for the treatment of myocardial infarction by studying the variation of the TFEB gene promoter sequence in patients with myocardial infarction and healthy people.Obj ective:To identify low-frequency genetic variants and single nucleotide polymorphisms of TFEB gene in patients with acute myocardial infarction and healthy controls by sequencing,and constructs pGL3-basic reporter vectors for promoter mutation sites.Investigate the effect of sequence variation and functional variation of the TFEB gene promoter on the development of acute myocardial infarction.Methods:1.Alarge sample of case-control was used,include 352 patients with acute myocardial infarction and 337 healthy controls who were examined at the same time.The clinical data of the two groups were collected.Whole genome DNA was extracted from peripheral blood of both groups.2.Query the promoter sequence of the TFEB gene in the NCBI gene database,then design primers,and use PCR to amplify the target gene fragment,and find the mutation sites by sequencing.The wild type target fragments and sequence variant sites of the TFEB gene promoter were constructed into the PGL3-basic reporter vector,respectively,and then the constructed pGL3 reporter gene vectors were transiently transfected into HEK-293 cells and H9C2 cells with the internal reference plasmid pRL-TK by liposome encapsulation.Collected cell lysates,and detected the activity of firefly luciferase and renilla luciferase in the transfected cells by a dual-fluorescein reporter gene assay kit through a dual fluorescent reporter gene assay kit.Detection of TFEB gene sequence variations results in changes in transcription factor activity.3.Analyze whether the low frequency variations of the TFEB promoter sequence affects certain putative transcription factor binding by the TRANSFAC program,then we compared the transcription factors that binds the normal sequence and the mutant sequence.4.The probes for designing the normal sequence and the mutated sequence of the TFEB promoter were labeled by biotin labeling method,and the effect of the mutation of the gene sequence of the binding transcription factors were analyzed by electrophoretic mobility shift assay.Result:1.A total of 15 DNA sequence variants(DSVs)were found in the experimental group and the control group.Tow new DSVs and four SNPs were found in the AMI group,namely g.41737144 A>G,g.41736544C>T and four SNPs,namely g.41737274 T>C(rs533895008),g.41736987C>T(rs760293138),g.41736806C>T(rs748537297),g.41736635T>C(rs975050638).Four new DSVs and two SNPs were found in the control group,namely g.41737451T>G,g.41736981A>G,g.41736407C>T,g.41736218T>G and two SNPs,namely g.41737034C>A(rs73733015)and g.41737005G>A(rs149166358).Two DSVs and one SNP were both found in the experimental group and the control group,respectively,g.41737034G>A,g.41736740C>A and one SNP,g.41737034G>C(rs73733015),no statistical significance(P>0.5).2.Construction of pGL3-basic reporter gene vector,namely pGL3-WT(wild type TFEB gene promoter),pGL3-41737451G,pGL3-41737274C,pGL3-41737144G.pGL3-41737034C.pGL3-41737034A,pGL3-41737005A,pGL3-41736987T,pGL3-41736981G,pGL3-41736806T,pGL3-41736635,pGL3-41736544T,pGL3-.41736407T and pGL3-41736218G.3.After constructing the pGL3-basic reporter gene vector,the constructed repor-ter gene vector and pRL-TK internal reference plasmid were co-transfected into HEK-293 cells and H9C2 cells in vitro to detect the relative luciferase activity of different DSVs.(1)Transfection of HEK-293 cells,we found that the presence of two DSVs and four SNPs in t.he AMI group significantly altered the transcriptional activity of the TFEB gene promoter,with the SNP[pGL3-41736987C>T(rs760293138)]significantly reduced the transcriptional activity of TFEB gene promoter the(P<0.01).And DSVs[g.41737144 A>G,g.41736544C>T]and SNPs[g.41737274T>C(rs533895008);g.41736987C>T(rs760293138),g.41736806C>T(rs748537297),g.41736635T>C(rs9750506380)]significantly increased the transcriptional activity of the TFEB gene promoter(P<0.01).Four DSVs[g.41737451T>G,g.41736981A>G,g.41736407C>T,g.41736218T>G],and two SNPs[g.41737005G>A(rs149166358),g.41737034C>A(rs73733015)were found in the control group and the SNP[g.41737034G>C(rs73733015)]presents both in the AMI group and the control group have not been found to affect the transcriptional activity of the TFEB gene promoter(P>0.05).(2)Transfection of H9C2 cells,we found that the results were consistent with the transfection results in HEK-293 cells.The two DSVs and four SNPs in AMI group significantly altered the transcriptional activity of the TFEB gene promoter,the SNP[pGL3-41736987C>T(rs760293138)]significantly reduced the promoter of the TFEB gene(P<0.01).DSVs[g.41737144 A>G,g.41736544C>T]and SNPs[g.41737274T>C(rs533895008),g.41736987C>T(rs76029313 8),g.41736806C>T(rs748537297).g.41736635T>C(rs9750506380)]significantly increased the transcriptional activity of the TFEB gene promoter(P<0.01).Four DSVs[g.41737451T>G,g.41736981A>G.g.41736407C>T,g.41736218T>G],two SNPs[g.41737005G>A(rs149166358),g.41737034C>A(rs73733015),found in the control group and the SNP g.41737034G>C(rs73733015)present both in the AMI group and the control group have not been found to affect the transcriptional activity of the TFEB gene promoter(P>0.05).4.We think that the DSVs and SNPs found in AMI patients can affect binding of the transcription factors.The TFEB gene promoter was analyzed by TRANSFAC program.It was found that the SNP[g.41737274T>C(rs533895008)]may abolish a binding site for the transcription factor HMGA factors and NF-Y,creat the binding site for the transcription factor NR-DR,TGF-7 and BRCA1.the DSV(g.41737144 A>G)may modify the binding site for BRCA1.The SNP[g.41736987C>T(rs760293 138)]may modity the binding site for CBF-1/RBP-J.The SNP[g.41736806 C>T(rs748537297)]may create a binding site for the transcription factor peroxisome PPARGAMMA.The SNP[g.41736635T>C(rs975050638)]may abolish a binding site for the transcription factor GFI1,and creat the binding site for the transcription factor GR-like receptors.The DSV(g.41736544C>T)may modify the binding sites for SREBP and GR-like receptors.5.Electrophoretic mobility shift assay results showed that SNP[g.41737274T>C(rs533895008)]created a binding site for transcription factor.DSV(g.41737144A>G),SNP[g.41736987C>T(rs760293138)]and DSV(g.41736544C>T)modified the binding sites for transcription factors.SNP[g.41736806C>T(rs74853729)]and SNP[g.41736635T>C(rs975050638)]did not affect the binding of transcription factors.So we think these DSVs and SNPs found in AMI patients may affect the transcriptional activity of the TFEB gene promoter by altering the binding sites for transcription factors.Conclusion:In this study,genetic and functional analysis of the TFEB gene promoter was performed both in AMI patients and healthy controls.Two DSVs and four SNPs were identified in AMI patients,which significantly altered the transcriptional activity of the TFEB gene promoter.Furthermore,two DSVs and two SNPs evidently affected the binding of transcription factors.Therefore,these DSVs and SNPs may change TFEB level,contributing to AMI development through diverse pathways as a low-frequency risk factor. |
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