Functional Genetic Variants Within The LAMP2 Gene Promoter In Acute Myocardial Infarction

Background: Myocardial infarction?MI?is the most common and major type of disease in coronary artery disease?CAD?.Its incidence is increasing year by year,and the age of onset is gradually younger.It has been an increasingly important medical and public health problem,and is the leading cause of mortality in world.It is currently believed that the main risk factors for CAD include smoking,hyperlipidemia,hypertension,and genetic factors.In recent years,with the constant deepening of Genetics,it is currently confirmed that the Genome-wide Association Study?GWAS?found that there are more than 200 CAD-related chromosomal loci.Lysosome associated membrane protein-2?LAMP2?is a novel Antineutrophil cytoplasmic antibody,which resides on the Xq24²5 chromosome.LAMP2 is a type 1 membrane protein that is present in lysosomes and plays a vital role in autophagy and degradation of lysosomal pro-ducts.In LAMP2-deficient fibroblasts,due to the disorder of cholesterol esterification,most of the unesterified cholesterol is accumulated in the late endosomes and lysosomes,leading to cholesterol aggregation,which induces the formation of atherosclerotic plaque.In the peripheral granulocytes of CAD patients,the expression level of LAMP2 gene is significantly increased,which causes abnormal chol-esterol metabolism and leads to atherosclerosis,promoting the occu-rrence and development of CAD.Objective: First of all,we studied the sequence variation of the LAMP2 gene promoters in patients with AMI and healthy control.And then,variants in LAMP2 gene promoters and wild type were subcloned into luciferase reporter vector?pGL3-basic?to construct expression vectors.At last,transfected into cultured cells,dual luciferase activities were examined to detect the level of the LAMP2 gene expression.We explore the role of LAMP2 in the occurrence and development of myocardial infarction by researching into the LAMP2 gene promoter's genetic and functional variation.Methods:1.In this research,we finally recruited 324 cases of AMI patients and 310 cases of ethnic-matched healthy controls,and then clinical data of the two groups were collected and the genomic DNA was extracted.2.PCR primers of LAMP2 human gene promoter sequences were designed based on NCBI database?NC₀00023.11?.The LAMP2 gene promoter was amplified by the technology of PCR,and then amplified PCR products were bi-directionally sequenced and compared with type LAMP2 gene promoter.3.Variant LAMP2 gene promoters and wild type were subcloned into luciferase reporter vector?pGL3-basic?to constrict reporter expression vectors,and vector pRL-TK was used as an internal control for transfection.Then the pGL3-basic luciferase reporter plasmid and the internal reference pRL plasmid were transiently transiently transfected into HEK293 cells and H9c2 cardiomyocytes using liposomes.After that,co-transfected into cultured cells by lipofectamine,dual-luciferase activities were examined by using dual-luciferase reporter assay system.Results:1.A total of 8 DSVs,including 6 single-nucleotide polymorphi s-ms?SNPs?,were found in the research.In the AMI group,there we re three SNPs: g.120469493G>T?Rs 868454097?,g.120469262T>C?R s1044167843?,g.120469507insG?Rs1192960118?,and one novel hetero zygous DSVs.In the healthy controls,one novel heterozygous insert ion DSV?g.120469183 insCTGCCGCCG?and one SNP(g.120469730T>C?Rs1395931999?were identified.Two SNPs(g.120469779A>C?Rs42900?,g.120469590T>G?Rs28603270?were found simultaneousl y in the AMI group and the normal control group.When the frequ encies of the two groups were compared,we found that one SNP`s?g.120469590T>G?Rs28603270??frequency of the two groups were s imilar,there was no statistical significance?p>0.05?,but the SNP`s?g.120469779A>C?Rs42900??was statistical significance?P<0.05?2.Variant LAMP2 gene promoters and wild type were cloned into luciferase reporter vector?pGL3-basic?to generate expression v ectors,including pGL3-basic?negative control?,pGL3-WT?wild type LAMP2 gene promoter?,pGL3-120469730 C,pGL3-120469493 T,pG L3-120469510 G,pGL3-120469262 T,pGL3-120469183 insCTGCCGC CG.3.Liposomes were transiently transfected into cell lines that were human embryonic-kidney cells?HEK-293?and rat cardiomyocyte cells?H9c2?.Then we started to collected the cells and assay dual-luciferase activities.At the last,the relative transcriptional activities of the LAMP2 gene promoters were calculated.After transfected into cultured cell lines?HEK-293 and H9c2?,t he DSVs?g.120469183 insCTGCCGCCG,g.120469730T>C?Rs1395931999??that were only found in the AMI group significantly decrease d the activity of the LAMP2 gene promoter?P<0.01?.the DSV?g.120469510A>G?only identified in the AMI group significantly increas ed activity of the LAMP2 gene promoter?P<0.01?.The DSVs?g.120469183 insCTGCCGCCG,g.120469730T>C?Rs1395931999??that were only identified in the control group did not alter activity of the LA MP2 gene promoter?P>0.05?significantly.The DSVs?g.120469779A>C?Rs42900?,g.120469590T>G?Rs28603270??were identified in the c ontrol group and the AMI group at the same time did not alter the activities of the LAMP2 gene promoter?P>0.05?significantly.Conclusion: Through the present research in which the LAMP2 gene promoter was genetically and functionally analyzed in AMI group and control group,the novel DSVs were in AMI group significantly altered the transcriptional activity of the LAMP2 gene promoter in cultured cardiomyocytes.Hence,the LAMP2 gene promoter DSVs may significantly alter the transcriptional activity of the LAMP2 gene promoter and change the level of LAMP2 expression,which plays an important role in the occurrence and development of AMI and provides new views for the pathological mechanism,diagnosis,treatment and prevention of AMI.