Functional Genetic Variants Within The SIRT2 Gene Promoter In Acute Myocardial Infarction

Background:Coronary artery disease?CAD?,including acute myocardial infarction?AMI?is the complication of atherosclerosis,and unstable atherosclerotic plaque rupture would contribute to AMI.Atheroscleros is a age-related disease,and the occurrence of atherosclerosis is involved in inflammation,oxidative stress,platelet function and other metabolic processes.Recently,genome-wide association studies have identified a large number of CAD-related genetic variants.However,only 10%of CAD cases could be explained.Low frequent and rare genetic variants have been recently proposed to be main causes for CAD.SIRT2 is a member of sirtuin family,NAD?+?-dependent class III deacetylases.SIRT2 is involved in genomic stability,metabolism,inflammation,oxidative stress and autophagy,as well as in platelet and vascular endothellium function.In the process of gene expression,the promoter controls gene expression level.Thus,we hypothesized that genetic variants in SIRT2 gene may contribute to AMI.Taken together,the DSVs identified in SIRT2 gene promoter may affect the transcriptional activity of SIRT2,contributing to the AMI development as a risk factor.Objective:Firstly,we discover the low frequency and rare genetic variants and single nucleotide polymorphisms?SNPS?in SIRT2 gene promoter by sequencing to investigate the relationship between these DSVs and AMI patients.Then,Wild type and variant SIRT2 gene promoters were subcloned into luciferase reporter vector?pGL3-basic?to construct expression vectors.After transfected into cultured cells,dual luciferase activities were examined to detect the functions of the SIRT2 gene variants in AMI patients and normal controls.We explore the role of SIRT2 in the pathogenesis of myocardial infarction by researching the SIRT2 gene promoter genetic and functional variation to provide the potential targets on prevention and treatment for myocardial infarction.Methods:1.In this study,we finally recruited 375 cases of AMI patients and 377 cases of ethnic-matched healthy controls,and then we collected clinical data of the two groups and extracted the genomic DNA.2.PCR primers were designed based on genomic sequence of the human SIRT2 gene?NCBI,NC₀00019.10?.PCR products were bi-directionally sequenced and then the DNA sequences were aligned and compared with the wild type SIRT2 gene promoter.3.Wild type and variant SIRT2 gene promoters were subcloned into luciferase reporter vector?pGL3-basic?to construct expression vectors and vector pRL-TK was used as an internal control for transfection.After co-transfected into cultured cells?HEK293 and H9c2?by lipofectamine,dual-luciferase activities were examined by using dual-luciferase reporter assay system on a Promega Glomax 20/20 luminometer.Results:1.A total of 17 DSVs,including 5 single-nucleotide polymorphisms?SNPs?,were identified in this study.Two novel heterozygous DSVs?g.38900270A>G and g.38899853C>T?and one heterozygous deletion DSV?g.38900888₉1delTAAA?,were identified in three AMI patients,but in none of controls.Five novel heterozygous DSVS?g.38900562C>T,g.3 8900413A>C,g.38900030G>A,g.38899925A>C and g.38899852C>T?were only found in healthy controls.In addition,three novel heterozygous DSVs?g.38900748T>A,g.38899903T>C and g.38899781C>G?,one deletion DSV?g.38901027₃0delTAAA?and five SNPs[g.38901007delT?rs10713585?,g.38900907A>G?rs4803006?,g.38900291C>G?rs2053071?,g.38900145C>T?rs116900177?and g.38899968A>C?rs112492606?]were found in both AMI patients and controls with similar frequencies?P>0.05?.2.Wild type and variant SIRT2 gene promoters were cloned into luciferase reporter vector?pGL3-basic?to generate expression vectors,including pGL3-WT?wild type SIRT2 gene promoter?,pGL3-38900907G,pGL3-38900888₉1del,pGL3-38900562T,pGL3-38900413C,pGL3-38900291G,pGL3-38900270G,pGL3-38900030A,pGL3-38899925C,pGL3-38899903C,pGL3-38899853T,pGL3-38899852T ? pGL3-38899781G.3.After transfected into cultured cell lines,human embryonic-kidney cells?HEK-293?and rat cardiomyocyte cells?H9c2?,the cells were collected and dual-luciferase activities were assayed and relative transcriptional activities of the SIRT2 gene promoters were calculated.?1?In HEK-293 cells,the DSVs?g.38900888₉1delTAAA and g.38900270A>G?that were identified only in AMI patients significantly decreased activity of the SIRT2 gene promoter?P<0.05 and P<0.01,respectively?.The DSV?g.38899853C>T?only identified in an AMI patient significantly increased activity of the SIRT2 gene promoter?P<0.01?.The DSVs?g.38900562C>T,g.38900413A>C,g.38900030G>A,g.38899925A>C and g.38899852C>T?that were found only in controls did not significantly alter activity of the SIRT2 gene promoter?P>0.05?.As expected,the SNPs[g.38900907A>G?rs4803006?and g.38900291C>G?rs2053071?]and the DSVs?g.38899903T>C and g.38899781C>G?that were found in both AMI patients and controls did not also alter activity of the SIRT2 gene promoter?P>0.05?.?2?In H9c2 cells,the DSVs?g.38900888₉1delTAAA and g.38900270A>G?significantly decreased the activity of the SIRT2 gene promoter?P<0.01?and the DSV?g.38899853C>T?significantly increased the activity of the SIRT2 gene promoter?P<0.01?.Similarly,the SNPs[g.38900907A>G?rs4803006?and g.38900291C>G?rs2053071?]did not significantly alter the activity of the SIRT2 gene promoter?P>0.05?.Conclusion:In the present study,the SIRT2 gene promoter was genetically and functionally analyzed in AMI patients and healthy controls.The novel DSVs identified in AMI patients significantly altered the transcriptional activity of the SIRT2 gene promoter in cultured cardiomyocytes.Therefore,the SIRT2 gene promoter DSVs may alter transcriptional activity of SIRT2 gene promoter and change SIRT2 level,contributing to AMI development as a risk factor.