Genetic And Functional Variants Of The SIRT4 Gene Promoter In Myocardial Infarction

Objective: To explore the relationship between AMI and SIRT4 gene,we detected the variation of SIRT4 gene promoter sequence in AMI patients and healthy people by sequencing.By comparing the changes of SIRT4 gene expression level regulated by DNA sequence variation to explore the relationship of AMI and SIRT4 gene.We try to explain the pathogenesis of AMI from the perspective of basic genetics,and provide a new direction for the prevention,diagnosis and treatment of AMI.Methods: The experimental group selected 466 patients who were diagnosed with AMI in the Affiliated Hospital of Jining Medical College from June 2014 to September 2016,and randomly selected 460 healthy subjects from the physical examination center of the hospital as the control group.The clinical data of the two groups were collected and the whole genome DNA was extracted.Primers were designed based on the human SIRT4 gene promoter sequence provided by the NCBI gene database.The SIRT4 gene promoter DNA sequence was amplified by PCR technology and sent to Shanghai Shenggong for sequencing.Then we analyzed the DNA sequence variation of the SIRT4 gene promoter based on the sequencing results.The wild type(WT)and the gene promoter fragment containing the DNA sequence variation site were respectively constructed into PGL3-basic vector,and then transfected into HEK293 cells and H9C2 cells along with internal reference plasmidPRL-TK.The relative activities of two luciferases in two types of cells were examined,and the influence of genetic variation of SIRT4 gene promoter on its transcriptional activity was statistically analyzed to explore the relationship between SIRT4 gene and AMI.Results: We found 13 DNA sequence variant sites(DSVs)in this study,including 8 single nucleotide polymorphisms(SNPs).There are 6DSVs found only in AMI patients,respectively g.120301113T>C(rs117917450),g.120301158T>C,g.120301223A>G(rs377133726),g.120301432G>T,g.120301646C>T,g.120301881C>G(rs140691657),which were not found in the control groups.And 5 novel heterozygous DSVs,respectively g.120301295G>A(rs746724456),g.120301563G>C,g.120301575C>T(rs375039725),g.120302349G>C,g.120302370G>T(rs765200215)were only found in the control groups,and were not found in AMI patients.The other two DSVs were SNPs,respectively g.120301100T>C(rs138194145),g.120301832T>G(rs146884386),which were found in both groups,but they were not statistically significant(P>0.05).The PGL3-basic reporter gene vector was constructed and co-transfected into HEK293 cells and H9c2 cells along with the internal reference plasmid PRL-TK.The relative expression activities of two luciferases in WT and SIRT4 gene promoters carrying different DSVs in two cells were detected,and the effect of genetic variation of SIRT4 gene promoter on its transcriptional activity was statistically analyzed.Wefound that the DSVs which were only found in the AMI groups,respectivelyg.120301113T>C(rs117917450),g.120301158T>C,g.120301223A>G(rs377133726),g.120301432G>T,g.120301646C>T,g.120301881C>G(rs140691657)significantly reduced the transcriptional activity of the SITR4 gene promoter(P<0.05),no other DSVs can alter its transcriptional activity(P>0.05).Conclusion: Through the genetic and functional analysis of the SIRT4 gene promoter,we found that the genetic variation of the SIRT4 gene promoter may change its transcriptional activity,then affect the expression level of SIRT4 gene and lead to the occurrence and development of AMI.