Genetic And Functional SIRT1 Gene Promoter Variants In Senile Degenerative Heart Valvular Disease

Background:Senile degenerative heart valvular disease(SDHVD)is characterized by degenerative heart valve function which occurs with aging.Up to now,surgical intervention is still the dominant treatment for SDHVD and there is no effective medicine.The risk factors of SDHVD include advanced age,hypertension,hyperlipidemia,diabetes and smoking,and genetic factors for SDHVD remain unclear.A large number of studies have shown that SDHVD is associated with chronic inflammation of valve tissue,lipid deposition,calcification and ossification and other pathological processes.SIRT1 is a deacetylase,which regulates a variety of physiological processes,including inflammatory reaction,lipid metabolism and calcification and others.Therefore,we speculated that the changes of SIRT1 gene expression may be related to SDHVD.Since gene promoters regulate gene expression levels,exploring genetic and functional variants of SIRT1 gene promoters may provide a new genetic basis for SDHVD.Objective:We aimed to find the DNA sequence variants(DSVs)of the SIRT1 gene promoter in SDHVD patients and healthy people,then constructed recombinant PGL3-basic expression vector according to the DSVs,and then transfected these plasmids into cells.At last,we tried to explore how the DSVs influence SIRT1 gene transcription level through dual-luciferase reporter gene system.Methods:1.SDHVD patients(n=236)diagnosed in the department of cardiology were recruited from the Affiliated Hospital of Jining Medical University.Healthy controls(n=285)were from the same hospital,and their ages were over 60 years old.Clinical data of the two groups were collected.Peripheral blood mononuclear cells were collected and genomic DNAs was extracted.2.Polymerase chain reaction(PCR)primers were designed according to the sequence of SIRT1 gene promoter,and then the target fragments were amplified by PCR for direct sequencing.DSVs were found by comparison with wild-type SIRT1 gene promoter,and statistical analysis was performed.3.Wild-type and DSVs of SIRT1 gene promoter were screened by PMD19-T vector.With plasmid extraction,double enzyme digestion,DNA purification,recombinant PGL3-basic expression vector with wild-type and DSVs SIRT1 gene promoter were constructed.Using liposome transfection method,recombinant PGL3 plasmids were transfected into cultured human embryonic kidney cells(HEK-293 cells)and rat cardiomyocytes(H9C2 cells).The transcription level of SIRT1 gene promoter was examined by the dual-luciferase reporter gene system.Through comparing with the transcription level of wild-type SIRT1 promoter,DSVs significantly affecting the transcription level of SIRT1 gene promoters were identified.Results:1.Twelve DSVs were found:only one new heterozygous DSVs was found in the SDHVD group:g.4843C>G.Six heterozygous DSVs were found in the control group:g.4206G>A,g.4667C>G,g.4860C>T,g.4912A>G,g.5042G>A,and g.5042G>T.There were 5 single nucleotide polymorphisms(SNPs)in the SDHVD group and the control group:g.4593A>G(rs3740051),g.4851A>C(rs932658),g.4874G>T(rs35995735),g.4969A>G(rs3740053),and g.4975G>C(rs2394443).2.Through dual-luciferase reporter gene system to detect SIRT1 gene promoter transcriptional activity,we found that g.4843C>G in HEK-293 cells and H9C2 cells significantly enhanced the SIRT1 gene promoter transcriptional activity(P<0.05),g.4206G>A,g.4667C>G,g.4912 A>G,g.5042G>A in HEK-293 cells and H9C2 cells significantly reduced the SIRT1 gene promoter transcription activity(P<0.05).While g.4860C>T and g.5042G>T only significantly reduced the transcriptional activity of SIRT1 gene promoter in H9C2 cells(P<0.05),but had no effect in HEK-293 cells(P>0.05),which may be caused by tissue differences.Conclusion:DNA sequence variants of SIRT1 gene promoter may change the transcriptional activity of SIRT1 gene,and thus affect its expression level.This study provided a new potential genetic target for the diagnosis and treatment of SDHVD.