| Objective:We aimed to investigate the genetic variants of LC3B gene promoter in patients with myocardial infarction(MI)and healthy controls by constructing the recombinant plasmid based on the sequence variants for the dual-luciferase reporter assay system.We tried to analyze the functional changes of LC3B gene promoter via checking the activity of firefly and renilla luciferases,explore the biological essece of relationship between genetic sequence variants and MI incidence and lay a theoretical foundantion for the genetic analysis in MI,further provide a new way of prevention,diagnosis and treatment for MI.Methods:In the study,the genetic sequence analysis and function assay of LC3B gene promoter was conducted in large case-controls,with MI patients(n=383)recruited from Jining Medical University Affiliated Hospital,and healthy controls(n=390)randomly chosen from Physical Examination Center in the same hospital during the same period as the patients were collected.There was no difference in gender and age between the two groups.Besides drawing on the clinical data,blood samples were collected from the two groups to extract genome DNAs.According to the genome DNA sequence of LC3B gene promoter,we designed and synthesized the PCR primer,and amplified the LC3B gene promoter by PCR and directly sequenced,through the results of which we found out the DNA sequence variants of both groups.LC3B gene promoters consisting of the sequence variants mentioned above were constructed into the pMD19-T vector for amplication,and were then enzyme cut and sequences detected.The amplified fragments were then cleaved with pGL3-basic vector,and along with PRL-TK vector,were transiently transfected into HEK293 cells and H9C2 cells.Dual-Luciferase reporter assay kit was exploited to measure the activity of the luciferases after gathering the cell lysate.We initially explored the effects of LC3B gene promoter sequences variants on the transcriptional activity,and the difference exsiting between patients with MI and healthy control group.Results:1.We totally identified twenty-five DNA sequence variants(DSVs)in this research population,including twelve single-nucleotide polymorphisms(SNPs).Seven novel DSVs were identified in MI patients,namely g.4107T>A,g.4325G>C,g.4445delG,g.4597C>G,g.4891C>A,g.4903G>T,g.4938C>T and three SNPs,g.4137C>T(rs575567442),g.4208C>T(rs111626199)and g.4853A>G(rs77019223).None of the above DSVs were found in controls.Five novel DNA sequence variants were discovered only in controls,namely g.4106C>T,g.4224T>C,g.4889C>G,g.4904C>G,g.5001A>C,and two SNPs,g.4175G>C(rs543058021),g.4494A>C(rs577616524),which were not found in MI patients.Besides,another eight DSVs,including seven SNPs,g.4068G>A(rs552929429),g.4280C>T(rs11117269),g.4282C>T(rs532744297),g.4433G>A(rs142738317),g.4631C>G(rs35227715),g.4774G>A(rs189170289),g.4883C>A(rs16944733),and one novel DSV,g.4488G>C were both existed in MI patients and controls,and were no different in frequency between the two groups(P>0.05).Interestingly,we found out g.4208C>T(rs111626199)and g.4853A>G(rs77019223)were closely linked together with the same frequencies.2.In HEK293 cells and H9C2 cells,the DSVs and SNPs in MI group,including g.4107T>A,g.4325G>C,g.4597C>G,g.4891 C>A,g.4903G>T,g.4938C>T,g.4137C>T(rs575567442)and g.4853A>G(rs77019223)significantly decreased the transcriptional activity of the LC3B gene promoter(P<0.05),while the other DSVs and SNPs were not identified the functional change of the LC3B gene promoter(P>0.05).Conclution:The LC3B gene promoter sequence variants(SNP,DSVs)identified in MI patients may influence LC3B gene expression by changing the transcriptional activity of LC3B gene promoter,and may exert a critical role in the occurrence and development of MI through distinct signal pathways. |
PDF Full Text Request