Genetic Variants And Functional Analyses Of The CTSL Gene Promoter In Acute Myocardial Infarction

Objective: By sequencing the promoter region of cathepsin L(CTSL)gene in the acute myocardial infarction(AMI)group and the control group,and comparing with the normal gene sequence in the gene library,the DNA sequence variations(DSVs)in the promoter region of CTSL gene were determined,and the p GL3 reporter gene vector containing the mutation site of CTSL gene promoter was constructed,By detecting the activity of double luciferase,functional analysis was carried out on the sequence variation of the CTSL gene promoter,to explore the effect of DSVs in the CTSL gene promoter region on the transcription activity of the CTSL gene promoter,and to explore the role of the CTSL gene promoter variation in AMI.Methods:1.Using the case-control method,228 patients in the AMI group and200 patients in the control group were included.The clinical data of the AMI group and the control group were collected,and the fasting peripheral blood of the two groups were collected and the whole genome DNA of the samples was extracted.2.Design polymerase chain reaction(PCR)primers according to the CTSL gene promoter sequence in NCBI,amplify the target segment of the CTSL gene promoter by PCR,sequence the target segment of the CTSL gene promoter by PCR by Sanger method,determine the DSVs and single nucleotide polymorphisms(SNPs)and conduct statistical analysis.3.Connect the wild-type target fragment and variant sequence of the CTSL gene promoter to the p MD19-T vector,extract the recombinant plasmid,construct the CTSL-p GL3 report gene vector after double enzyme digestion,and co-transfect the p RL-TK and the constructed CTSL-p GL3 report gene vector to HEK-293 cells and neonatal rat cardiomyocytes(NRCMs),and detect the activity of double luciferase in HEK-293 cells and NRCMs,respectively,To explore the effect of CTSL gene promoter DSVs on transcriptional activity.4.Use the TRANSFAC database to predict the binding of DSVs and transcription factors that affect the transcription activity of the CTSL gene promoter.Results:1.A total of 7 DSVs,including 5 SNPs,were found by sequencing the promoter region of the CTSL gene in 428 subjects.g.87726026 G>A,g.87726001 C>A(rs41307457),g.87725078 C>T(rs1009810799),g.87726151 C>T(rs41312184),and g.87725317 G>A were only found in the AMI group,but not in the control group.g.87725838 G>C(rs187982055)and g.87725948 C>A(rs3118869)appeared in both AMI group and control group.2.The p GL3 reporter gene vector(p GL3-WT,p GL3-87726026 A,p GL3-87726001 A,p GL3-87725078 T,p GL3-87726151 T,p GL3-87725317 A,p GL3-87725838 C and p GL3-87725948A)of the CTSL gene promoter wild type and mutation sequence site was constructed,and the internal reference plasmid p RL-TK and the constructed p GL3 reporter gene vector were co-transfected to HEK-293 cells and NRCMs,It was found that g.87725078 C>T(rs1009810799)and g.87725317 G>A increased the transcriptional activity of the CTSL gene promoter,while the remaining DSVs failed to change the transcriptional activity of the CTSL gene promoter.Conclusion: In this study,we studied the genetic variants and functional analyses of the CTSL gene promoter in patients with AMI and healthy people,and found that DSVs [(g.87725078 C>T(rs1009810799)and g.87725317 G>A)] that only appeared in AMI patients affected the transcriptional activity of the CTSL gene promoter.Therefore,this study speculates that rare mutations in the CTSL gene promoter in AMI patients may affect the transcriptional activity of the CTSL gene promoter,thereby affecting the expression of the CTSL gene,which may have an impact on the occurrence and development of AMI.