The Study Of Obesity-related Single Nucleotide Polymorphism On The Gene Of Adenylate Cyclase 3

Obesity,which is caused by mutation of the adenylate cyclase 3(AC3),is the first single-gene obesity disease.Although a large number of Genome-wide association study(GWAS)have shown that there are multiple obesity-related SNPs on human AC3,it is unclear whether these SNPs influence the expression and function of AC3.The CRISPR/Cas9 can be used for single base editing,therefore,we firstly analyzed the obesity-related SNP sites in AC3 using bioinformatics,and then the SNP sites of AC3 gene were edited by CRISPR/Cas9 technology to explore the effect of SNPs on AC3 gene expression and its mechanism in 293 T cells in this paper.1.Bioinformatics analysis of the obesity-related SNP sites in AC3 gene: 1)Predictive analysis of the post-translational modification of the protein and the binding sites of the miRNAs on the 3’ UTR.The results showed only the SNPs on exon1 and exon17 might be phosphorylated,and there was no miRNA binding at the SNP site on the 3’ UTR;2)predicting whether the SNP sites on the AC3 gene has transcription factor binding.The results showed there were many transcription factors bindings at the SNP site,such as EMX family,OTX family,KLF family,to lay the foundation for studying the mechanism of SNP regulating AC3 expression.2.To investigate the effects of exon13 SNP(rs2289092),exon17 SNP(rs1127568),3’UTR SNP(rs1044040)and intron1 SNP(rs6545814)on AC3 expression: 1)Firstly,the sequences of the SNP locus was knocked out to explore the role of the sequence of the SNP locus in AC3 expression.To detect the indels,Cas9-sgRNA plasmids targeting different SNP sites were constructed and then transfected into 293 T cells,and the best gRNA which produced the highest indel(%)would be obtain.The expression of AC3 has detected after knocked out the target sequences,and the results showed the expression of AC3 was decreased after knocked out the exon13 or exon17 target sequences;2)To further clarify the effect of single base mutation at SNP locus on AC3 expression,Cas9(D10A)-sgRNA plasmid and a template donor for homologous targeted repair of exon13 SNP site(Donor-HR plasmid)were transfected and obtained the stable transfected cells.The expression level of AC3 was detected after the point mutation at the exon13 SNP site.The results showed that the expression of AC3 hardly changed.3.To explore the mechanism which transcription factors bind to SNP site to regulate AC3 expression.The luciferase reporter plasmids containing the wild-type exon13 or point mutation exon13 were constructed,and the transcription factors EMX2 and OTX1 were transfected into 293 T cells with the above two plasmids,respectively.After 48 h,the luciferase signal was detected using a reporter gene detection system to investigate the binding of the transcription factors EMX2 and OTX1 to the exon13-SNP site.The results showed that the binding of EMX2 to T was stronger than G.OTX1 could bind to T,while might not bind to G.Then EMX2 and OTX1 were co-transfected with wild-type mouse AC3 and exon13-SNP mutant mouse AC3 to 293 T cells,respectively,and the expression level of wild-type mouse AC3 transfected with EMX2 was significantly decreased.The results lay the foundation for verifying the regulation of EMX2 on AC3 expression in vivo.Conclusion :1.Only the SNPs on exon1 and exon17 may be phosphorylated;There are many transcription factors binding at the SNP sites,such as EMX family,OTX family,KLF family.2.The SNP site on the AC3 gene exon13 has no direct effect on the expression of AC3 protein.3.The transcription factor EMX2 may be involved in the regulation of AC3 expression through sequence-dependent binding to the exon13 SNP site.