Relationship Between Polymorphism Of Chromosome 9q22.33 And Susceptibility To Ovarian Cancer And Its Function Study

ObjectOvarian cancer,one of the most common cancers in women,is a complex polygenic disease in which genetic factors play an important role.In the previous study,our research group found that the single nucleotide polymorphism（SNP）site rs1413299 located in area 9q22.33 was associated with the risk of epithelial ovarian cancer in Chinese Han women.The SNP site is located on intron 6 of COL15A1 gene.The TCGA data of the United States showed that the expression of COL15A1 gene was different between ovarian cancer tumor tissues and adjacent normal tissues,but the influence of SNP in this region on the occurrence and development of ovarian cancer was not clear.Therefore,this study intends to re-sequence the highly-linked unbalanced region of rs1413299 and verify it in the case control study,screen out the possible functional SNP sites and make functional prediction,and to clarify the molecular mechanism of SNP locus affecting the risk of ovarian cancer.MethodsHaploview software was used to analyze the linkage disequilibrium of the ovarian cancer susceptibility site rs1413299 to obtain the linkage disequilibrium area where the site was located.The high-throughput second-generation sequencing method was used to re-sequence the genomic DNA of 192 normal female blood to find the mutation sites in this region of Chinese Han women.MAF>0.05 and high linkage with rs1413299 SNP locus were screened for case-control study,to find out the locus with differences in genotype frequency of SNP locus in the case control study.In1099 cases of ovarian cancer and 1591 healthy controls,the Sequenom i Plex and Wafer Gen platform were used for genotyping of 19 SNP sites,and 101 cases of ovarian cancer tissues were selected.Real-time quantitative PCR method was used to detect COL15A1 gene expression,and the differences between different SNPs genotypes and COL15A1 gene expression were compared.Using bioinformatics sites such as Haplo Reg and Regulome DB to predict the biological function of diverse SNP sites.Electrophoretic Mobility Shift Assay（EMSA）and DNA pulldown assay were used to verify the differences of functional SNP site binding protein ability.Luciferase reporter gene assay was performed to verify the interaction between functional SNP site and COL15A1 promoter region.Results13 SNP sites were screened by resequencing of chromosome 9:101,730,000-101,860,000 regions,and 6 SNP sites were predicted on SNAP website.A case-control validation study of these 19 SNP sites found that 4 SNP sites were significantly correlated with the incidence of ovarian cancer,respectively rs7027650(Intron 2,Additive:OR:0.76,95%CI:0.68-0.84,P=2.09×10^-7),rs1413298(Intron 36,Dominant:OR:1.18,95%CI:1.01-1.39,P=4.31×10^-2),rs1889268(Intron 9,Additive:OR:1.13,95%CI:1.01-1.28,P=3.72×10^-2),rs10819566(Intron 7,Additive:OR:1.15,95%CI:1.02-1.31,P=2.84×10^-2).The results of e QTL analysis showed that COL15A1gene expression was significantly different between rs7027650（P=0.028）and rs1889268（P=0.014）.Electrophoretic Mobility Shift Assay（EMSA）results showed that the binding capacity of these SNP loci was different with different genotypes,and SNP rs7027650 showed the more obvious difference.We finally screened rs7027650 for transcription factor prediction,and the results showed that the sequence of rs7027650 may bind to AR,NR3C1,NR3C2,SRF and other transcription factors.DNA pulldown assay verifies the difference between A and T genotype binding proteins of rs7027650.Luciferase reporter gene assay confirmed that rs7027650 upstream and downstream 500bp gene fragment could interact with the promoter region of COL15A1 to reduce its activity,but the difference between different genotypes on transcriptional inhibition was not statistically significant.ConclusionThrough resequencing of 9q22.33 region and case-control study,we found that rs7027650 was a possible functional SNP in this region.Preliminary functional experiments confirmed that different genotypes at rs7027650 have different ability to bind AR and NR3C1 protein.Moreover,the 500bp upstream and downstream gene fragment of rs7027650 may inhibit the activity of COL15A1 promoter region,but the transcriptional regulation mechanism of rs7027650 on COL15A1 gene is still not clear,and further functional experiments are needed to clarify its mechanism.