Molecular Mechanism Of Genetic Markers Rs7297051, Rs72755295 And Rs2736108 Associated With Breast Cancer

Breast cancer is the most common malignant tumor in women.The incidence of breast cancer is increasing year by year and the onset age is younger,posing a serious threat to women's health.There are obvious genetic factors in breast cancer onset,and one of the methods to study this is genome-wide association study(GWAS).In previous GWASs,genetic markers rs7297051,rs72755295 and rs2736108 were proposed to be significantly associated with breast cancer.The functional causal SNPs might be not the tag SNPs but other SNPs in linkage disequilibrium(LD)with tag ones.Moreover,the pathogenic mechanism of these SNPs was not disclosed by GWAS.rs7297051 is located at the non-coding region of 12p11.22.Through the LD analysis on the genotype data from the 1000 Genomes Project for three representative populations in the world,CEU(Utah Residents(CEPH)with Northern and Western European Ancestry),CHB(Han Chinese in Beijing,China)and YRI(Yoruba in Ibadan,Nigeria),it was proposed that there are no other SNPs in strong LD with rs7297051,thus indicating that this SNP is the causal one.The?1.5 kb surrounding region centered rs7297051 was amplified and ligated with pGL3-promoter,and the plasmid containing the corresponding allele was constructed by mutagenesis.Then the two plasmids were transfected into breast cancer cell line MCF-7 and MDA-MB-231 for dual-luciferase assay.The results showed that the luciferase activity between the plasmids with the two alleles had no significant difference in both breast cancer cells,indicating that rs7297051 is not with the function to regulate gene expression in breast cell.The molecular mechanism between rs7297051 and breast cancer was still unclear.rs72755295 is located in 1q43 region.In previous study,through the LD analysis of the 1000 Genomes Project,our laboratory found that rs4149909 was the only SNP in LD with rs72755295.rs72755295 could change the activity of enhaner,while rs4149909 had no function of regulating gene expression.The surrounding region of rs72755295 could bind with transcription factor PAX6(paired box 6).In this study,the results of chromatin conformation capture indicated that the target gene of the enhancer region containing rs72755295 was EXO1(exonuclease 1).Through electrophoretic mobility shift assay(EMSA),it was observed that the two alleles of rs72755295 had different ability to bind nucleoprotein.rs2736108 is located in 5p 15.33 region.Through the LD analysis for the 1000 Genomes Project data,it was observed that there were 4 SNPs,rs2736098,rs2853669,rs2736109 and rs2736107,are in LD with rs2736108.Among them,rs2736098 is a synonymous mutation for TERT(Telomerase reverse transcriptase)while rs2853669,rs2736109,rs2736107 and rs2736108 are all located in the promoter region of TERT.Previous studies reported that rs2736108,rs2736109 and rs2736107 were non-functional SNPs,while rs2853669 could change the activity of TERT promoter.Searching in ENCODE project data indicated that rs2736098 adjacent region contained strong H3K4mel modification,an common marker of the active enhancer.Therefore,it might be hypothesized that rs2736098 might be located in the enhancer and could regulate the expression of TERT gene by changing the activity of the enhancer.Therefore,the?1,5 kb segment containing rs2736098 was amplified and ligated with pGL3-promoter and the plasmid with corresponding allele was generated by mutagenesis.The two plasmids were transfected into MCF-7 cells,and the dual-luciferase results showed that the relative luciferase activity of T allele of rs2736098 was 1.52 times higher than C allele(P=2.58×10-7),which indicated that rs2736098 had the function of changing the activity of enhancer.Chromatin immunoprecipitation results indicated that the transcription factor USF1(upstream transcription factor 1)could bind to the surrounding region of rs2736098.EMSA showed that the two alleles of rs2736098 had different ability to bind nucleoprotein.Our results identified a novel mutation that could regulate TERT expression and disclosed its molecular mechanism.Our work confirmed the real pathogenic SNPs in these regions,and investigated the expression regulation of related genes,identified the molecular mechanisms of the genetic markers rs72755295 and rs2736108,which could broaden the understanding of the genetic mechanism for breast cancer and provide more insight into the screening,prevention and treatment of this disease.