Molecular Mechanism Of SNPs In The 12q22 Region Of The Human Genome Associated With Breast Cancer

Breast cancer is the most common malignancy in women and is a malignant and complex disease induced by multiple factors.Due to its high morbidity and mortality,difficult treatment and high cost,it poses a great threat to women’s physical and mental health.The etiology of breast cancer is relatively complex,and genetic factors are an imporant risk factor.With the deeping of research,more and more breast cancer susceptibility genes have been discovered.At the same time,it has been proposed that the polymorphism of breast cancer susceptibility genes also has a significant impact on its occurrence and development.Genome-wide Association Study（GWAS）is a commonly used method for studying the genetic susceptibility of complex diseases,and numerous breast cancer susceptibility loci have been disclosed by this approach.The recent GWAS indicated that the single nucleotide polymorphism（SNP）rs 17356907 locating at 12q22 is a genetic marker for this disease.Due to the limited number of the SNPs genotyped by microarray in GWAS,the risk SNP of breast cancer might be not rs17356907 but other SNP in linkage disequilibrium（LD）with it.Moreover,the mechanism between this marker and breast cancer had never been scrutinized.In the study of this problem,we mainly get the following results:（1）On the basis of the existing results of GWAS,we analyzed the 1000 genomes data for three representative populations,CHB（Han Chinese in Beijing）,CEU（Utah Residents with European Ancestry）and YRI（Yoruba in Ibadan）in rs17356907 surrounding region.It could be observed that rs11108165,rs61938093 and rs11836367 are in strong LD with rs17356907.We futher studied the functional genomics of these mutations,and screened the pathogenic loci to determine the real pathogenic mutations and reveal the relationship between related genes and the occurrence of breast cancer.（2）We constructed the recombinant pGL3-promoter plasmids including the wild-type and mutant alleles of these SNPs,and transfected them into MCF-7 cell to detect the expression of dual luciferase.The results indicated that the two alleles of rs61938093,rs11836367 and rs17356907 presented significant differences in relative luciferase activity,suggesting that three SNPs could regulate gene expression in breast cell.In contrast,rs11108165 was not functional in breast cell.（3）Since the functional SNPs are located in the non-coding region,and might regulate gene expression in the mode of enhancer.Chromatin conformation capture assay was utilized to determine the target genes of enhancer.The results showed that the regulation target of the enhancer containing the three functional SNPs was not NTN4（netrin 4）,but USP44（ubiquitin specific peptidase 44）.（4）Considering location of these three mutations was hypothesized that the three functional SNPs loci might be near transcription factor binding sites,and affect gene transcription by changing the transcription factor binding affinity.The transcription factor database was utilized to make bioinformatics prediction,and chromatin immunoprecipitation to validate this conjecture.The transcript factor MYC（MYC proto-oncogene,bHLH transcription factor）was recognized to bind the region spanning rs17356907,but the potential transcription factors binding to the other two functional loci have not been identified yet,which needs futher research.（5）Moreover,the results of electrophoretic mobility shift assay showed that the binding affinity with nuclear proteins was significantly different between two alleles of rs61938093,while the binding affinity SNPs was relatively small for the alleles of other two functional SNPs.（6）By constructing MYC transcription factor overexpression vector and transfecting MCF-7 cells,detecting the relative luciferase activity,and the down regulated gene expression of MYC transcription factor was determined.Our efforts confirmed that rs61938093,rs11836367 and rs17356907 in 12q22 region are functional causal SNPs for breast cancer,and elucidated the mechanism of rs17356907 and breast cancer.Rs17356907 could influence MYC binding affinity,alter enhancer activity and further regulate USP44 gene expression,which conferred breast cancer risk.Our work is of great guiding significance to understand the genetic mechanism,development process,prevention,diagnosis and treatment of breast cancer.