Association Study And Bio-informatic Analysis Of SNPs Within MicroRNA Binding Sites And Breast Cancer Risk

MicroRNAs are noncoding small RNAs that regulate gene expression by Watson-Crick base-pairing to target mRNA. They are involved in most biological and pathological processes, including tumorigenesis. The binding of microRNA to mRNA is critical for regulating the mRNA level and protein expression. However, this binding can be affected by single-nucleotide polymorphisms that can reside in the microRNA target site, which can either abolish existing binding sites or create illegitimate binding sites.Therefore, polymorphisms in microRNA can have a differing effect on gene and protein expression and represent another type of genetic variability that can influence the risk of certain human diseases. Different approaches have been used to predict and identify functional polymorphisms within microRNA binding sites. Among the SNPs identified, the researchers give great emphasis to the SET8 SNP and IQGAP1 SNP about their potential biological relevance in cancer. Therefore, we conducted a large case-control study and bio-informatic analysis to evaluate the association of these polymorphisms in predicted microRNA binding sites and the risk of breast cancer.PurposeMicroRNAs regulate gene expression by binding to the 3'-untranslated region (3'-UTR) of target genes.Single nucleotide polymorphisms (SNPs) that reside in the microRNA seed target sites of critical genes may affect such regulation, resulting in cancer development. We have identified a SNP within the seed-binding region for miR-502 in the 3'-UTR of the SET8 gene that codes for a methyltransferase for histone H4.SET8 methylates TP53 and thus regulates cell proliferation and genome stability. This study is to investigate the role for this SNP and its interaction with the TP53 codon 72 SNP in the propensity for breast cancer development. IQGAP1 knockout mice develop gastric cancer, but the IQGAP1 protein is associated with advanced-stage human glioma. IQGAP1 expression is regulated by a microRNA, miR-124, through a binding site at the 3'-untranslated region, where a single nucleotide polymorphism (SNP) exists in the core binding region. We ask whether IQGAP1 expression is associated with breast cancer development and whether genetic variants at the miR-124 binding site are important,In the bio-informatic study, we performed a thoroughly searching and analyzing of the bio-information about SNPs within microRNA binding sites, aims at providing references for the future systematic investigation of the association of this group of bio-marker and cancer susceptibility.MethodsWe conducted a case-control study of 1,110 breast cancer cases and 1,097 controls. We also measured the relative expression levels of SET8 mRNA and miR-502 in breast cancer tissues of 51 cases with different SET8 genotypes.We evaluated the association between IQGAP1 expression and survival in breast and gastric cancer, and glioma using transcriptome data. We genotyped the IQGAP1 SNP rs1042538 A/T in 1,541 breast cancer cases and 1,598 controls and analyzed the frequency of the variant and interactions with major risk factors in these populations. We modulated IQGAP1 expression in non-cancerous MCF10A breast epithelial cells and MCF7 breast cancer cells. We selected databases "Patrocles" and "polymiRTS", as well as other related databases for searching of SNPs within microRNA binding sites.According to the critaria setted before seaching about the type of seed region and the limits of population frequency, we screened for the high-frequency SNPs among Chinese population within microRNA binding sites of cancer-related genes. We then compared the searching results of the two databases for common SNPs predicted by both. Finally, we analyzed the functional characteristics of these common SNPs and formulated their possible interaction model.ResultsOur analysis revealed that the SET8 CC and TP53 GG genotypes were independently associated with earlier age onset of breast cancer in an allele-dose dependent manner. Moreover, individuals with both SET8 CC and p53 GG genotypes developed cancer at age of 47.74 years compared with 54.55 years for individuals with both SET8 TT and TP53 CC genotypes. We further showed that the SET8 CC genotypes were associated with decreased transcript levels of SET8,but not miR-502 levels, in 51 breast cancer tissue samples tested.Increased IQGAP1 expression was associated with shorter survival in glioma but not in breast or gastric cancers. The IQGAP1 TT genotype, compared with the AA genotype, was associated with a significantly lower risk of developing breast cancer (P=0.049, OR=0.78; 95% CI,0.61-0.99).In case-only analyses, the TT, compared with the AA, genotype was associated with progesterone receptor-positive subjects (OR,1.35;95%CI,1.00-1.83).Decreased IQGAP1 expression resulted in increased cell growth in normal but not breast cancer epithelial cells.In the initial searching, we found 1,742 validated SNPs within microRNA binding sites from the Patrocles database. Through searching of the dbSNP database, we found 404 SNPs with a high frequency among Chinese population. Further, after pubmed database searching, we found 154 SNPs located within cancer-related genes. Among the 154 SNPs,131 SNPs were independent SNPs, the other 23 were parallel SNPs, that is two or more SNPs located within a small distance in one gene. Eight of the parallel SNPs were cis-acting SNPs and the other two were trans-acting SNPs. After a validating search of the PolymiRTS database, among the 154 cancer related SNPs,42 SNPs were shared by the two databases. Functional characterization of these SNPs showed that, among the 42 SNPs,22 SNPs destoried the original microRNA targets,3 SNPs created new targets, and the other 17 transitted the target from one microRNA to another microRNA.ConclusionsThese data suggest that the miR-502-binding SNP in SET8 may modulate SET8 expression and contribute to early development of breast cancer either independently or together with the TP53 codon 72 SNP. Larger studies with multi-ethnic groups are warranted to validate our findings.IQGAP1 plays various roles in carcinogenesis in different tissue types. The presence of SNP at the miR-124 binding site may be a marker for predicting breast cancer risk and prognosis. We strongly advocate systematic study of the SNPs within microRNA binding sites and cancer susceptibility in Chinese population. In the general evaluation of the contribution of this group of bio-marker to cancer risk, we should also consider the functional characteristics and interaction model of these SNPs.