A Functional Polymorphism In The MiR-367 Binding Site In RYR3 3'UTR Affects Breast Cancer Risk, Calcification And Prognosis

Breast cancer is one of the most common malignant disease in women. Single nucleotide polymorphisms (SNPs) have been shown to impact both cancer risk and cancer prognosis although the underlying mechanism is largely obscure. MicroRNAs (miRNAs) are evolutionarily conserved, endogenous, single-stranded, non-coding RNA molecules that are reported to be involved in many biological processes, including cell proliferation, apoptosis, and tumorigenesis, through their regulation of gene expression. Most miRNAs bind to target sequences located within the 3'-untranslated region (3'UTR) of mRNAs by base pairing, resulting in the cleavage of target mRNAs or repression of their translation. An increasing number of 3'UTR SNPs located in miRNA binding sites have been reported to be associated with cancers and drug response, presumably due to the differential binding affinities of the SNPs for miRNA. Polymorphisms in these miRNA binding sites in the 3'UTRs of target genes represent a group of genetic variations that modulate the regulatory loop between miRNAs and their target genes. Sequence-based bioinformatic predictions have identified a number of these types of SNPs, which has provided candidates for experimental verifications and case-control studies to determine their importance.ObjectiveRecently, a new class of single nucleotide polymorphisms (SNPs) that directly affect gene regulation by microRNAs (miRs) has emerged as important for cancer risk and prognosis. In this study, we interrogated a SNP, rs 1044129, that is located in the miR-367 binding site at the 3'UTR of the calcium release channel ryanodine receptor 3 (RYR3) gene and determined whether this SNP was associated with breast cancer risk, calcification and prognosis. We then functionally validated SNP rs1044129, which is located in the miR-367 binding site in the 3'UTR of RYR3. Finally, we studied the important role of RYR3 in breast cancer cells and the significance in clinical medicine.MethodsIn a case-control study, rs 1044129 was genotyped in 1,532 breast cancer cases and 1,605 healthy Chinese women. Crude and adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were calculated with an unconditional logistic regression model. Kaplan-Meier curves and Cox regression analysis were used to evaluate the relationship between genotype and survival. A thermodynamic model and the reporter gene assay were used to functionally validate the role of rs 1044129 in miR367-RYR3 regulation in breast cancer. In RYR3 functional studies, we used MTT assay to detect cell proliferation, wound healing assay in migration experiment, Fluo-4AM to evaluate the intercellular calcium concentration.ResultsThe rs 1044129 G allele genotypes were associated with increased breast cancer risk (aOR,1.26; 95%CI,1.02 to 1.54) and increased incidence of mammographically detected microcalcifications (OR,1.52; 95%CI,1.21 to 1.90). Kaplan-Meier survival curves showed that G allele genotypes were significantly associated with poor progression-free survival in cases(P=0.036). A thermodynamic model based on base-pairing and the secondary structure of the RYR3 mRNA and miR-367 miRNA showed that miR-367 had a higher binding affinity for the A genotype than G genotype. We confirmed that miR-367 regulates the expression of a reporter gene driven by the RYR33'UTR and that the regulation was affected by the RYR3 genotype. We provided evidence that the ryanodine receptor gene (RYR3), which encodes a large protein that forms a calcium channel, is important for the growth, migration, intracellular calcium concentration and morphology of breast cancer cells.ConclusionThus, rs 1044129 is a novel SNP that resides in a miRNA-gene regulatory loop that affects breast cancer risk, calcification, and survival.