The role of miRNA-mediated cis-regulation in breast cancer susceptibility

Cis-regulation of gene expression is believed to be central in breast cancer (BC) predisposition. Here we aimed to unravel the contribution of allele-specific miRNA regulation to BC risk. We screened the effect of 223 published BC genome wide association studies (GWAS) -significant single nucleotide polymorphisms (SNPs) (and their 2668 unique proxies in high linkage disequilibrium) on differential miRNA-regulation. We filtered these SNPs based on location in miRNA genes and/or messenger RNA (mRNA) of protein-coding genes. Selected SNPs were then evaluated for putative differential miRNA-binding using TargetScan and miRanda, two distinct miRNA-target prediction algorithms, modified to analyse sequences carrying SNP alleles. Results were filtered for miRNAs with evidence of expression in breast tissue, and for genes displaying differential allelic expression (DAE), a hallmark of cis-regulation. To validate our findings, we prioritized the candidate SNPs for functional characterization, by combining TargetScan’ and miRanda’ predictions. Interestingly, none of the SNPs mapped to miRNA genes, thus suggesting that miRNA biogenesis and target-binding alteration, via seed sequence modification, are mechanisms unlikely to be involved in BC risk. Of the SNPs located in mRNA sequences we found 93 out of 3891 that were predicted to alter the miRNA-mRNA binding in 27 BC-associated risk loci. From our predictions, we found rs4245739 in MDM4 and rs11540855 in ABHD8, already functionally validated by others to cause allele-specific miRNA-binding. We carried in vitro functional characterization of rs6884232 in ATG10, one of the best candidates identified by both TargetScan and miRanda algorithms. The predicted specific binding of hsa-miR-21-3p to the G allele of this SNP was evaluated using a dual-luciferase system, with constructs carrying either the A or the G allele, and in combination with miRNA mimics and inhibitors in a breast adenocarcinoma cell line. However, no allele-specific specific differences in luciferase activity ...