In the present study, we identified a new pathogenetic mechanism to explain the association of certain SNPs with BC susceptibility by using the latest knowledge on post-transcriptional gene regulation by miRNAs. Evidence of the effect of SNPs on miRNA binding derived from our integrative analysis delineates a novel role for SNPs in gene expression modulation. Supporting this theory, a recent work suggested that differences in SNP allele frequency among ethnic groups account for differences in gene expression (23
). Two out of the 11 cis
SNPs identified by Spielman et al. (23
) were actually transcribed SNPs located in 3′ UTRs, and by bioinformatics analysis, we found that both of the SNPs were qualified to induce MFE absolute changes greater than 8% with multiple putative interacting miRNAs (Supplemental Table S9
). Therefore, we speculate that cis
SNPs modulate phenotypic gene expression diversities, at least in part, through alteration of miRNA target binding capability; ultimately, leading to differences in the susceptibility to complex genetic diseases, such as BC. Notably, among the target SNPs identified as differentially expressed in BC, we found rs334348 located in the 3′ UTR of TGFBR1
. The association of this SNP with germline allele specific expression of TGFBR1 was recently found, and it was shown to confer an increased risk of colorectal cancer (24
). This phenotype could be explained, for example, by an altered miRNA interaction, and therefore strongly supports our hypothesis that SNPs affecting miRNAs function contribute to allele specific protein expression and consequently play a role in tumor susceptibility. A recent study by Kim and Bartel (25
) based on transcriptome analyses of heterozygous mouse strains, showed that polymorphic miRNA target sites determine differences in gene expression, which further supports our hypothesis.
The focus of this study is to address the effect of transcribed SNPs on miRNA::mRNA interactions and thereby the regulation of gene expression by these SNPs with possible implications in tumorigenesis. We found that numerous target SNPs can effectively interfere with miRNA target recognition, and the distribution of these SNPs significantly vary among BC and control populations, suggesting a potential role in BC susceptibility. It is interesting to note that the BC-associated rs1982073-TGFB1 variant had been previously linked with low expression levels of TGFB1 during the initial phases of tumorigenesis, while higher expression levels were found in more advanced metastatic BC stages (26
). This switch in TFGB1 expression could be due to concomitant changes of miR-187
expression levels and could be favored by carrying the [T] variant. Additionally, the described model, together with TGFB1 pro -or anti-tumorigenic activities in different phases of tumorigenesis, can explain the conflicting results observed for rs1982073-TGFB1 association with BC. Another interesting observation is that the stronger binding occurring between miRNA and active allele does unequivocally mean repression; in fact, as shown for rs1799782-XRCC1, miR-187
stabilizes XRCC1 expression when carrying the active allele. This finding is in line with other evidence showing that miRNAs can positively modulate the protein expression (28
). Furthermore, by searching the complete PCG transcripts (5′ UTR, CDS, 3′ UTR) for miRNA target SNPs, we have identified miR-638
as regulator of BRCA1 via binding to its target site inside CDS (to our knowledge the first miRNA regulating BRCA1 published so far). Although a conserved interaction site was predicted by TargetScan in the 3′ UTR of BRCA1, we found that it is not a functional site in the conditions we tested. Recently, Tay and coworkers also proved the existence and abundance of miRNA targets located inside the CDS of PCGs (3
), confirming the possibility that miR-638
regulates BRCA1 in a similar way. Although large case-control studies have not reached a unifying conclusion on the relevance to BC susceptibility of the miRNA target SNP rs7799917 inside BRCA1
), its association with tumor susceptibility has been recently highlighted in a SNP analysis of DNA repair genes in glioblastomas multiforme samples (32
). In the present study, we found that rs7799917 [T] allele is associated with a weaker miR-638
dependent BRCA1 reduction, and is linked with BC (). Further investigations are underway to explain this apparent inconsistency. We speculate that this is due to concomitant variations during tumor development of miR-638
expression levels. Moreover, we observed that low frequency CDS mutations (both synonymous and non synonymous, data not shown) might exert on miRNA gene regulation effects similar to target SNPs (33
). This provides an innovative insight on the importance of synonymous mutations, which in the future should be considered and investigated with renewed attention.
Supported by the data presented here, we highlight the possibility that both genetic variants and a microRNA expression patterns can account for gene expression differences among distinct populations. Therefore, the newly recognized class of target SNPs may contribute to cancer susceptibility not per se (or as tags for specific haplotypes (30
)), but in concert with miRNA expression patterns, which are becoming more easily available in this new era of miRNA profiling.
In summary, considering the multifactorial model of BC susceptibility, we propose that transcribed allelic variants can alter miRNA PCG regulation and thus increase miRNA abnormalities. These contribute to tumor susceptibility and tumor general risk by a mechanism of subtle gene regulation still not fully appreciated. As the lists of miRNAs and target SNPs keep growing, the need to confirm the effect of target SNPs on gene expression becomes more pressing and will eventually help us achieve a deeper insight into many variables of the molecular pathways relevant to the development of BC and other human malignancies.