After analyzing fifteen SNPs identified from the public SNP database in DNA samples extracted from patients with incident breast cancer and controls, we report here a significant association between a common sequence variant in miR-196a-2 and predisposition to breast cancer. This association remains significant after correcting for multiple comparisons, and is corroborated by in vitro evidence, including: 1) The variant negatively influences endogenous processing of the miRNA precursor to its mature form; 2) The variant has a phenotypic impact on target gene expression, as fewer genes were altered following introduction of the pre-miR-196a-T vector relative to the pre-miR-196a-C vector; 3) A pathway analysis revealed 137 cancer-related transcripts with significantly altered expression following introduction of pre-miR-196a; and 4) Cell cycle response to mutagen challenge in the form of G2 delay was directly affected in cells transfected with pre-miR-196a-C, and this effect was abrogated in cells transfected with pre-miR-196a-T.
In addition to the in vitro
findings, a methylation analysis of a CpG island located upstream of the miRNA indicated that increased methylation was associated with decreased breast cancer risk, as cases had significantly higher methylation levels than controls. However, since chemical and radiation therapy may influence methylation patterns, only samples from untreated cases could be used for this analysis, thus significantly reducing the available sample size. In addition, RNA samples were not available for these subjects, and thus expression levels cannot be directly associated with level of methylation. However, a previous study reported a strong correlation (r=0.80) between methylation levels near a miRNA gene and miRNA expression in a subgroup of breast cancer samples (19
). If higher degree of methylation correlates with decreased miRNA expression, these data would be consistent with all other findings, which suggest that miR-196a has oncogenic properties. Further study will be necessary in order to confirm whether methylation at this site does in fact correlate with pre-miR-196a expression, and to determine whether this area may indeed serve as a novel epigenetic biomarker for breast cancer susceptibility.
A previous study of 5 common miRNA SNPs conducted in China also identified the same polymorphism (rs11614913) as significantly associated with breast cancer risk (25
). Interestingly, although the same allele was associated with risk (C allele), this was the rare allele in the Chinese population, while it was the common allele in our, predominantly Caucasian, population. In another study investigating the same five miRNA SNPs and non-small cell lung cancer survival in China, the same group identified the C allele of rs11614913 as significantly associated with poor survival, indicating the possibility that this miRNA may have implications for prognosis, in addition to cancer risk (26
). This study also suggests that the pathways influenced by miR-196a are not relevant only for breast cancer, but are also important for at least one other unrelated cancer type.
Several members of the HOX gene family are known to be targeted by miR-196a (27
). While many of these genes showed up at poor intensity in our array, four members of the HOX family, HOXB2, HOXB3, HOXC13, and HOXB5 were significantly downregulated in cells treated with pre-miR-196a-C, with average fold changes of -10.21 (FDR<0.001), -3.23 (FDR<0.001), and -1.88 (FDR=0.001), and -2.18 (FDR=0.038), respectively. Of these, only HOXB2 was significantly downregulated in pre-miR-196a-T treated cells, although the magnitude of the effect was much smaller than that observed in the pre-miR-196a-C population (fold change=-3.69), again suggesting that the SNP results in decreased target gene repression.
The network analysis demonstrated the potential for miR-196a-2 to be operative in a number of biological pathways, including a high confidence network (P=1.0E-48
) with relevance for cancer. Genes in this network included at least two tumor suppressors which were significantly downregulated following pre-miR-196a-C introduction but were not significantly altered in the pre-miR-196-T cells. GADD45G, which was downregulated more than two-fold, is epigenetically silenced in a number of tumors, and acts as a tumor suppressor by negatively regulating cell proliferation (28
). INHBB, which was also downregulated more than two-fold, is a negative regulator of growth which has been proposed as a tumor suppressor for some cancer types, and is expressed in normal breast tissue (29
). TP63, which was more than six-fold upregulated following pre-miR-196a-C introduction may act as an oncogene, despite having significant sequence similarity to p53 (31
). In addition, two genes encoding calcium-binding proteins (S100A8 and S100A9) were also significantly upregulated, by fifteen- and 47-fold, respectively. These proteins form a heterodimer which has been implicated in tumor promotion, possibly mediated by its role as a proinflammatory cytokine (32
). In addition, these genes are upregulated in a number of tumors, and their overexpression has been associated with poor prognosis in invasive ductal cell carcinoma of the breast (33
). Downregulation of tumor suppressors and upregulation of oncogenes by introduction of pre-miR-196a-C would be consistent with overall oncogenic activity, and the diminished regulatory activity of pre-miR-196a-T is consistent with a protective role for the T allele of the SNP.
Although our findings demonstrate that the SNP does have a phenotypic impact, both in terms of pre-miRNA processing and target gene regulation, these data are not useful in determining which genes are direct targets of mature miR-196a and miR-196a*, and which are indirectly influenced. As such, an exploration into the nature of the physical interactions between these miRNAs and their potential targets should be the subject of future investigations. Furthermore, given the earlier finding demonstrating a role for this SNP in lung cancer prognosis, similar studies are warranted to determine whether there are prognostic implications for this variant among patients with breast cancer.