Polymorphisms in miRNA genes and their target sites are a novel class of variation in the human genome that are rapidly being identified and investigated in human cancers including HNSCC (13
). Motivated by several recent publications linking the MIR196A2
mature miRNA SNP to cancer susceptibility and prognosis (15
), we tested the hypothesis that this SNP (rs11614913) is associated with susceptibility to, and prognosis of HNSCC; finding a significantly decreased risk of HNSCC, and significantly reduced survival in cases with pharyngeal disease. Notably, the prevalence of control genotypes was completely consistent with another ethnically similar (Caucasian) population-based study (15
). However, unlike some other reports (15
) when we assessed relative expression of mature miRNAs 196a and 196a* we did not detect significantly different expression by MIR196A2
genotype in either normal head and neck tissues or tumors.
Comparing expression levels of the mature miR-196a among MIR196A2
genotypes Hu et al.
observed significantly lower expression of miR-196a in non-small cell lung tumor samples with C/C genotype (n=6) compared to C/T and T/T individuals (n=17) (19
). These authors also measured endogenous levels of pri- and pre-miRNA sequences, but did not observe differential expression of these precursor molecules based on genotype (19
). In a separate report, Hoffman et al.
expression of mature miR-196a in breast cancer cells transfected with pre-miR-196a-C compared to cells transfected with miR-196a-T relative to empty vector control (15
), but did not observe differential expression of the precursor miRNA in their transfection experiment, suggesting that MIR196A2
genotype may result in altered processing of the pre-miR (15
). Relative to previous work, our expression results were from a large number of samples (n=83) affording sufficient statistical power to detect even subtle differences, and although the miR-196a expression results appear to be conflicting, cell-type-specific differential expression of miRNAs could explain these results. A recent examination of differential miRNA expression in several cancer types and adjacent normal tissues (including breast and lung, though not head and neck) showed a wide range of miR-196a expression across different normal tissues, tumor types, and between tumor and normal tissues from common sites (GSE14985) (27
If miRNA SNPs directly affect the expression of mature miRNA species, then the functional relationship between these polymorphisms and disease susceptibility or prognosis may seem clear: altered miRNA expression leads to altered regulation of target mRNAs important in tumorigenesis. Nonetheless, even in the absence of altered mature miRNA expression levels, variation in the mature miR-196a* sequence itself could result in differential regulation of target mRNAs. Mature miR-196a* in variant allele form could have either an increased or decreased affinity for targets of wild-type miR-196a*. A determinant of the impact of miRNA sequence variation is cell-type-specific expression levels of both miRNAs and their targets: cell-type-specific transcriptomes. In addition, it is important to note that tumorigenesis-related alterations in a cell's transcriptome may also affect the regulatory capacity of miRNAs to the extent that a miRNA variant allele may have a protective effect for disease as well as predict poor prognosis. Functional characterization of miRNA target transcripts is far from complete and there is currently little data available to speculate on cell-type-specific regulation of mRNAs by miR-196a and miR-196a*.
Though there is conflicting data for the association between MIR196A2
genotype and miR-196a expression, the variant allele is in the other mature miRNA product of this gene, miR-196a*. Consistent with our data, Hoffman et al. did not observe an effect of the variant on expression of mature miR-196a* (15
). In addition, studies that report significant associations between the MIR196A2
polymorphism and cancer risk; breast, gastric, and non-small-cell lung cancers (in both Caucasian and Asian populations) all show the T allele to be associated with decreased risk of disease (15
). Collectively, these results strongly suggest that reduced risk associated with MIR196A2
variant genotype is not mediated by differential expression of the gene's mature miRNAs.
In the context of carcinogenesis, the dynamic relationship between a miRNA and its targets may be modified by etiologic exposures. Compounded with cell-type-specific transcriptomes, there are varying etiologic exposures associated with different cancers: in HNSCC etiologic exposures may differ between individuals (within tumor site), and are known to have different magnitudes of effect by tumor site. For instance, HPV16 seropositive individuals have a higher risk of developing pharyngeal disease compared to other sites (3
). Further, HPV16 and other important etiologic exposures in HNSCC have been associated with specific molecular alterations that may have broader implications for a cell's transcriptome, and hence, may differentially impact regulation by miRNAs. Nonetheless, we did not observe any effect modification by exposures for genotype associations in this study, suggesting that the precise mechanism(s) by which genotype confers risk is too dynamic and complex to fully elucidate. In addition to more complete characterization of the miRNA targets, further study of additional cancers with similar risk factors (e.g. cervical cancer and HPV and lung cancer and tobacco smoking) might also be helpful in illuminating precisely how these variant genotypes confer risk.
This work suggests that patients carrying a variant allele in MIR196A2 have a significantly decreased risk of HNSCC. In addition, homozygous variant cases with pharyngeal disease also had significantly poorer survival, suggesting that the protective effect of the variant allele does not extend to disease severity. Additional studies are warranted to further confirm these findings and explore the relationship between MIR196A2 genoytpe and risk of other forms of cancer.
Translational Relevance: We find that carriers of a variant allele in MIR196A2 have a significantly decreased risk of developing head and neck cancer compared to wildtype individuals. Further, homozygous variant carriers with pharyngeal disease have significantly reduced survival. Importantly, our evaluation of a mature miRNA polymorphism is an example of a directed approach to investigating a growing category of polymorphic variants in miRNAs and miRNA target sites which will likely have tremendous implications for disease risk and prognosis in all types of human cancer.