MicroRNAs are highly conserved, single-stranded, short, noncoding RNAs of about 22 nucleotides that regulate gene expression through completely or partially base pairing with target mRNAs at the 3′-untranslated region, resulting in mRNA cleavage or translational suppression [11]. To date, emerging evidence has demonstrated that microRNAs play key roles in a broad range of physiologic and pathologic processes [12], [13]. Although the biologic functions of microRNAs remain largely unclear, previous studies have indicated that microRNAs may participate in tumorigenesis, functioning as tumor suppressors and/or oncogenes [14]–[18], and affect the etiology, diagnosis, and prognosis of various cancers [17], [19]–[25]. Furthermore, microRNAs may be involved in regulation of the immune and inflammatory response systems [26], [27], which may control HPV clearance and escape from immune surveillance. Therefore, inherited genetic alterations of microRNAs may affect susceptibility to HPV-associated OSCC.

MicroRNAs act as omnipresent regulators of gene expression and are involved in many cellular processes, including inflammation and immune responses. Recent studies have demonstrated that microRNAs participate in mediating inflammatory and cytokine signaling as well as innate and acquired immune response to viral infection by targeting critical elements in inflammatory signaling pathways [37], [38]. MicroRNAs also modify viral-host interactions, which play central roles in the development and progression of infection-associated tumors.

Therefore, functional genetic polymorphisms of microRNAs may lead to individual variations in immune function, inflammation, and apoptosis that modify viral immune escape, antiviral defense, and evasion of apoptosis, leading to modification of the risk of infection-associated cancers. Numerous studies have shown that some microRNAs are up-regulated, while other microRNAs are down-regulated in head and neck cancers [39]–[41]. Such increased or decreased expression of microRNAs may be associated with the development, progression, and prognosis of head and neck cancers [24], [42].

Recently, several authors have reported associations between microRNA SNPs and the risk of head and neck cancers. One study indicated that the miR196 rs11614913 variant reduced the risk of head and neck cancers [32]. Another study indicated that miR146 rs2910164, miR149 rs2292832, and miR196 rs11614913 did not modify the risk of head and neck cancers independently of HPV infection but that miR499 rs3746444 moderately reduced the risk of head and neck cancers [33]. The conflicting results may be attributed to many factors, such as different anatomical tumor sites, lack of information about other confounders, and small sample sizes. For example, the previous studies, although larger than our study, had mixed tumor sites without stratification by HPV infection status, whereas in our current study, we assessed the joint effects of microRNA SNPs and HPV16 infection on OSCC risk. HPV16 infection has recently been identified as one of the primary etiologic factors for causes of SCCOP, but oral cavity and laryngeal cancer mainly result from tobacco and alcohol consumption. In our current study, although we had a smaller study size, we found that microRNA SNPs and HPV16 seropositivity may function jointly in the development of OSCC, particularly in patients with SCCOP and in never smokers.

Although the precise mechanism by which the common microRNA genetic variants and HPV16 infection jointly play a role in the development of OSCC has not been fully clarified, a joint effect of microRNAs and HPV16 infection on susceptibility to OSCC is biologically plausible. HPV16 is oncogenic: it encodes viral oncoproteins E6 and E7, which inhibit p53 and Rb cell cycle tumor suppressors. MicroRNA variants might functionally affect expression of genes involved in myriad cellular processes, including inflammation and immune response pathways, subsequently controlling the host's ability to clear HPV and HPV's ability to escape immune surveillance [37]. Therefore, our data suggest that HPV and microRNA variants might act jointly in the development of OSCC. However, further studies are required to validate the hypothesized functionality of these SNPs.

In the present study, our analysis stratified by tumor site showed that the effect modification was more pronounced for SCCOP than for oral cavity SCC, reinforcing the concept that there are differences in the etiology of cancers of the oropharynx and oral cavity. Additionally, the analysis further stratified by smoking status for each SNP showed that the joint effect of microRNA SNPs and HPV seropositivity on the risk of OSCC was much stronger in never smokers than in ever smokers. These data further support the notion that risk genotypes of the four common SNPs of microRNAs may be involved in the development of HPV-associated OSCC in non-Hispanic white never smokers. However, the modifying effect of each of microRNA SNPs on risk of OSCC associated with HPV16 was not statistically significant. This lack of significance could be either because there was no such interaction effect in these subgroups or because the small sample sizes in each substratum limited the statistical power to detect a significant interaction effect. Therefore, the significance and degree of such interaction in each subgroup needs to be further investigated in future studies with larger sample sizes.

Although the present study minimized potential confounding factors, several limitations should be considered. First, a possible selection bias in patient recruitment cannot be ruled out as our study was a hospital-based case-control study and as the cases and controls were not selected from the same target population. In addition, only non-Hispanic white subjects were included in our study. It is therefore uncertain whether these results are generalizable to other ethnic populations. Second, the effects of potential demographic confounding factors were minimized by adequate frequency matching on age, sex, smoking status, and alcohol use, but stratified analyses included a limited number of individuals in each subgroup. As such, the role of these SNPs and HPV16 in OSCC will require confirmation in larger studies in different populations. Third, because individual patients may have had differences in their immune response to HPV16 infection or antibody instability, HPV16 seropositivity may not reflect the tumor's true HPV16 status, leading to possible misclassifications of some patients as HPV16 seronegative who had tumors that actually were HPV16 DNA positive. This misclassification could result in a major selection bias for the estimates of association. Finally, lack of information on changes in sexual practices in current study did not allow us to evaluate its potential influence on risk of HPV-associated OSCC, particularly SCCOP. Therefore, our future studies should include the patients with tumor HPV status and sexual behaviors to further evaluate our findings once such information become available. Nevertheless, the use of serologic status may provide a possibility to include a cancer-free control group for the present case-control study design.