In this relatively large hospital-based case–control study of 1,584 cervical cancer cases and 1,394 cancer-free female controls, we validated two previously reported significant miRNA-related SNPs involved in the LAMB3-miR-218 pathway for the risk of cervical carcinoma in Chinese populations . We found that the pri-miR-218 rs11134527 variant GG genotype was significantly associated with a decreased risk of cervical carcinoma compared with the AA and AA/AG genotypes, and our sample size had a statistical power of 94.9% to detect such an association. Further RNAfold prediction analysis showed a MFE changed from −182.5 kcal/mol to −126.0 kcal/mol, when the nucleotide at the pri-miR-218 rs11134527 locus changed from A to G, indicating that this variant may act as a functional SNP, which affects the miRNA binding process and contributes to cervical cancer susceptibility. However, for the other SNP (i.e., LAMB3 rs2566), our data did not have statistical evidence to support its association with cervical cancer risk. Our sample size had 100% statistical power to detect an OR of 1.57 that was reported by Zhou et al. . The inconsistency for the LAMB3 rs2566 SNP between Zhou’s study and ours may be caused by differences in selection of subjects, different catchments of the hospitals and residential regions as well as different sample sizes.
Recent studies have demonstrated that miRNAs may function as tumor suppressors and/or oncogenes in human cancers [21, 22], because elevated or decreased expression of miRNAs has been found in various tumor types, which may alter the regulation of mRNA expression. It is of note that miRNAs regulate gene expression by the sequence-specific binding to the target mRNA, and these binding processes may be affected by SNPs located in the miRNA complementary site . Therefore, it is important to understand the functional and evolutionary significance of related genetic variations in determining expression of miRNAs and mRNAs that interact with each other as well as with environmental risk factors in the related biological processes [23, 24].
It is well known that genetic variants may modify cancer risk associated with environmental factors. Although there were no two-factor interactions between genotypes and environmental factors, using the MDR analysis , we further explored high-order multiple-factor interactions in associations with cervical cancer risk and found that age at primiparity was the strongest risk predictor among all the risk factors considered. Meanwhile, the interaction between the variant genotypes and other risk factors appeared to modify the risk of cervical carcinoma, with the five-factor model being the best model.
MiR-218, is encoded by an intron of the SLIT2 tumor suppressor gene , is known to be associated with the development and progression of several cancers [21, 22]. The decreased level of the miR-218 expression has been observed in cancers of the breast, ovary, lung and stomach [22, 26, 27], and its low expression level was also correlated with tumor stage, LN metastasis and poor prognosis in gastric cancer . Recently, Martinez et al. reported a decreased expression level of miR-218 (> 2 fold) in HPV-16 or 18 positive cervical cancer cell lines (i.e., SiHa, CaSki and HeLa) as well as in cervical tumor tissues . They also demonstrated miR-218 as a specific cellular target of high-risk HPV types , suggesting that the down-regulation of miR-218 is likely linked to the process of HPV-associated tumorgenesis. Based on the Microcosm Targets tool software (http://www.ebi.ac.uk/enright-srv/microcosm/), the mature miR-218 was found to have an effect on the mRNA expression regulation through more than 900 target genes, including LAMB3, RICTOR, ROBO1 and BIRC5, that may play important roles in cervical carcinogenesis. These genes were reported to participate in a number of cancer signaling pathways, such as the Wnt/β-catenin, ERK/MAPK and Notch pathways . Laminin-5 has been found as a sensitive marker of early invasion of cervical lesions . LAMB3 that expressed in many epithelial tissues could induce carcinogenesis by increasing carcinoma cell migration and disturbing tumor microenvironment . Moreover, LAMB3 increased expression levels of the HPV16 E6 oncoprotein in cervical cancer cells and this process might be mediated by miR-218, which indicates a possible mechanism of the LAMB3-miR-218 pathway involved in the development of cervical carcinoma.
It is known that the mRNA secondary structure is critical for mRNA-miRNA interactions and gene functions . To investigate whether the pri-miR-218 rs11134527 SNP could alter the local second structure of the pri-miR-218 mRNA, we performed the RNAfold prediction analysis and found an obviously changed mRNA structure from rs11134527 allele A to G. These findings further suggest that germline genetic variations of pri-miR-218, such as rs11134527, may lead to an alteration of miR-218 expression and affect the miRNA binding process and thus are associated with cervical cancer susceptibility.
Several limitations of our study need to be addressed. Firstly, this hospital-based case–control study may have selection bias and information bias, which may be minimized by frequency-matching cases and controls as well as the adjustment for potential confounding factors in the final analyses. Secondly, only two miR-218-related SNPs involved in the LAMB3-miR-218 pathway (i.e., one in pri-miR-218 and the other in miR-218 binding site) were investigated in this study. Cancer is a complex and multifactorial disease, and any single SNP may not be sufficient for the prediction of the overall risk . Future studies should include more genes and more SNPs, especially functional ones, associated with cervical cancer risk. Finally, we did not have enough information on other risk factors, especially HPV infection. This was because the hospital did not perform HPV and related subtype detection for the diagnosis of all cervical cancer cases, let alone for the female controls. A recent meta-analysis found that high-risk HPV16, 18 and 45 types accounted for a greater or equal proportion of HPV infections in cervical cancer, but not other high-risk HPV types, such as HPV33, 51 and 58 . Therefore, HPV types could be confounders in estimating the risk associated with genetic factors.