Quantitative Assessment of the Association between Genetic Variants in MicroRNAs and Colorectal Cancer Risk

Background. The associations between polymorphisms in microRNAs and the susceptibility of colorectal cancer (CRC) were inconsistent in previous studies. This study aims to quantify the strength of the correlation between the four common polymorphisms among microRNAs (hsa-mir-146a rs2910164, hsa-mir-149 rs2292832, hsa-mir-196a2 rs11614913, and hsa-mir-499 rs3746444) and CRC risk. Methods. We searched PubMed, Web of Knowledge, and CNKI to find relevant studies. The combined odds ratio (OR) with 95% confidence interval (95% CI) was used to estimate the strength of the association in a fixed or random effect model. Results. 15 studies involving 5,486 CRC patients and 7,184 controls were included. Meta-analyses showed that rs3746444 had association with CRC risk in Caucasians (OR = 0.57, 95% CI = 0.34–0.95). In the subgroup analysis, we found significant associations between rs2910164 and CRC in hospital based studies (OR = 1.24, 95% CI = 1.03–1.49). rs2292832 may be a high risk factor of CRC in population based studied (OR = 1.18, 95% CI = 1.08–1.38). Conclusion. This meta-analysis showed that rs2910164 and rs2292832 may increase the risk of CRC. However, rs11614913 polymorphism may reduce the risk of CRC. rs3746444 may have a decreased risk to CRC in Caucasians.


Introduction
Colorectal cancer (CRC) is the third leading cause of cancer death for both men and women in the USA [1]. In Europe, CRC represents one of the primary causes of cancer deaths [2], and, in Asia, CRC is the fourth leading cause of mortality by cancer, and its incidence is increasing [3]. Epidemiological evidences have suggested that the risk of colorectal neoplasm is affected by multiple factors including a positive family history, excessive meat consumption, smoking status, and alcohol consumption, as well as genetic alterations, such as genetic polymorphisms [2].
However, the mechanism of colorectal carcinogenesis is still not fully understood. Compared with other complex diseases, CRC may be caused by both genetic and environmental factors [4]. Because well-recognized genetic predisposition syndromes account for less than 3% of CRC, low-penetrance genetic factors alone or in combination with environmental factors probably contribute to the development of CRC [5].
MicroRNAs (miRNAs) are a large class of small noncoding RNAs, which participate in various biological processes and may regulate tumor suppressor genes or oncogenes [6]. Single nucleotide polymorphisms (SNPs) in miRNAs may alter the expression, processing, and transcription of miRNAs and thus contribute to cancer development [7]. Recently, many epidemiological studies have demonstrated that some SNPs in the miRNA genes could alter miRNA expression and/or maturation and also be associated with the susceptibility and progression of cancer [8]. Rs2910164 in hsa-mir-146a locus resides at position +60 relative to the first nucleotide of the pre-miR-146a gene. The previous studies have shown that miR-146a plays an important role in cell proliferation and metastatic ability in cancers and the variation of miR-146a may be involved in carcinogenesis 2 BioMed Research International [9,10]. MiR-196a2 includes two different mature miRNAs (miR-196a-5P and miR-196a-3P) and rs11614913 located in the mature sequence of miR-196a-3P. Rs11614913 could influence the expression levels of mature miR-196a and may have an impact on the expression of its target gene, which may play a role in regulating carcinogenesis [10]. It has been identified that rs3746444 is located in the stem region of the mir-499 gene. And several studies have identified miR-499 rs3746444 as a possible biomarker for some cancers [11][12][13]. Compared with other three genes, there were few studies of rs2292932 in miR-149 up to now. But the T allele in miR-149 might be masked by the presence of other unidentified causal genes involved in cancer development [14]. Furthermore, several Genome-wide association studies have reported that these four common SNPs may be associated with cancer risk [8]. However, results of these studies remain inconsistent. To provide a more precise estimate of the association of miRNA polymorphisms with CRC risk, we performed a meta-analysis on all eligible published case-control studies and evaluated the effect of the four SNPs on CRC risk.

Publication Search.
Relevant articles were independently searched by two authors (Wang M and Dai ZJ) in PubMed, Web of Science, and Chinese National Knowledge Infrastructure (CNKI). The keywords were as follows: colorectal cancer/colorectal carcinoma, microRNA 146a/149/196a2/499, and polymorphism/genotype/variant/ SNP. All qualified studies prior to December 20, 2014, were included. The eligible literature must be published in English or Chinese. Furthermore, reference lists of main reports and review articles were also reviewed manually to identify additional relevant publications.

Selection Criteria.
The following criteria were used to select eligible studies for further meta-analysis: (1) casecontrol design; (2) full-text study; (3) studies that evaluated the associations between miRNA polymorphisms and CRC risk; and (4) studies that included detailed genotyping data.

Data Extraction.
Articles were reviewed independently by two reviewers and data with discrepancies were discussed by all authors. For each included study, the following information was collected: first author, year of publication, country of origin, ethnicity, source of control, total number of cases and controls, and genotyping methods as well as number of cases and controls with the different genotypes. Different ethnic groups were categorized as Caucasian, Asian, African, and "mixed. " All the case and control groups were well controlled. 11 studies are systematic review comments.

Statistical
One study was excluded because the article's lack of detailed genotyping data.
15 studies were included in this meta-analysis. The meta-analysis assessed association by using 5 different genetic models: homozygous genetic model, heterozygote genetic model, dominant genetic model, recessive genetic model, and allelic model. Hardy-Weinberg equilibrium (HWE) among the control subjects was tested by the Chi-square test. Statistical heterogeneity among studies was assessed with the and 2 statistics. If the value of heterogeneity test was more than 0.05 ( > 0.05), the pooled OR estimate of the study was calculated by the fixed-effects model. Otherwise, the random-effects model was used. The value of the index was used to assess the degree of heterogeneity ( 2 < 25%: no heterogeneity; 25% < 2 < 50%: moderate heterogeneity; 50% < 2 < 75%: high heterogeneity; and 2 > 75%: extreme high heterogeneity). Publication bias was evaluated by the funnel plot. All statistical analyses were carried out with the review manager version 5.1 (Revman; The Cochrane Collaboration, Oxford, UK).

Characteristics of Studies.
According to the inclusion criteria defined above (Figure 1), fifteen studies on miRNA polymorphisms with CRC risk were identified [8], including 5,486 CRC patients and 7,184 cancer-free controls. All the included eligible studies were published in English or Chinese. Among the eligible fifteen studies, eleven studies were based on Asian backgrounds which were carried out in China and Korea. Only four studies were based on Caucasian, having been carried out in Greek, Lithuania, Italy, and Czech, respectively. Simultaneously, there were seven hospital based studies and eight population based studies. Main characteristics of the included studies were listed in Table 1. Table 2, the frequencies of the minor allele varied widely across the eligible studies, ranging from 0.14 to 0.80 (rs2910164), 0.33 to 0.82 (rs2292832), 0.34 to 0.66 (rs11614913), and 0.14 to 0.68 (rs3746444). The average frequencies of the minor allele in the four polymorphisms were 0.47, 0.66, 0.51, and 0.27, respectively. The distributions of genotypes in the controls were all in agreement with HWE except four studies [8,[15][16][17].

Meta-Analysis Results. As shown in
The main results of this meta-analysis were presented in Table S1, in Supplementary Material available online at http://dx.doi.org/10.1155/2015/276410. There were 11 studies with 3,937 cases and 5,120 controls for rs2910164. miR-146a rs2910164 polymorphism has no association with CRC risk in the overall population (C versus G: OR = 0.96, 95% CI = 0.82-1.12, = 0.62, Figure  And there was also no association existing in any genetic models after we rejected the studies which were not in agreement with HWE. There was only one study based on Caucasian. When excluding the Caucasian study, the null association remained in Asians. We further made stratified analysis based on the source of control. And we observed significant associations in population based studies in the recessive genetic model (TT versus CC + CT: OR = 1.18, 95% CI = 1.08-1.38, = 0.04).
Ten studies with 2,906 cases and 4,150 controls were used to evaluate the relationship between rs11614913 polymorphism and CRC risk. No significant association was detected        Caucasians (GG versus AA: OR = 0.57, 95% CI = 0.34-0.95, = 0.03, Figure 5).

Publication Bias.
In this meta-analysis, we performed funnel plot to access the publication bias. As showed in Figure 6, the funnel plots failed to reveal any obvious asymmetry in all genotypes in overall population. Therefore, the results indicated that publication bias was not significant in this meta-analysis.

Discussion
Allelic variants in the sequence of mature miRNAs represent a particularly interesting potential source of phenotypic diversity of genetic diseases, which may contribute directly to disease susceptibility [18]. Accumulating evidence has shown that miRNAs regulate the expression of roughly 30% of the all human genes through posttranscriptional mechanisms [19]. SNPs in miRNA genes could function through three ways: firstly, the transcription of the primary transcript; secondly, pri-miRNA and pre-miRNA processing; and thirdly, effects on miRNA-mRNA interactions [7]. Genetic effects connected to SNPs at the level of miRNA genes may have a significant relationship with the expression and clinical features in CRC [16]. A large number of studies had highlighted several associations between SNPs in miRNAs and the risk of CRC [8]. However, controversial experimental data were obtained. For example, there was no significant association between rs2910164 and CRC in Hezova et al. 's study [20], whereas Chae et al. reported that miR-146a rs2910164 polymorphism of genotype CC may contribute to a higher risk of CRC [21]. Min et al. from Korea [22] demonstrated that miR-196a2 rs11614913 polymorphism had a decreased risk to CRC, whereas Lv et al. [8] reported that T allele in rs11614913 was associated with an increased risk of CRC compared with the C allele.
The present meta-analysis, including 5,486 CRC patients and 7,184 cancer-free controls from 15 case-control studies, was conducted to evaluate the association between the four common SNPs in miRNAs (miR-146a rs2910164, miR-149 rs2292832, miR-196a2 rs11614913, and miR-499 rs3746444)   and CRC risk. In this study, we found that rs3746444 polymorphism has no association with CRC risk in the overall population. However, accumulative data from two studies based on Caucasian background (317 cases and 477 controls) showed significantly decreased cancer risks in the homozygous model. Considering the limited sample size, we need more large well-designed studies to evaluate the result. Simultaneously, we failed to find any significant correlation between the rs2910164, rs2292832, and rs11614913 polymorphisms and risk of CRC in overall studies or in different ethnic groups for all genetic models. But significant association between rs2910164 and CRC was observed in hospital based studies in the recessive model. And we found that rs2292832 polymorphisms may be associated with increased the risk of CRC in population based studies for recessive model. While T allele of rs11614913 was a protect factor of CRC in the homozygous model when we excluded the studies which were not in agreement with HWE.
Although the biological mechanism of SNPs in miRNAs contributing to the regulation of cancer susceptibility and development remains unknown, a large quantity of metaanalyses has been engaged in the newly developed field in the past few years [23]. A previous meta-analysis by K. Srivastava and A. Srivastava [23] was demonstrated that miR-196a2 rs11614913 polymorphisms have significant associations with overall cancer risk. In Xu et al. 's meta-analysis [24], TT genotype of rs11614913 polymorphism was associated with decreased cancer risk. Rs2910164 C allele was associated with decreased overall cancer risk especially for cervical cancer and prostate cancer risk in Chinese population. Rs3746444 G allele was a risk factor in Chinese population, especially for breast cancer. Different results were also presented in several  meta-analyses which evaluated the relationship between miRNAs and CRC risk [25]. Wan et al. 's study demonstrates that miR-196a2 rs11614913 most likely contributes to decreased risk of CRC, whereas miR-146a rs2910164 may not be associated with the susceptibility to CRC [25]. While the pooled data from Du et al. 's meta-analysis supported that the miR-196a2 rs11614913 and miR-149 rs2292832 polymorphisms may contribute to susceptibility to CRC [26]. The latest metaanalysis, which related to the associations between miRNA polymorphisms and colorectal cancer, performed by Wu et al. indicated that SNP rs11614913 but not SNP rs2910164 and SNP rs2292832 may contribute to susceptibility to CRC in an Asian-specific manner [27]. This result was unconformable with our results. Compared with Wu's meta-analysis which included 9 relevant studies, our research totally identified 15 studies including 5,486 CRC cases and 7,184 controls. Thus, our meta-analysis contained the newest data and largest sample size on the study of the relationship between miRNAs and colorectal cancer. Additionally, we performed subgroup analyses by different data information in more detail. Some limitations still existed in this meta-analysis. Firstly, the included studies are mainly based on Asian background.
There were only five studies based on Caucasian background and no studies on African background. Secondly, some detailed information (such as sex, age, life-style, and environmental factors) was not considered. Further large scale multicenter studies based on Caucasian or African will be needed to clarify the possible roles of these polymorphisms in CRC. Though many investigators have been devoted to identify the roles of the miRNAs in carcinogenesis, the mechanisms were still unclear. Further biologically functional studies are warranted to explain the molecular mechanisms.
In summary, this meta-analysis showed that there were no associations between the four polymorphisms and CRC risk in the overall population. Further analysis stratified by the source of control showed that rs2910164 polymorphism is associated with CRC risk in hospital based studies, and rs2292832 polymorphism may contribute to the susceptibility of CRC in population based studies. In the pooled analysis from all studies which are in agreement with HWE revealed that rs11614913 TT carriers may have a decreased CRC risk. Moreover, in the stratified analysis according to ethnicity, rs3746444 polymorphism may reduce the risk of CRC in Caucasians.