Lung cancer is the most common malignant cancer, which causes approximately 1.59 million cases deaths, with less than 15% of the 5 years survival rate according to World Health Organization (WHO) (1,2). For Chinese, lung cancer is the second common malignant tumor in female and the most frequent malignant tumor in male. Tumorigenesis is a very complex process (3). And the etiology of lung cancer is also strongly affected by genetic or environmental factors and the interaction of gene-environment. Molecular epidemiological studies also indicated that a large number of genetic variants might be associated with the risk of lung cancer (4). The GWAS studies indicated that there were many disease-associated loci in non-coding RNAs (5,6). MicroRNAs played pivotal roles in non-coding RNAs and were associated with cancer cell proliferation, invasion and cell cycle regulation. Many studies shown that miRNAs could influence the initiation of cancer or the progression of malignant tumor (7). Thus, the abnormal expressions of miRNAs may be the cause of lung cancer (8). Single nucleotide polymorphisms (SNPs) in microRNAs could affect the cancer risk by changing the expressions of miRNAs (9). In the past, many studies shown that the three common polymorphisms [miR-146a (rs2910164), miR-196a2 (rs11614913) and miR-499 (rs3746444)] could influence the susceptibility of lung cancer, but the results were contradictory. The common variants [minor allele frequency (MAF) >0.05] were concerned in previous studies, such as rs2910164, rs11614913 and rs3746444 (10).
MiR-146a (rs2910164) is exits on chromosome fifth LOC285628, and the mature miR-146a is located in the second exon (11,12). MiR-146a played an important role in a majority of disease (13) and it also can influence the occurrence and development of tumor by disturbing cell invasion and migration (14,15). Many studies have conducted to estimate the potential association between miR-146a and lung cancer in humans. According to the search strategy, we found six studies to analysis the association between miR-146a and lung cancer (16-21).
MiR-196a-2 (rs11614913) located the mature miRNA complementary region of pre-miR-196a-2, and a large number of studies have shown that the SNP was associated with the risk of lung cancer by influencing the expression and maturation of miRNAs (19-24).
MiR-499 gene is also exiting in miRNA mature region. MiR-499 plays an important role in the regulation of cell differentiation. Some studies have indicated that miR-499 (rs3746444) polymorphism was associated with the risk of lung cancer (7,19,20) (Qiu F, 2012, unpublished data).
The aim of this meta-analysis was to achieve a combined risk estimate including most recently published studies before Apr 2017. And we were evaluated the relationship between these three polymorphisms (miR-146a, miR-196a2, and miR-499) with lung cancer risk.
We conducted a comprehensive and systematic search to analyze the association between these three common polymorphisms and lung cancer in PubMed, Wanfang, VIP, CNKI database. Last search was conducted on April 2017. We used the subject headings and keywords such as, miRNAs, cancer, tumor, gene, polymorphism, variation, miR-499 (rs3746444), miR-146a (rs2910164), miR-196a2 (rs11614913) and supplemented by literature tracing methods to collect relevant studies.
The Inclusion criteria were: (I) published all over the world about miR-146a (rs2910164), miR-196a2 (rs11614913) and miR-499 (rs3746444) polymorphisms and lung cancer risk from case-control study; (II) providing the number of the case group and the control group; (III) obtaining the full text of the literature; (IV) providing enough data to calculate the statistical index of odds ratio (OR) 95% confidence interval (CI); (V) the similar method and assumption of each research; (VI) getting the consensus for each research from two inspectors.
The exclusion criteria were: (I) duplicated data; (II) meeting, case report and literature review; (III) involving gene expression, meta-analysis, cell lines; (IV) the Newcastle-Ottawa scale (NOS) quality assessment less than five stars (25).
Two investigators independently extracted data. The following data were extracted: the first author’s name, published time, country, race, control source, number of cases and controls, the number of cases and controls of each genotype, the detection method of miRNA, Hardy-Weinberg equilibrium (HWE), NOS (Tables 1-3). If difference was existed after data collection, the third author needs to ensure the data. HWE was used to evaluate the gene frequency and genotype frequency by the goodness of fit χ2 test or Chi-square test in each study control groups. The disequilibrium was exited, when P<0.05. We assessed the association between the three SNPs and lung cancer risk by using ORs with 95% CIs (P<0.05). We analyzed the pooled ORs using five different genetic models: dominant model, homozygote comparison, recessive model, allelic comparison and heterozygote comparison, respectively. Furthermore, subgroup analyses carried out by race and control source. Heterogeneity between the eligible studies was assessed using Chi-square-based t-test. When I2≤50%, heterogeneity is not apparent. We can choose fixed effects model; whereas I2>50%, we think the heterogeneity exiting between studies, we should choose the random effect model (26,27). Further, to test the stability of the results, an accurate sensitivity analysis was needed by omitting one by one. We used quantitative method to determine publication bias in our meta-analysis (Egger’s test) (28,29). All analyses results were performed by using Stata software.
The selection of eligible studies
According to the search strategy, we got 519 articles from PubMed database, CNKI, Wanfang database and Chinese biomedical literature database. We removed 450 records by primary screening of titles. Figure 1 shows the screening process of studies. We excluded 58 articles (17 studies were duplicate records, 8 studies were prognosis, 3 studies were involved in cells, 9 studies were gene expression, 14 studies were meta-analyses, 2 studies were review, 4 studies did not have the data which we needed, 1 study was cell line). Finally, there were 11 studies in our meta-analysis with 9,231 lung cancer cases and 9,280 controls. Six studies about miR-146a (rs2910164) polymorphism were included. Six studies about miR-196a2 (rs11614913) polymorphism were analyzed. Four studies (5 data) about miR-499 (rs3746444) were entered.
The results of quantitative analysis
In this study, we set five different genetic models of miR-146a (CG vs. CC, GG vs. CC, GG + CG vs. CC, GG vs. CG + CC, G vs. C). Table 4 showed all the detailed results. The analysis revealed that miR-146a polymorphism was significantly associated with the risk of lung cancer (CG vs. CC: OR =0.859, 95% CI: 0.781–0.945, P=0.002; GG vs. CC: OR =0.846, 95% CI: 0.751–0.953, P=0.006; GG + CG vs. CC: OR =0.855, 95% CI: 0.782–0.935, P=0.001; G vs. C: OR =0.910, 95% CI: 0.859–0.964, P=0.001). In the subgroup analysis by source of controls, statistically decreased lung cancer risk was found in hospital- based groups (Table 4). No significant association between miR-146a and lung cancer risk was found in the population-based groups. Stratified analysis by ethnicity, the miR-146 a polymorphism was significantly associated with lung cancer risk among Asians (Table 4). Figure 2 shows the forest plot of miR-146a (CG vs. CC).
In this study, we set the heterozygote comparison of miR-196a2 (CT vs. TT), homozygote comparison (CC vs. TT), dominant model (CC + CT vs. TT), recessive model (CC vs. CT + TT), and allelic comparison (C vs. T). The analysis revealed that miR-196a2 polymorphism was significantly associated with increased lung cancer risk (CC vs. TT: OR =1.200, 95% CI: 1.056–1.364, P=0.005; CC + CT vs. TT: OR =1.117, 95% CI: 1.011–1.235, P=0.029; CC vs. CT + TT: OR =1.123, 95% CI: 1.009–1.251, P=0.034; C vs. T: OR =1.089, 95% CI: 1.022–1.161, P=0.008). In the stratified analysis by ethnicity, the miR-196a2 polymorphism was significantly associated with lung cancer risk in Asians (Table 5). In the subgroup analysis by source of controls, we found a statistically association between miR-196a2 and the lung cancer risk among hospital-based groups (Table 5). The forest plot of miR-196a2 (CC vs. TT) was shown in Figure 3.
Table 6 listed the results of each genetic model. The analysis showed that miR-499 polymorphism might have association with lung cancer risk in the five different genetic models (AG vs. AA: OR =1.131, 95% CI: 1.022–1.252, P=0.018; GG vs. AA: OR =1.702, 95% CI: 1.093–2.650, P=0.019; AG + GG vs. AA: OR =1.207, 95% CI: 1.042–1.398, P=0.012; GG vs. AG + AA: OR =1.640, 95% CI: 1.066–2.523, P=0.024; G vs. A: OR =1.226, 95% CI: 1.026–1.464, P=0.025). Results of the stratified analysis by ethnicity suggested that the significant effect for miR-499 polymorphism was observed in the risk of lung cancer in among Asian (Table 6). Figure 4 shows the forest plot of miR-499 (AG vs. AA).
The analysis of sensitivity
We analyzed the sensitivity analysis by excluding study one by one. The analysis of sensitivity was suggesting that no obviously effects were exited from each article. That was say, our results were stable.
Publication bias analysis
We performed quantitative analysis by Egger’s test. We found P>0.05 and 95% CI including 1 in this study, suggesting no meaningful bias in this meta-analysis.
In the past years, majority studies have shown that miRNAs play vital roles in cancer risks (30-32). And the newly miRNAs have enjoyed a high level of concern in medical science. MiR-219-1 (rs213210 and rs107822) might be associated with lung cancer risk (33). A significant association between the miR-608 (rs4919510) polymorphism and lung cancer risk was also observed (34). Rs12740674 in miR-1262 was significantly related with increased risk of lung cancer (35). Many studies have proved that miR-146a (rs2910164), miR-196a2 (rs11614913) and miR-499 (rs3746444) were related to the incidence of lung cancer. Compared with other SNPs, these three common polymorphisms in microRNAs were more popular and hot. MiR-146a (2910164), miR-196a2 (rs11614913) and miR-499 (rs3746444) polymorphism may have different function in the variety of tumors (36-38).The down-regulation of miR-146a contributed to COX-2 expression levels in lung cancer cells, including A549 cells (39). By influencing the expression and maturation of miRNAs, miR-196a2 was associated with the risk of lung cancer (19). MiR-196a2 was related with a wide variety of malignancies, including non-small-cell lung cancer (40). MiR-499 might play an important role for early detection non-small cell lung cancer (NSCLC) (41). For lung cancer, previous meta-analysis did not get the same results about miR-146a and miR-196a2 in some aspects. There was a meta-analysis found that miR-146a was not associated with lung cancer by Xu et al. (42). Xu et al. found that miR-196a2 was associated with lung cancer risk. Ren et al. (43) indicated that miR-146a might have significant association with lung cancer risk, particularly in Asians and the control from hospital-base. For lung cancer, miR-196a2 was a dangerous factor in Asians and the control from population-base.
It was worth noting that there were several problems in the above meta-analyses. As we can see, the association of miR-146a, miR-196a2 and miR-499 with lung cancer susceptibility could not be clear observed by previous meta-analysis. They got the different results might be limited the number of articles. Recently, we found more new researches in these fields and the sample size was large. Also, our own laboratory has been engaged in this research. Therefore, an update meta-analysis was needed to show the clear association between three common polymorphisms and lung cancer risk. Comparing with the previous meta-analysis, this meta-analysis including 9,231 cases and 9,280 controls combined previously published articles and an academic dissertation was conducted for the aim of estimating the real relation between three common polymorphisms and the risk of lung cancer. For all we know, this is the largest meta-analysis to evaluate the relationship between the three common polymorphisms in miRNAs and lung cancer risk.
In the overall analysis, results implied that miR-146a (rs2910164) had a significant correlation with the risk of lung cancer. And compared with homozygous CC, homozygous GG might be a protective factor for lung cancer. Compared with allele C, allele G was found to be associated with decreased lung cancer risk. Subgroup analyses based on ethnicity, results indicated that significantly affected lung cancer risk was found for miR-146a in Asians when subgroup analysis by sources of controls, it performed that miR-146a (rs2910164) had obvious correlation with lung cancer risk in the hospital-based study but not in population-based study. And compared with homozygous CC, homozygous GG might be associated with decreased the risk of lung cancer. Compared with allele C, allele G could decrease the risk of lung cancer. In the overall analysis, results presented that miR-196a2 (rs11614913) had obvious correlation with the risk of lung cancer. Homozygous CC might be the risk factor for lung cancer compared with homozygous TT. By observing recessive model, we could find the homozygous CC increased the risk of lung cancer. The subgroup analysis by race, individuals with homozygous CC had the higher risk of lung cancer in Asian population compared with homozygous TT. When subgroup analyzed by sources of controls, homozygous CC/heterozygous CT could be associated with increased lung cancer risk compared with homozygous TT in the hospital-based study. In the overall analysis, results presented that miR-499 had correlation with the risk of lung cancer. MiR-499 might be related with increased lung cancer risk in five genetic models. The subgroup analyzed by race, the results of five genetic models showed the significant association between miR-499 and the risk of lung cancer.
Although our research efforts are sufficient to implement a comprehensive analysis, there are still some limitations. Firstly, the different genetic backgrounds and living environments were exited in these conducted studies (44,45). Then, these case control studies only included Asian (Chinese and Korean populations) and Caucasian (Italy population). So the results could not be used as a model for other countries. Due to the limited number of Caucasians study, the results could not be used as a model for Caucasians. Secondly, publication bias might be existed, but the results of publication bias did not have statistical significance. Third, since the original studies did not have the smoking status, it was not possible to conduct subgroup analysis on smoking. Finally, we only analysis the published studies but not include unpublished articles.
In conclusion, this meta-analysis provides evidence that miR-146a (rs2910164), miR-499 (rs3746444) and miR-196a2 (rs11614913) polymorphisms might contribute to associating with lung cancer risk. As we know, this is the first time to get the significant relationship between miR-499 and lung cancer risk. Also, the result needs to be confirmed by a series of further experiments.
In this meta-analysis, miR-146a (rs2910164), miR-196a2 (rs11614913) and miR-499 (rs3746444) may be associated with the risk of lung cancer.
Funding: This study was supported by Natural Science Foundation of Liaoning Province (No. 201602870).
Conflicts of Interest: The authors have no conflicts of interest to declare.
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