RESEARCH MiR-34b/c rs4938723 Polymorphism Significantly Decreases the Risk of Digestive Tract Cancer: Meta-analysis

Background: Previous studies investigating the association between miR-34b/c rs4938723 polymorphism and cancer risk showed inconclusive. Here, we performed meta-analysis to investigate the association between miR-34b/c rs4938723 polymorphism and digestive cancer risk. Materials and Methods: Literature database including PubMed, OVID, Chinese National Knowledge Infrastructure (CNKI) were searched for publications concerning the association between the miR-34b/c rs4938723 polymorphism and digestive cancer risk. Results: A total of 6 studies consisting of 3246 cases and 3568 controls were included in this meta-analysis. The combined analysis suggested the miR-34b/c rs4938723 polymorphism significantly reduced digestive cancer risk under allelic model, homogeneous co-dominant model and recessive model (C vs T: OR=0.88, 95%CI=0.82-0.95, p -value=0.001; CC vs TT: OR =0.67, 95%CI=0.57-0.80, p -value=0.000; CC vs TT/TC: OR=0.68, 95%CI=0.58-0.80, p -value=0.000). Q-test and I2 test revealed no significant heterogeneity in all genotype comparisons. The Begger’s funnel plot and Egger’s test did not show significant publication bias. Conclusions: The current evidence supports the conclusion that the miR-34b/c rs4938723 polymorphism decreases an individual’s susceptibility to digestive cancers. promoter region of miR-34b/c is associated with renal cell cancer risk in a Chinese population.


Literature search
In order to identify the relevant papers about miR-34b/c rs4938723 polymorphism and digestive cancer risk, we performed a systematic search from PubMed, OVID, Chinese National Knowledge Infrastructure (CNKI) databases, with a combination of the following keywords: " miR-34b/c, microRNA-34b/c, pre-miR-34b/c" ; "rs4938723"; "allele mutation or polymorphism" (last search was updated on 2 Mar. 2015). References of previous meta-analyses and reviews were also manually searched to identify additional studies. We evaluated potentially relevant publications by examining their titles and abstracts and all studies matching the eligible criteria were retrieved.

Inclusion and exclusion criteria
Studies included in the current meta-analysis must conform to all the following criteria: (a) evaluation of miR-34b/c rs4938723 and digestive cancer risks, (b) use a case-control design, (c) sufficient published data for estimating an odds ratio (OR) with 95% confidence interval (CI).
The exclusion criteria were as follows: (a) study pertaining to other SNP of miR-34b/c and cancer risk, but not rs4938723; (b) review or meta-analysis concerning miR-34b/c rs4938723 and cancer risks. (c) Study on miR-34b/c rs4938723 and non-digestive cancer risk.

Quality assessment
The quality of each study was assessed according to the quality assessment criteria recommended by Xue et al (Supplemental Table 1). Quality scores of studies ranged from 0 (lowest) to 15 (highest). Studies with score less than 9 were considered as low quality, while those scores equal to or greater than 9 were categorized into high quality (Xue et al., 2015).

Data extraction
Two investigators independently extracted the data, finally reached consensus on all items. For each study, the following parameters were extracted: the first author's last name, year of publication, country of origin, ethnicity, the numbers of genotyped cases and controls and genotyping methods.

Statistical analysis
OR corresponding to 95% CI was used to assess the strength of association between rs4938723 polymorphism and digestive cancer risk. The significance of the pooled OR was determined by the Z-test, and p-value 0.05 was considered as statistically significant. We explored the association between rs4938723 and digestive cancer risk using allelic model (C vs T), co-dominant model (homogeneous co-dominant model: CC vs TT, heterogeneous co-dominant model: TC vs TT), recessive (CC vs TC + TT), over-dominant (TC vs TT + CC) and dominant (CC + TC vs TT) genetic models. A chi-square-based Q-statistic test and an I2-test test were both carried out to evaluate the heterogeneity of the studies. By heterogeneity test, if p-value 0.10 for the Q-test, the pooled OR was calculated by the fixed effects model (Mantel and Haenszel, 1959). Otherwise, the random-effects model (DerSimonian and Laird, 1986). Hardy-Weinberg equilibrium (HWE) in the control group was estimated using Fisher's exact test and a p-value 0.05 was considered significant. Leave-one-study-out sensitivity analysis was performed to reflect the influence of the individual data-set to the pooled OR (Normand, 1999). Publication bias was evaluated using the funnel plot and Egger's test (Egger et al., 1997). p-value0.05 indicate the presence of potential publication bias. All statistical tests were performed with Stata ES 12.0 (Stata Corporation, College Station, TX, USA).

Study characteristics
The Processes of studies searching and selecting are illustrated in the flow diagram ( Figure 1). A total of 204 articles were achieved by literature search,

Table 1. Characteristics of Studies Included in the Meta-analysis
Author, year Country Ethnicity Cancer type  from PubMed, OVID, Chinese National Knowledge Infrastructure (CNKI) databases, using different combination of key terms. 11 articles were obtained by browsing the reference of meta-analysis or articles pertaining to miR-34b/c rs4938723 polymorphism and cancer risk. 47 records were excluded for duplicate, 144 records were excluded improper titles and/or abstracts. Then, 24 eligible studies were retrieved for detailed evaluation. During the extraction of data, 18 articles were excluded (ten were meta-analysis, one study evaluated mir-34b rs2187473 (T/C) polymorphisms and oral carcinoma risk, but not miR-34b/c rs4938723, 7 studies evaluated the relationship between mir-34b rs2187473 (T/C) polymorphisms and non-digestive cancer). As a result, a total of 6 publications including 3246 cases and 3568 controls were included in the meta-analysis [16][17][18][19][20][21].
The characteristics of the included were summarized in Table 1. In the eligible studies, there were 2 studies of esophageal cancer, 2 studies of gastric cancer and 2 studies of colorectal cancer. 5 studies come from china, 1 study from Korea. The controls of 5 studies came from hospital-based gender and age matched healthy population; the gender of control did not matched to that of cases in one study. The distribution of genotypes in the controls in all eligible studies did not deviate from HWE (Table 1). Besides, each study included in the meta-analysis was regarded as high quality according to the quality assessment criteria recommended by xue et al (Supplemental Table 1) (Xue et al., 2015).

Meta-analysis results
In overall population, there was significant heterogeneity in miR-34b/c rs4938723 for all model comparison including allelic model, homogeneous codominant model, heterogeneous co-dominant model, recessive model comparisons and dominant model comparison, and over-dominant model (  Figure 2 and 4). However, we did not found the association between rs4938723 polymorphism and digestive cancer risk in overall population under heterogeneous co-dominant model, dominant model comparison and over-dominant model (Table 2). Meanwhile, there was no significant heterogeneity in all genetic models comparison (Table 2 and Figure 1).

Publication bias
Begger's funnel plot and Egger's test were performed  (Table 2). However, there were publication bias under heterogeneous co-dominant model, dominant model comparison and over-dominant model comparison which were not found to be related to the susceptibility of digestive cancer (Table 2).

Sensitivity analysis
We deleted one single study from the overall pooled analysis each time to check the influence of the removed data set to the overall ORs. The pooled ORs and 95% CIs were not significantly altered when any part of the study was omitted, which indicated that any single study had little impact on the overall ORs (data not shown).

Discussion
In current study, meta-analysis between miR-34b/c rs4938723 and digestive cancer risk was performed. There was not significant heterogeneity under all genetic models (Table 2). And the pooled analysis indicted the variant C allele and CC homozygote was associated with a lower digestive cancer risk compared to the T allele, TT wildtype homozygote and TC/CC genotype. However, recent studies and meta-analysis suggested the T to C shift of the rs4938723 polymorphism could increase certain cancer risk including hepatocellular, renal and nasopharyngeal carcinoma; was not linked to certain cancer susceptibility such as breast cancer (Xu et al., 2011;Li et al., 2013;Wang et al., 2013;Yi et al., 2014;Zhang et al., 2014b). Moreover, Zhang et al (Zhang et al., 2014b) further experimentally ascertained the expression of miR-34b/c was higher in in normal renal tissues with TT+TC genotypes than in those with CC genotypes, the luciferase activities with rs4938723 T allele in 293-T cells was higher than that with C allele. Therefore, C allele of rs4938723 polymorphism has different effects on different cancer types by affect the expression of miR-34b/c.
It was proposed that mechanisms involving in the down-regulation of miR-34b/c included inactivating mutations of p53, hyper-methylation and mutation of its encoding genes (Lujambio et al., 2008;Toyota et al., 2008;Corney et al., 2010;Suzuki et al., 2010). MiR-34b/c SNP rs4938723 locates in a typical CpG island, may affect predicted GATA-X transcription factors binding and methylation status of miR-34b/c CpG islands. It is plausible that the miR-34b/c polymorphism rs4938723 might affect cancer susceptibility by creating the predicted GATA-binding site or the CpG methylation (Gao et al., 2013;Zhang et al., 2014b). Furthermore, other cancer risk factors such as age, alcohol consumption, and TP53 Arg72Pro polymorphisms might alter the effect of miR-34b/c rs4938723 polymorphism on cancer risk synergistically or antagonistically (Xu et al., 2011;Li et al., 2013;Zhang et al., 2014a;Pan et al., 2015). So, different combinations of multiple mechanisms adopted Supplemental Table 1. Score of Quality Assessment Criteria and Score Gao, 2013Oh, 2014Yin, 2013Zhang, 2014Yang, 2014Pan, 2015 Representativeness of case Selected from population cancer registry 2 Selected from hospital Sensitivity analysis also showed that omission of any single study did not have significant impact on the combined ORs. In addition, funnel plot did not reflect obvious asymmetry, and Egger's test further indicated no considerable publication bias under allelic model, homogeneous co-dominant model and recessive model comparison. The distribution of genotypes in the controls in all eligible studies did not deviate from HWE. Furthermore, all studies included in the meta-analysis were regarded as high quality according to the quality assessment criteria recommended by Xue et al (S1 Table  1). Therefore, the results were very robust and reliable.
To a certain extent, our meta-analysis still includes some limitations. First, the numbers for each type of digestive cancer were relatively small, so there is insufficient statistical power to investigate the association between mir-34b/c polymorphism and each type cancer; second, the lack of detailed original data of factor influencing the effect of miR-34b/c rs4938723 polymorphism on cancer risk, such as the age of the populations, alcohol consumption, methylation status of mir-34b/c gene CpG islands and TP53 Arg72Pro polymorphisms in the eligible studies may hinder our further analyses (Han et al., 2013;Li et al., 2013;Zhang et al., 2014a;Pan et al., 2015). Third, there were still not eligible studies to analyses miR-34b/c rs4938723 and digestive cancer risk in African &Caucasian. Therefore, more studies are needed to explore the potential relationship between miR-34b/c rs4938723 polymorphisms and different types of digestive cancer susceptibility in ethnically diverse populations to consolidate our findings.
Taken together, our study provides evidence that T to C shift of the rs4938723 polymorphism reduce individual susceptibility to digestive cancer, Future studies with larger sample size and more ethnic groups on the association between miR-34b/c rs4938723 polymorphism and digestive cancers are required to confirm current findings.