Pri-miR-124 rs531564 polymorphism and colorectal cancer risk

MiR-124 functions as a tumor suppressor and plays an important role in tumorigenesis. A common polymorphism (rs531564, C>G) in the pri-miR-124 has been recently studied in connection with cancer risk. The aim of the present study was to investigate the association between pri-miR-124 rs531564 polymorphism and the risk and clinicopathological characteristics of colorectal cancer (CRC). Two case-control studies involving 900 CRC patients and 1110 cancer-free controls showed that pri-miR-124 rs531564 polymorphism was significantly associated with the decreased risk of CRC in Xuzhou population [GG vs. CC: OR = 0.25, 95%CI = 0.09–0.67, P = 0.003; (CG+GG) vs. CC: OR = 0.73, 95%CI = 0.56–0.94, P = 0.01; GG vs. (CC+CG): OR = 0.27, 95%CI = 0.10–0.70, P = 0.004; G vs. C: OR = 0.70, 95%CI = 0.56–0.89, P = 0.003], Bengbu population [GG vs. CC: OR = 0.20, 95%CI = 0.04–0.90, P = 0.02; GG vs. (CC+CG): OR = 0.21, 95%CI = 0.05–0.95, P = 0.03; G vs. C: OR = 0.72, 95%CI = 0.54–0.98, P = 0.03] and pooled population [GG vs. CC: OR = 0.26, 95%CI = 0.11–0.59, P<0.001; (CG+GG) vs. CC: OR = 0.76, 95%CI = 0.62–0.93, P = 0.008; GG vs. (CC+CG): OR = 0.27, 95%CI = 0.12–0.62, P < 0.001; G vs. C: OR = 0.71, 95%CI = 0.59–0.85, P<0.001]. Additionally, pri-miR-124 rs531564 polymorphism was significantly associated with the decreased risk of poor differentiation and lymph node metastasis of CRC. Our results suggest that pri-miR-124 rs531564 polymorphism may be a genetic modifier for developing CRC. However, further studies are needed to validate our findings.

C > G) in the pri-miR-124 has been identified and shown to influence the expression of mature miR-124 11 . In addition, pri-miR-124 rs531564 polymorphism was reported to be significantly correlated with the risk of various cancers, including cervical cancer and esophageal squamous cell carcinoma (ESCC) [12][13][14] . Thus, we hypothesized that the potentially functional polymorphism also contributed to the risk of CRC.
In the current study, we firstly conducted two independent case-control studies in Chinese population to investigate the associations, and then examined the clinicopathological characteristics of CRC for different genotypes.

Results
Pri-miR-124 rs531564 polymorphism and CRC risk. The selected characteristics of enrolled subjects are listed in Table 1. No significant difference was found in distributions of age and gender. Association of the pri-miR-124 rs531564 polymorphism with CRC risk was shown in Table 2 Clinicopathological characteristics of CRC for different genotypes. The association between pri-miR-124 rs531564 polymorphism and clinicopathological characteristic of CRC patients was conducted in two independent studies (Table 3). Significant associations were found in status of differentiation and lymph node metastasis. In CRC patients from Xuzhou city, the pri-miR-124 rs531564 polymor-

Discussion
Similar to the protein-coding genes, miRNAs are transcribed for the majority by RNA polymerase II as long primary miRNA (pri-miRNAs) transcripts with stem-loop structure 15 . The pri-miRNAs are processed to produce miRNA precursors (pre-miRNAs) and then further cleaved into mature miRNAs which act mainly through annealing to 3′ untranslated regions (3′ UTRs) of gene transcripts, leading to inhibition of further steps of gene expression. MiR-124 is a highly conserved miRNA and plays an important role in neural processes, from nervous system development to normal neuronal cell function 16 . Nowadays, miR-124 has been reported to be a potential tumor suppressor and an independent prognostic marker in many cancers, including colorectal cancer, prostate cancer, and nasopharyngeal carcinoma [17][18][19] . The miR-124 gene is located in 8p23 and has an identified polymorphism (rs531564) which Continued affects miR-124 expression 11 . The associations of rs531564 polymorphism and cancer risk have been reported; however, the results of previous studies are controversial. Most studies showed that rs531564 polymorphism was associated with decreased risk of cancer, such as cervical cancer and esophageal squamous cell carcinoma; however, one study indicated no association between rs531564 polymorphism and cancer risk including esophageal cancer, which was probably due to difference in genotyping method, cancer type or source of controls [12][13][14]20 .
In two independent case-control studies from Xuzhou population (577 CRC patients and 690 healthy controls) and Bengbu population (323 CRC cases and 420 healthy subjects), we found that pri-miR-124 rs531564 polymorphism was significantly associated with the decreased risk of CRC, which is similar to the results of the recent meta-analysis 21 . Analysis based on bioinformatics and gene expression showed that the G allele in the rs531564 polymorphism changed the formation of a ring-shaped structure in miR-124, which was beneficial to the increase of the amount of mature miR-124 11 . Therefore, we speculated that the role of pri-miR-124 rs531564 polymorphism in CRC susceptibility was associated with the stability of the pri-miRNA, or the efficiency of processing of pri-miRNA into mature miRNA. Furthermore, our results showed that pri-miR-124 rs531564 polymorphism was significantly associated with clinicopathological characteristics of CRC patients, including poor differentiation and lymph node metastasis. To the best of our knowledge, this is the first molecular epidemiological study to investigate the associations of pri-miR-124 rs531564 polymorphism with the risk and clinicopathological characteristics of CRC. However, we could not determine the interaction between pri-miR-124 rs531564 polymorphism and potential risk factors, such as drinking, red meat consumption and body mass index, because of a lack of enough individual data in case-control studies.
Taken together, our findings suggest that pri-miR-124 rs531564 polymorphism contributes to the decreased risk of CRC, poor differentiation and lymph node metastasis in Chinese population, possibly by affecting miR-124 expression. However, further research in this field is warranted to complete understanding of carcinogenesis associated with the pri-miR-124 rs531564 polymorphism.  DNA extraction and genotyping. The genomic DNA of each subject was isolated from peripheral blood samples using genomic DNA extraction kit (Qiagen) according to the manufacturer's protocol. DNA fragments containing rs531564 were amplified with the following primers (forward: 5-TCTTCTACCCACCCCTCTTCC-3; reverse: 5-AATCTGCACACACAAGCACTC-3). The PCR products were sequenced in forward direction. The SNP genotypes were determined by DNAMAN and BLAST.

Materials and Methods
Statistical Analysis. Differences in general characteristics were examined using Student's t-test or χ 2 test. The Hardy-Weinberg equilibrium (HWE) was tested by a goodness-of-fit χ 2 test to compare the expected genotype frequencies with observed genotype frequencies in cancer-free controls. Logistic regression was used to analyze the association between the rs531564 polymorphism and the risk and clinicopathological characteristics of CRC, adjusted by confounding factors including age, gender, family history of cancer, tumor site, differentiation, TNM and/or lymph node metastasis when appropriate. In Table 3, all the comparisons are done using the second characteristic as reference. P < 0.05 was used as the criterion of statistical significance. All statistical analyses were implemented in the SPSS statistical software (version 19.0).