Genetic variant in miR‐21 binding sites is associated with colorectal cancer risk

Abstract Single nucleotide polymorphisms (SNPs) within binding sites of microRNAs (miRNAs) could modify cancer susceptibility by changing the binding affinity of miRNAs on their target mRNA 3ʹ‐untranslated regions (UTRs). MicroRNA‐21 (miR‐21) is involved in the development of colorectal cancer. However, the relationship between SNPs within the binding sites of miR‐21 and colorectal cancer risk has not been widely investigated. A case‐control study including 1147 patients and 1203 controls was performed to evaluate the association of SNPs in miR‐21 binding sites and colorectal cancer risk. Dual‐luciferase reporter assays and functional assays were performed to evaluate the effects of miR‐21. The SNP rs6504593 C allele conferred an increased risk of colorectal cancer compared with the T allele in an additive model (odds ratio [OR] = 1.19, 95% confidence interval [CI] = 1.04‐1.36, P = 0.011). Dual‐luciferase reporter assays demonstrated that the rs6504593 T allele negatively post‐transcriptionally regulated IGF2BP1 by altering the binding affinity of miR‐21. Additionally, colorectal cancer cells transiently transfected with miR‐21 mimics promoted cell proliferation and suppressed apoptosis, whereas inhibition of miR‐21 decreased cell growth. These data suggest that the miR‐21 binding site SNP rs6504593 in the IGF2BP1 3ʹ‐UTR may alter IGF2BP1 expression and contribute to colorectal cancer risk.

site SNP rs6504593 in the IGF2BP1 3ʹ-UTR may alter IGF2BP1 expression and contribute to colorectal cancer risk.

K E Y W O R D S
3ʹ-UTR, colorectal cancer, genetic variants, miR-21

| INTRODUCTION
Colorectal cancer is one of the main causes of cancer-related mortality in the United States. They are projected to be 140 250 new colorectal cancer cases and 50 630 deaths in 2018. 1 In China, colorectal cancer is also a public health concern due to the rapid increase in morbidity in recent decades. 2 Colorectal cancer is known as a complex disease because of environmental and genetic factors and their interactions. Despite enormous efforts that have been devoted to exploring the pathogenesis of colorectal cancer, the precise etiology remains to be elucidated.

| Study population
We conducted a case-control study including 1147 colorectal cancer individuals and 1203 cancer-free controls. In brief, the participants were consecutively recruited from The First Affiliated Hospital and Nanjing First Hospital of Nanjing Medical University beginning in September 2010 and were histopathologically confirmed. The cancer-free controls were randomly selected from individuals who participated in physical examinations and were frequency-matched by age (±5 years) and sex. This study protocol was approved by the Institutional Review Board of Nanjing Medical University. Previous studies have described the detailed information of the study subjects. 18

| SNPs selection
We focused on both the miR-21 target genes dataset and 3ʹ-UTR dataset using the UCSC browser (http://www.genome.ucsc.edu).

| SNPs genotyping
Assay System (Promega) was used to measure the luciferase activity, and the Renilla luciferase activity was normalized to Firefly luciferase activity.

| Quantitative reverse transcription polymerase chain reaction
Colorectal tumor tissues and their paired adjacent normal tissues were collected from colorectal cancer subjects. The clinical characteristics are provided in Table S2. Total RNA was extracted from colorectal cancer cells and tissue specimens using TRIzol (Invitrogen) according to the manufacturer's protocol. Reverse transcription was performed with Primescript RT Master Mix (TaKaRa) and RNA PCR Kit (AMV) (TaKaRa). Real-time polymerase chain reaction (PCR) assay with SYBR Green Master Mix reagent kit (TaKaRa) was performed using a 7900 Real-time PCR system (ABI). The relative expression of miR-21, ALPP and IGF2BP1 normalized to U6 and ACTINB was calculated using the 2 −ΔCt method. The primer sequences are listed in Table S1.

| The cancer genome atlas and eQTL analysis
We obtained miRNA and mRNA expression data and genotypic data of colorectal samples from the cancer genome atlas (TCGA) database. Expression quantitative trait loci (eQTL) analysis was performed to evaluate the associations between SNP genotypes and mRNA expression levels. Only samples that have miR-21 rs6504593 genotyping data were included for eQTL analysis.

| Statistical analysis
Student's t test for continuous variables and Pearson's χ 2 test for categorical variables were used to analyze the differences in the distribution of demographic and clinical characteristics between colorectal cancer cases and controls. Hardy-Weinberg equilibrium for the selected SNP allele frequencies was evaluated using a goodnessof-fit χ 2 test in the control group. Multivariate logistic regression analysis with adjustments for age, sex, smoking and drinking status was used to evaluate the association of the selected SNPs with colorectal cancer risk by computing adjusted odds ratios (ORs) and 95% confidence intervals (CIs). All experimental results are shown as the mean ± SD, and the statistical comparisons between groups were tested by t test, Mann-Whitney U test and ANOVA. SAS 9.4.0 (SAS Institute) was used for all the statistical analyses, and statistical significance was set at P < 0.05.

| Demographic and clinical characteristics of subjects
The demographic and clinical characteristics of cases and controls are presented in Table S3. No significant differences were detected between colorectal cancer individuals and cancer-free controls for age (P = 0.751), gender (P = 0.116), smoking status (P = 0.097) and alcohol drinking status (P = 0.113). However, a higher proportion of family history of cancers was observed in colorectal cancer cases than in controls (P < 0.001

| Association between selected SNPs and colorectal cancer susceptibility
The baseline characteristics of the selected SNPs are summarized in

| miR-21 and its target genes' expression in colorectal cancer tissues
We measured the expression of miR-21 in colorectal tumor and adjacent normal tissues and found that miR-21 expression was significantly upregulated in tumor tissues compared to normal tissues (P < 0.001) ( Figure 1A). This result was consistent with the data obtained from TCGA (P < 0.001) ( Figure 1B). IGF2BP1 expression showed no significant difference between colorectal cancer and adjacent normal tissues (P = 0.177) ( Figure 1C), whereas TCGA data showed that IGF2BP1 expression in colorectal cancer tissues was significantly higher than that in normal tissues (P < 0.001) ( Figure 1D).
The expression level of ALPP in colorectal cancer and adjacent normal tissues was almost undetectable, which was supported by data in TCGA. Therefore, IGF2BP1 was retained for further analysis.
Moreover, a negative association between IGF2BP1 and miR-21 expression was discovered in both colorectal tumor tissues (r = −0.348, P = 0.029) and normal tissues (r = −0.401, P = 0.010), although no significant correlation was found by using TCGA data ( Figure S1).

3ʹ-UTR interaction
To demonstrate whether rs6504593 regulates IGF2BP1 expression by miR-21, we performed dual-luciferase reporter assays by constructing psiCHECK-2 vectors containing wild-type or mutated-type IGF2BP1 3ʹ-UTR (Figure 2A). The specific binding sites of IGF2BP1 3ʹ-UTR and miR-21 were provided by bioinformatics tools (Figure 2B). The results indicated that constructs containing the rs6504593 T allele significantly reduced luciferase activity compared with the C allele in HCT116 and SW620 cells (P = 0.003 and P = 0.012, respectively) ( Figure 2C). These data suggested that miR-21 may directly target the IGF2BP1 3ʹ-UTR with the rs6504593 T allele.

| eQTL analysis of IGF2BP1
To understand whether rs6504593 can regulate the expression of IGF2BP1, we performed an eQTL analysis in TCGA data. We found that rs6504593 genotypes had no influence on IGF2BP1 expression in either colorectal cancer tissues or normal tissues (P = 0.705 and P = 0.774, respectively) ( Figure S2).

| DISCUSSION
In this study, we investigated the association between genetic variants in miR-21 binding sites and colorectal cancer susceptibility in a case-control study. Our finding shows that rs1049109 and rs6504593 were new susceptibility SNPs for colorectal cancer, which have not been reported in previous studies.
IGF2BP1, belonging to the family of RNA-binding proteins, plays key roles in many biological processes through binding to the key motif in RNA structures. 20 The abnormal expression of IGF2BP1 may cause a series of diseases, including malignant tumors. 21

| CONCLUSIONS
Here, we have demonstrated that the SNP rs6504593 was associated with colorectal cancer susceptibility. Moreover, the rs6504593 T allele negatively regulated the post-transcription of IGF2BP1 by altering the binding affinity of miR-21. Furthermore, we found that miR-21 mimics promoted cell proliferation and suppressed apoptosis, whereas an inhibitor of miR-21 decreased cell growth. Thus, our study provides a novel view of miR-21-induced colorectal cancer development. The miR-21 binding site SNP rs6504593 may be used as a new potential biomarker for the diagnosis of colorectal cancer.