A Functional Polymorphism in miRNA-196a2 Is Associated with Colorectal Cancer Risk in a Chinese Population

Abstract

Single-nucleotide polymorphisms in microRNAs (miRNAs) may alter miRNA expression levels or processing and, thus, may contribute to cancer development. We hypothesized that miRNA-196a2 polymorphism is associated with risk of colorectal cancer (CRC). In a case-control study of 573 patients with CRC and 588 cancer-free controls frequency matched by age and sex, we genotyped the functional polymorphism rs11614913 (T>C) and assessed its association with the risk of CRC in a Chinese population. We found that the CT/CC genotypes were associated with a significantly increased risk of CRC (odds ratio [OR]=1.44, 95% confidence interval [CI]=1.10–1.88), compared with the TT genotype. Further, the polymorphism was significantly associated with the risk of patients with advanced stage tumor (Dukes C and D) (OR=1.65, 95% CI=1.11–2.46). Our results suggest that the functional polymorphism rs11614913 in miRNA-196a2 is involved in the etiology of CRC and, thus, may be a marker for genetic susceptibility to CRC.

Introduction

C olorectal cancer (CRC) is one of the most common cancers and a major problem worldwide (Parkin et al., 2005). The incidence rate and mortality rate attributed to CRC have been increasing in recent years in China (Yang et al., 2005). Sex, age, diet, body mass index, and genetic factors are major risk factors for CRC (Haggar and Boushey, 2009). Recently, examinations of microRNA (miRNA) expression profiles in CRC have highlighted the importance of miRNA expression alterations in CRC diagnosis, prognosis, and susceptibility (Dong et al., 2011).

miRNAs are an abundant class of small noncoding RNAs that function as negative gene regulators (Ambros, 2004; Bartel, 2004). miRNAs regulate the expression of roughly 10%–30% of all human genes through post-transcriptional mechanisms, and their abnormal expression has been linked with many human diseases including cancer (Lu et al., 2005; Barbarotto et al., 2008). miRNAs participate in human carcinogenesis as tumor suppressor or oncogenes (Esquela-Kerscher and Slack 2006; Stahlhut Espinosa and Slack 2006). Single-nucleotide polymorphisms (SNPs) or mutations in miRNA sequence may alter miRNA expression and/or maturation (Saunders et al., 2007; Chen et al., 2008).

Recently, several studies identified that a functional polymorphism rs11614913 (T>C) in miRNA-196a2 is associated with cancer risk (Hoffman et al., 2009; Hu et al., 2009). Further, previous studies have revealed that this polymorphism affects the processing of the pre-miRNA into its mature, regulatory form (Hu et al., 2008; Hoffman et al., 2009). Three investigators have performed a meta-analysis of all eligible studies, and found that rs11614913 raises the risk of cancer, especially among Asians and breast cancer (Chu et al., 2011; Wang et al., 2011; Xu et al., 2011). However, their meta-analyses did not estimate the effect of rs11614913 on CRC risk. Since miRNA-196a2 is important in the CRC carcinogenic process, we hypothesized that the miRNA-196a2 rs11614913 polymorphism is associated with the risk of CRC. To test this hypothesis, we investigated the association of the miRNA-196a2 rs11614913 polymorphism with the risk of CRC in a case-control study of 573 CRC cases and 588 cancer-free controls in a Chinese population.

Materials and Methods

Study subjects

The case-control study of CRC was approved by the institutional review board of Nanjing Medical University. This study included 573 patients with CRC and 588 cancer-free controls. The patients with CRC were consecutively recruited starting from September 2010 at The First Affiliated Hospital of Nanjing Medical University, Nanjing, China, without restriction regarding age and sex. All patients were newly diagnosed incident cases and histopathologically confirmed as colorectal adenocarcinoma, and the participation rate of the cases was ∼95%. The pathological stage of CRC at the time of diagnosis was classified into Dukes A, B, C, and D (Corman, 1980). Tumor grade was classified into low (well differentiated), intermediate (moderately differentiated), and high (poorly differentiated). The control subjects were genetically unrelated to the cases without individual history of cancer, and frequency matched to patients based on sex and age. Each participant was scheduled for an interview after written informed consent had been obtained, and a structured questionnaire was administered by interviewers to collect information on demographic data and environmental exposure history. Those subjects who smoked daily for >1 year were defined as smokers. Individuals who consumed one or more alcoholic drinks per week for at least 1 year were considered drinkers. After interview, about 5 mL of venous blood sample was collected from each subject.

Genotyping

Blood samples were collected in EDTA containing tubes. Genomic DNA was extracted from white-blood-cell fractions by using the Qiagen Blood Kit (Qiagen). Genotyping was performed with the TaqMan SNP Genotyping Assay by using the 384-well ABI 7900HT Real-Time PCR System according to the manufacturer's instructions (Applied Biosystems). The sequences of primer and probe for each SNP are available on request. A total of 10 negative controls and 8 duplicates were included for each SNP as a quality-control measure. Genotype analysis was performed by two persons independently in a blind fashion. About 10% of the samples were randomly selected for repeated genotyping for confirmation, and the results were 100% concordant.

Statistical analyses

Differences in the distributions of demographic characteristics, selected variables, and frequencies of genotypes of miRNA-196a2 rs11614913 polymorphism between the cases and controls were evaluated by using the Student's t-test (for continuous variables) or χ ²-test (for categorical variables). The associations between the rs11614913 genotypes and risk of CRC were estimated by computing odds ratios (ORs) and their 95% confidence intervals (CIs) from unconditional logistic regression analysis with the adjustment for possible confounders. Hardy-Weinberg equilibrium (HWE) was tested by using a goodness-of-fit χ ²-test. p<0.05 was considered statistically significant, and all statistical tests were two sided. All the statistical analyses were performed with the software SAS 9.1.3 (SAS Institute).

Results

The baseline characteristics of CRC cases and controls are shown in Table 1. As expected, no significant difference was found between the cases and controls with regard to age and sex (p=0.136 for age, and 0.824 for sex), which suggested that age and sex were matched adequately. Similarly, there were no statistically significant differences in smoking status and alcohol use between the case and control groups (p=0.191 for smoking status, and 0.082 for alcohol use). However, 22.7% of CRC cases had a family history of cancer, which was significantly higher than that of the controls (7.1%) (p<0.001). Of the 573 patients with CRC, 48.7% of patients had colon cancer, whereas 51.3% of patients had rectum cancer. The Dukes A, B, C, and D stages were 9.1%, 40.6%, 35.1%, and 15.2%, respectively. For histological differentiation, 6.5%, 74.9%, and 18.6% were classified as low, intermediate, and high grade, respectively.

Table 1.

Distribution of Selected Variables Between Colorectal Cancer Cases and Controls

	Cases (n=573)		Controls (n=588)
Variables	n	%	n	%	p
Age (mean±SD)	60.3±12.5		59.3±9.8		0.136
Sex					0.824
Male	354	61.8	367	62.4
Female	219	38.2	221	37.6
Smoking status					0.191
No	377	65.8	408	69.4
Yes	196	34.2	180	30.6
Drinking status					0.082
No	414	72.3	451	76.7
Yes	159	27.7	137	23.3
Family history of cancer					<0.001
No	443	77.3	546	92.9
Yes	130	22.7	42	7.1
Tumor site
Colon	279	48.7
Rectum	294	51.3
Dukes stage
A	52	9.1
B	233	40.6
C	201	35.1
D	87	15.2
Tumor grade
Low	37	6.5
Intermediate	429	74.9
High	107	18.6

SD, standard deviation.

As shown in Table 2, the frequencies of the TT, CT, and CC genotypes were 22.7%, 52.9%, and 24.4%, respectively, among the cases, and 29.2%, 50.2%, and 20.6%, respectively, among the controls (p=0.028). The rs11614913 C allele frequency was 0.509 among the cases and 0.457 among the controls, and the difference was statistically significant (p=0.012). The observed genotype frequencies among the controls were in agreement with the HWE (p=0.790). Logistic regression analysis revealed that the CT and CC genotypes were associated with a significantly increased risk of CRC, compared with the TT genotype (OR=1.39, 95% CI=1.05–1.84 for CT vs. TT, and OR=1.57, 95% CI=1.12–2.20 for CC vs. TT). The C allele was associated with the increased risk of CRC in a dose-response manner (p _trend=0.011). Further, a significant increased risk of CRC was found in the combined genotypes CT/CC compared with the TT genotype (OR=1.44, 95% CI=1.10–1.88).

Table 2.

Distribution of Genotypes of miRNA-196a2 rs11614913 and Their Associations with Risk of Colorectal Cancer

	Cases (n=573)		Controls (n=588)
Genotypes	n	%	n	%	p	OR (95% CI)	OR (95% CI) ^a
TT	130	22.7	172	29.2		1.00	1.00
CT	303	52.9	295	50.2	0.031	1.36 (1.03–1.80)	1.39 (1.05–1.84)
CC	140	24.4	121	20.6	0.012	1.53 (1.10–2.14)	1.57 (1.12–2.20)
CT/CC	443	77.3	416	70.8	0.011	1.41 (1.08–1.84)	1.44 (1.10–1.88)
C allele	0.509		0.457		0.012
p _trend					0.011

Adjusted for age, sex, smoking status, and alcohol use in logistic regression model.

CI, confidence interval; OR, odds ratio.

The effect of miRNA-196a2 rs11614913 polymorphism was further evaluated based on the clinical characteristics of patients with CRC (Table 3). A significant increased risk was observed between the CT/CC genotypes and patients with CRC with Dukes C and D stage (OR=1.65, 95% CI=1.11–2.46). However, no significant associations were found between rs11614913 and other clinical characteristics including age and sex (data not shown).

Table 3.

Association Between miRNA-196a2 rs11614913 Genotypes and Clinical Characteristics of Colorectal Cancer

	TT		CT/CC			CT/CC versus TT
Variables	n	%	n	%	p	OR (95% CI)	OR (95% CI) ^a
Tumor site
Colon	63	48.5	216	48.8		1.00	1.00
Rectum	67	51.5	227	51.2	0.953	0.99 (0.67–1.46)	0.96 (0.65–1.43)
Tumor grade
Low/intermediate	107	82.3	359	81.0		1.00	1.00
High	23	17.7	84	19.0	0.744	1.09 (0.65–1.81)	1.11 (0.66–1.85)
Dukes stage
A/B	77	59.2	208	46.9		1.00	1.00
C/D	53	40.8	235	53.1	0.014	1.64 (1.10–2.44)	1.65 (1.11–2.46)

Adjusted for age, sex, smoking status, and alcohol use in logistic regression model.

Discussion

In this case-control study, we investigated the association of miRNA-196a2 rs11614913 polymorphism and risk of CRC in a Chinese population. We found that the CT/CC genotypes were associated with a significantly increased risk of CRC, and the association was also evident in patients with advanced stage CRC. To the best of our knowledge, this is the first study of the association of miRNA-196a2 polymorphism with CRC risk.

miRNAs play important roles in the etiology of many human diseases by post-transcriptionally regulating the expression of the approximately one-third of all human genes (Lewis et al., 2005). miRNA expression profiles have been frequently reported to be correlated with the etiology, classification, progression, and prognosis of multiple human cancers, including CRC (Dong et al., 2011). Although the role of miRNA genetic variants in cancer susceptibility largely remains unknown, the importance of miRNA SNPs has been implicated in many cancers. SNPs in miRNAs may affect miRNA target expression and functions and, thus, may contribute to cancer risk (Duan et al., 2007; Yu et al., 2007). Genetic polymorphism of miRNA-196a2 has been shown to alter the expression of mature miRNA-196a2 and binding activity of target mRNA (Hoffman et al., 2009). In the present study, we found that individuals who carried the rs11614913 CT/CC genotypes had an increased risk of CRC compared with the TT genotype. A possible explanation is that miRNA-196a2 rs11614913 polymorphism may affect miRNA expression and its potential targets, and, therefore, might play a role in regulatory processes during CRC development (Ryan et al., 2010).

Recently, three meta-analyses investigated the association between miRNA-196a2 rs11614913 polymorphism and risk of cancers (Chu et al., 2011; Wang et al., 2011; Xu et al., 2011). Chu et al. (2011) found that individuals with the rs11614913 TC/CC genotypes were associated with higher cancer risk than those with the TT genotype. In the stratified analyses, they observed that the significant risk was more evident in breast cancer and lung cancer. However, Xu et al. (2011) demonstrated that the rs11614913 most likely contributes to decreased susceptibility to cancer, especially in Asians and breast cancer. In addition, Wang et al. (2011) indicated that miRNA-196a2 rs11614913 polymorphism is associated with cancer susceptibility, especially with breast cancer and in Chinese and Indian populations. In this study, we found that miRNA-196a2 rs11614913 polymorphism was significantly associated with increased risk of CRC in a Chinese population. These conflicting results showed a wide range of miRNA-196a2 expression across different normal tissues, tumor types, and between tumor and normal tissues from common sites. Further, the differences might also reflect the fact that different ethnic and environmental exposures may have modified the risk of cancer.

In the subgroup analysis with clinical characteristics of CRC, we found that the rs11614913 polymorphism was associated with the patients with advanced stage (Dukes C and D). Advanced stage is correlated to worse prognosis; therefore, our results suggested that miRNA-196a2 rs11614913 polymorphism may contribute to the progression of CRC. Although the grade of differentiation is also an important variable after the stage, and rectal and colon cancer differ in their histology, diagnosis, sensitivity to radiotherapy, and prognosis (Kapiteijn et al., 2001), we did not find any significant association between the rs11614913 polymorphism and CRC risk among the tumor site and grades, which may be due to our moderate sample size, and the limited statistical power of the study.

In conclusion, in this case-control study, we observed some evidence of an association between miRNA-196a2 rs11614913 polymorphism and the risk of CRC in a Chinese population. Additional, large studies with ethnically diverse populations are warranted to further confirm the impact of miRNA-196a2 polymorphism on CRC susceptibility.

Footnotes

Acknowledgment

This study was partly supported by National Natural Science Foundation of China (30872084 and 30972444), the Key Program for Basic Research of Jiangsu Provincial Department of Education (08KJA330001), the Postdoctoral Science Foundation of China (20100481164), the Key Project of Nanjing Medical University (2010NJMUZ20), and the Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

Disclosure Statement

No competing financial interests exist.

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