A Functional Polymorphism in Pre-miR-146a Is Associated with Susceptibility to Gastric Cancer in a Chinese Population

Abstract

MicroRNAs play an important role in regulating gene expression at the post-transcriptional level and are involved in numerous physiological processes. Aberrant expression of MicroRNAs is considered to participate in occurrence and progression of human cancers. A G>C polymorphism, rs2910164, which is located in the sequence of miR-146a precursor, could alter mature miR-146a expression and has been suggested to influence cancer risk. The present study was aimed to investigate whether this polymorphism has effects on susceptibility to gastric cancer in the Chinese population. We genotyped the miR-146a rs2910164 polymorphism using the TaqMan method in a two-stage case–control study comprising a total of 1686 gastric cancer patients and 1895 cancer-free subjects. The logistic regression was used to assess the genetic associations with gastric cancer risk. We found a significant association between rs2910164 polymorphism and increased gastric cancer risk [p=0.038, odds ratio (OR)=1.26, 95% confidence interval (CI)=1.01–1.56; GG vs. CC/CG]. Similar results were observed in a follow-up replication study. Combined data from the two studies generated a more significant association [p=0.001, OR=1.27, 95% CI=1.10–1.46; GG vs. CC/CG]. Besides, the increased risk associated with the rs2910164GG genotype was more evident in younger subjects (OR=1.51, 95% CI=1.25–1.81) rather than in older subjects. Our results suggest that the rs2910164 polymorphism in the sequence of miR-146a precursor may influence the susceptibility to gastric cancer in our Chinese population.

Introduction

G astric cancer is the fourth most common cancer and the second leading cause of cancer-related deaths all over the world (Parkin, 2004; Parkin et al., 2005). It is estimated that ∼40% of gastric cancer cases occur in the Chinese population (Parkin et al., 2005). Although the exact causes of gastric cancer have not been identified yet, accumulating epidemiologic studies have showed that diet, smoking, alcohol, and Helicobacter pylori infection are well-known causes of gastric cancer (Uemura et al., 2001; Crew and Neugut, 2004). However, only a fraction of exposed individuals eventually develop gastric cancer, which suggests that genetic susceptibility also plays a role in the etiology of gastric cancer. Recently, emerging evidence suggests that genetic polymorphisms in candidate genes were associated with susceptibility to gastric cancer (Yoshida et al., 2010; Li et al., 2011; Shibata et al., 2011).

MicroRNAs (miRNAs) are a class of small noncoding RNA molecules composed of about 22 nucleotides and act as post-transcriptional that bind to complementary sequences in the 3′ untranslated regions of target messenger RNA transcripts, leading to translational repression and gene silencing (Bartel, 2004, 2009; Filipowicz et al., 2008). Numerous studies have demonstrated that aberrant expression of miRNAs plays an important role in cancer development through regulating the expression of oncogenes and/or tumor suppressor genes (Bartel, 2004; Calin and Croce, 2006; Esquela-Kerscher and Slack, 2006). Aberrant expression of miR-146a was observed in a variety of human malignancies, such as prostate cancer (Lin et al., 2008), breast cancer (Garcia et al., 2010), cervical cancer (Wang et al., 2008), and pancreatic cancer (Li et al., 2010). As for gastric cancer, Hou et al. (2011) demonstrated that miR-146a was down-regulated in gastric cancer and that low expression of miR-146a was correlated with increased tumor size and poor differentiation stage, which was further supported by the results of another study (Kogo et al., 2011).

Accumulating evidence suggests that single-nucleotide polymorphisms in human miRNA genes may affect miRNA biogenesis pathway and influence the susceptibility to complex diseases such as cancer (Liang et al., 2010; Nicoloso et al., 2010). The miRNA-146a rs2910164 G>C polymorphism causes change from a G:U pair to a C:U mismatch in the stem structure of miR-146a precursor (Hu et al., 2008). Jazdzewski et al. (2008) have demonstrated that this polymorphism could lead to lower expression of mature sequence and eventually influence the susceptibility to papillary thyroid carcinoma. To date, there are several studies that have investigated the associations between the miRNA-146a rs2910164 polymorphism and different types of cancer (Xu et al., 2011; Wang et al., 2012), such as lung cancer (Tian et al., 2009), breast cancer (Hu et al., 2009), hepatocellular carcinoma (Xu et al., 2008; Akkiz et al., 2011), prostate cancer (Xu et al., 2010), and gastric cancer (Okubo et al., 2010; Zeng et al., 2010). However, the results of these studies have been inconclusive. The role of rs2910164 polymorphism in affecting the susceptibility to gastric cancer has been previously investigated in the Chinese population (Zeng et al., 2010) and the Japanese population (Okubo et al., 2010), but the results of these two studies also remained inconsistent. Zeng et al. (2010) suggested that the rs2910164GG genotype was a risk factor for gastric cancer in the Chinese population, whereas Okubo et al. (2010) revealed an opposite results in the Japanese population. Hereby we conducted a two-stage case–control study that comprised a total of 1686 gastric cancer patients and 1895 cancer-free subjects to further assess whether the rs2910164 polymorphism has effect on gastric cancer risk in the Chinese population.

Materials and Methods

Study subjects

The study was approved by the institutional review board of the Nanjing Medical University. The design of the study was hospital based, and two independent sets of subjects were included in the present study. Overall, 750 gastric cases and 835 age- and sex-matched controls recruited at the second affiliated hospital of Nanjing Medical University, Nanjing, China, and Cancer Hospital of Nantong City, Nantong, China, from March 2006 to January 2010 were included in the test set, and 936 cases and 1060 controls enrolled from Yixing People's Hospital, Yixing, China, from January 1999 to December 2006 were included in the validation set. All subjects in the study are ethnic Han Chinese coming from different families and have no blood relationship. All the patients were newly diagnosed with histopathologically confirmed, incident gastric cancer without history of other cancers, or previous chemotherapy or radiotherapy. Those patients that had previous cancer, metastasized cancer from other or unknown origin, or previous radiotherapy or chemotherapy were excluded. Those control subjects who had previous history of cancer were excluded. At recruitment, written informed consent was obtained from all participants involved in this study.

DNA extraction and genotyping

Whole-genome DNA was isolated and purified from leucocytes of peripheral blood by proteinase K digestion and phenol/chloroform extraction. The rs2910164 polymorphism was genotyped by the TaqMan MGB technology (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Amplifications were performed on the 384-well ABI 7900HT real-time polymerase chain reaction (PCR) system (Applied Biosystems), and SDS 2.4 software was used for allelic discrimination. Four blank controls were included in each plate to ensure accuracy of the genotyping. About 10% of the samples were randomly selected for repeated assays, and the results were in agreement with the results of the first assays.

Statistical analyses

The allele frequencies of the rs2910164 polymorphism in the controls were tested against departure from the Hardy–Weinberg equilibrium using the goodness-of-fit χ ²-test before analysis. Differences in the distributions of demographic characteristics, selected variables, and frequencies of genotypes of rs2910164 polymorphism between the cases and controls were evaluated using the χ ²-test. Genetic associations between rs2910164 genotypes and risk of gastric cancer were estimated by computing odds ratios (ORs) and 95% confidence intervals (CIs) from logistic regression analysis with the adjustment for age and sex. All the statistical analyses were performed with software SAS 9.1.3 (SAS Institute, Cary, NC), and two-sided p<0.05 was considered statistically significant.

Results

Characteristics of the study population

The frequency distributions of characteristics and clinical features of two sets of study group are presented in Table 1. The cases and controls were matched on age and sex (p=0.383 and 0.424 for age and sex in the test set, and p=0.164 and 0.061 for age and sex in the validation set, respectively). There were more older subjects (>65) and women in the test set than in the validation set. However, no significant differences in age and sex between cases and controls were observed when we combined the two populations, as shown in the Table 1. In the test set, there were 295 cardia gastric cancer patients and 455 noncardia gastric cancer patients; 406 patients were found to have diffuse type of gastric cancer and 299 as intestinal type. Clinical TNM stage is categorized according to tumor size (T), lymph node metastasis (N), and distant metastasis (M). The percent of TNM stage of patients in the test set from I to IV were 26.8%, 22.0%, 35.5%, and 15.7%, respectively. Of the 936 patients in the validation set, 358 were cardia-type gastric cancer (CGC) and 578 noncardia-type gastric cancer (NCGC); 539 patients were diagnosed as diffuse type of gastric cancer, and 397 as intestinal type. Approximately 27.7%, 19.8%, 42.2%, and 10.3% of the patients were found to have TNM stage I, II, III and IV diseases, respectively.

Table 1.

Patient Characteristics and Clinical Features

	Test set, n (%)			Validation set, n (%)			Combined, n (%)
Variables	Cases	Controls	p^a	Cases	Controls	p^a	Cases	Controls	p^a
Age (years)
≤ 65	430 (57.3%)	460 (55.2%)	0.383	601 (64.2%)	712 (67.2%)	0.164	1031 (61.2%)	1172 (61.9%)	0.655
> 65	320 (42.7%)	374 (44.8%)		335 (35.8%)	348 (32.8%)		655 (38.8%)	722 (38.1%)
Sex
Male	510 (68.0%)	552 (66.1%)	0.424	721 (77.0%)	778 (73.4%)	0.061	1231 (73.0%)	1330 (70.2%)	0.061
Female	240 (32.0%)	283 (33.9%)		215 (23.0%)	282 (26.6%)		455 (27.0%)	565 (29.8%)
Site
Cardia	295 (39.3%)			358 (38.3%)			653 (38.7%)
Noncardia	455 (60.7%)			578 (61.7%)			1033 (61.3%)
Histological types
Diffuse	406 (57.6%)			539 (57.6%)			945 (57.6%)
Intestinal	299 (42.4%)			397 (42.4%)			696 (42.4%)
Depth of invasion
T1	120 (17.4%)			147 (15.7%)			267 (16.4%)
T2	120 (17.4%)			199 (21.3%)			319 (19.6%)
T3	350 (50.7%)			543 (58.0%)			893 (55.0%)
T4	101 (14.5%)			45 (5%)			146 (9.0%)
Lymph node metastasis
N0	333 (44.4%)			371 (39.6%)			704 (41.8%)
N1/N2/N3	417 (55.6%)			565 (60.4%)			982 (58.2%)
Distant metastasis
M0	616 (87.3%)			878 (93.8%)			1494 (91.0%)
M1	90 (12.7%)			58 (6.2%)			148 (9.0%)
TNM stage
I	189 (26.8%)			259 (27.7%)			448 (27.9%)
II	155 (22.0%)			185 (19.8%)			340 (21.2%)
III	250 (35.5%)			395 (42.2%)			609 (37.9%)
IV	111 (15.7%)			97 (10.3%)			208 (13.0%)

Two-sided X² test.

TNM: T, tumor size; N, lymph node metastasis; M, distant metastasis.

Association between the miRNA-146a rs2910164 C>G polymorphism and gastric cancer risk

Genotype distributions of the miRNA-146a rs2910164 C>G polymorphism among the patients and controls in the test and validation set are shown in Table 2. The observed genotype frequencies in the controls of the test set, validation set, and the combined set were all conformed to the Hardy-Weinberg equilibrium (p=0.544, 0.929 and 0.641, respectively). In the test set, we found that the genotype distribution of the rs2910164 polymorphism was statistically significantly different between case and control subjects (p=0.021). Further logistic regression analyses revealed that a significant increased gastric cancer risk was associated with the rs2910164GG genotype, compared with the CC or CC/GC genotypes (OR=1.50, 95% CI=1.12–2.01 and OR=1.26, 95% CI=1.01–1.56). In the replication study, we found similar associations between the rs2910164GG genotype and an increased gastric cancer risk (OR=1.42, 95% CI=1.10–1.84 for GG vs. CC and OR=1.27, 95% CI=1.05–1.53 for and GG vs. GC/CC). There was no statistical heterogeneity between the two studies (data not shown), and a more significant association was observed when we combined data from the two studies (p<0.001; OR=1.46, 95% CI=1.20–1.76 for GG vs. CC and OR=1.27, 95% CI=1.10–1.46 for GG vs. GC/CC).

Table 2.

Genotype and Allele Frequencies of MicroRNA-146 a rs2910164 Polymorphism Among the Cases and Controls and Their Associations with Risk of Gastric Cancer

Genotypes	Cases, n (%)	Controls, n (%)	p^a	OR (95% CI) ^a	Adjusted OR (95% CI) ^b
The test set
CC	122 (16.3)	175 (21.0)	0.021	1.00 (reference)	1.00 (reference)
CG	380 (50.7)	424 (50.8)		1.29 (0.98–1.68)	1.28 (0.98–1.68)
GG	248 (33.0)	236 (28.3)		1.51 (1.13–2.02)	1.50 (1.12–2.01)
CG+GG	628 (83.7)	660 (79.0)	0.017	1.37 (1.06–1.76)	1.36 (1.05–1.75)
CC+CG	502 (67.0)	599 (71.7)	0.038	1.00 (reference)	1.00 (reference)
GG	248 (33.0)	236 (28.3)		1.25 (1.01–1.55)	1.26 (1.01–1.56)
G allele	876 (58.4)	896 (53.7)	0.007	1.09 (1.02–1.16)	-
The validation set
CC	164 (17.5)	218 (20.6)	0.021	1.00 (reference)	1.00 (reference)
CG	442 (47.2)	527 (49.7)		1.15 (0.88–1.41)	1.12 (0.88–1.42)
GG	330 (51.3)	315 (29.7)		1.39 (1.08–1.80)	1.42 (1.10–1.84)
CG+GG	772 (82.5)	842 (79.4)	0.084	1.22 (0.97–1.53)	1.21 (0.97–1.52)
CC+CG	600 (64.7)	745 (70.3)	0.008	1.00 (reference)	1.00 (reference)
GG	330 (51.3)	315 (29.7)		1.29 (1.07–1.55)	1.27 (1.05–1.53)
G allele	1102 (58.9)	1157 (54.6)	0.006	1.08 (1.02–1.14)	-
Combined
CC	286 (17.0)	393 (20.7)	<0.001	1.00 (reference)	1.00 (reference)
CG	822 (48.8)	951 (50.2)		1.19 (0.99–1.42)	1.19 (1.00–1.42)
GG	578 (34.2)	551 (29.1)		1.44 (1.19–1.75)	1.46 (1.20–1.76)
CG+GG	1400 (83.0)	1502 (79.3)	0.004	1.28 (1.08–1.52)	1.28 (1.08–1.51)
CC+CG	1108 (65.8)	1344 (70.9)	<0.001	1.00 (reference)	1.00 (reference)
GG	578 (34.2)	551 (29.1)		1.27 (1.11–1.47)	1.27 (1.10–1.46)
G allele	1978 (59.5)	2053 (54.2)	<0.001	1.10 (1.06–1.14)	-

Two-sided χ ² test

Adjusted for age and sex.

OR, odds ratio; CI, confidence interval.

Stratified analysis of miRNA-146a rs2910164 C>G polymorphism and gastric cancer risk

We then evaluated the effect of the miRNA-146a rs2910164 C>G polymorphism on gastric cancer stratified by age and sex. As shown in Table 3, we found that the increased risk associated with the carriers of the rs2910164GG genotype was more evident among younger subjects (<65 years) (OR=1.51, 95% CI=1.25–1.81). No significant difference was observed in the stratification of sex, tumor site, and histological types. Besides, we also investigated whether the polymorphism was associated with clinical characteristics of the patients such as TNM stage, distant metastasis, lymph node metastasis, and depth of tumor infiltration; however, no positive result was observed (data not shown).

Table 3.

Stratification Analyses Between MicroRNA-146 a rs2910164 Polymorphism and Gastric Cancer Risk in the Combined Group

	Rs2910164 genotypes
	CC+CG, n (%)		GG, n (%)
Variables	Cases	Controls	Cases	Controls	p^a	Adjusted OR (95% CI) ^b
Age
≤65	682 (66.2)	876 (74.7)	349 (33.8)	296 (25.3)	<0.001	1.51 (1.25–1.81)
>65	426 (65.0)	468 (64.7)	229 (35.0)	255 (35.3)	0.905	1.00 (0.80–1.25)
Sex
Male	804 (65.3)	927 (69.7)	427 (34.7)	403 (30.3)	0.018	1.25 (1.06–1.47)
Female	304 (66.8)	417 (73.8)	151 (33.2)	148 (26.2)	0.015	1.36 (1.03–1.79)
Site
Cardia	419 (64.2)	1344 (70.9)	234 (35.8)	551 (29.1)	0.001	1.34 (1.11–1.62)
Noncardia	689 (66.7)	1344 (70.9)	344 (33.3)	551 (29.1)	0.018	1.23 (1.04–1.44)
Histological types
Diffuse	627 (66.4)	1344 (70.9)	318 (33.6)	551 (29.1)	0.013	1.25 (1.06–1.48)
Intestinal	449 (64.5)	1344 (70.9)	247 (35.5)	551 (29.1)	0.002	1.31 (1.09–1.58)

Two-sided χ ² test.

Were adjusted for age, sex.

Discussion

In the present study, we evaluated the associations between the functional polymorphism (rs2910164) in the sequence of miR-146a precursor and risk of gastric cancer. We found that individuals with the rs2910164 variant genotype (GG) had a significantly increased gastric cancer risk compared with those carrying the CC or CC/CG genotypes. Furthermore, in the stratified analysis, we found that the increased risk associated with the rs2910164GG genotype was more evident in younger subjects (<65 years). These findings may contribute to the efforts directed to a better understanding of the etiology of gastric cancer.

Accumulating evidence intensively suggests that aberrant regulation of specific miRNAs and their targets in various kinds of cancer is correlated with tumor growth, angiogenesis, and metastasis (Esquela-Kerscher and Slack 2006, Croce, 2009). Previous studies have showed that down-regulation of miR-146 was involved in the development and progression of several types of cancer, including papillary thyroid carcinoma (Jazdzewski et al., 2008), prostate cancer (Lin et al., 2008), and gastric cancer (Tchernitsa et al., 2010; Hou et al., 2011; Kogo et al., 2011). In gastric cancer, Tchernitsa et al. (2010) first demonstrated that miR-146a expression was lower in gastric cancer tissue than in non-neoplastic tissue by using microarray analysis and semi-quantitative real-time PCR. Then, Hou et al. (2011) showed that miR-146a was down-regulated in four gastric cancer cell lines (MKN-28, SGC-7901, MKN-45, and BGC-823 cells). They also found that low expression of miR-146a was associated with increased tumor size and poor differentiation stage. The influence of the rs2910164 polymorphism on the expression level of miR-146a has been investigated by several studies (Shen et al., 2008; Yue et al., 2010). Shen et al. (2008) and Yue et al. (2010) both have suggested that the rs2910164 G-to-C change resulted in elevated expression of mature miR-146a. Considering that miR-146a is down-regulated in gastric cancer, it is possible that individuals carrying the rs2910164 genotype, which is associated with decreased level of miR-146a (i.e., GG), have an increased risk of gastric cancer. Therefore, it is biologically plausible that the polymorphism confers individuals' susceptibility to gastric cancer.

To date, several molecular epidemiological studies have been conducted to investigate the associations between the miR-146a rs2910164 polymorphism and the risk of various cancers. However, the results from these studies are inconclusive. Most recently, two meta-analyses of studies were conducted to derive a more precise estimation of the relationship (Xu et al., 2011; Wang et al., 2012). Wang et al. suggested that the polymorphism was associated with the increased risk for overall cancer in the domain model (GG+CG vs. CC: OR=1.18, 95% CI: 1.03–1.35), especially in Asians. Xu et al. suggested the C allele of rs2910164 was associated with protection from digestive cancer (C vs. G: OR=0.86, 95% CI=0.77–0.96). The results of our study in the Chinese population, which also revealed a significant association of the polymorphism and increased gastric cancer risk, are in line with their findings. As we mentioned before, the role of the rs2910164 polymorphism in affecting the susceptibility to gastric caner has been previously investigated in the Chinese population (Zeng et al., 2010) and the Japanese population (Okubo et al., 2010). Our results are also consistent with the study conducted in the Chinese population that demonstrated that the rs2910164 variant genotypes (CG/GG) were associated with an increased gastric cancer risk (OR=1.58, 95% CI: 1.12–2.22); however, these are inconsistent with the study conducted in the Japanese population showing that the rs2910164CC genotype was associated with an increased risk of gastric cancer (OR=1.30, 95% CI: 1.02–1.66). Although the two populations all belong to the Asian ethnic groups, ethnic differences also exist. Therefore, we speculate that the discrepancy may be due to the differences of the ethnic variation. For instance, the frequencies of the G allele of the rs2910164 polymorphism among the two ethnicities are as follows: 0.556 in the Chinese Han population in Beijing and 0.367 in the Japanese population in Tokyo (HapMap database). The G allele frequency in our controls from the combined set was 0.586, which is similar to the reported allele frequency in the Chinese Han population.

In the stratification analysis, our results showed that carrying the rs2910164GG genotype was at a significantly increased risk for gastric cancer in younger subjects (<65 years), whereas not in older subjects. This result is also supported by the study of Zeng et al. (2010), in which a similar phenomenon was observed. As age increases, a large amount of DNA damage and genomic alterations accumulate in the body, which would facilitate carcinogenesis (Vijg et al., 2005; Maslov and Vijg, 2009). Besides, deleterious alterations also occur to the immune response with aging (Burns and Leventhal, 2000), which further promote cancer development. It has been widely accepted that age is the most important risk factor for cancer. Therefore, the increased risk associated with the rs2910164GG genotype in the older subjects may be overwhelmed by the presence of more genomic aberrations and the deleterious alterations of immune system in them, which may partly contribute to the age difference we observed. Further analysis denied significant association between the polymorphism and clinical characteristics of the patients such as TNM stage, distant metastasis, lymph node metastasis, and depth of tumor infiltration, suggesting that the polymorphism may not influence the progression of gastric cancer.

Helicobacter pylori infection status, gastroesophageal reflux, and tobacco smoking are well-known causes of gastric cancer. Genetic polymorphisms may interact with these factors to influence gastric caner risk. Unfortunately, we did not have available information on these factors in our study, and therefore we were unable to perform further analysis. Further studies with more detailed data on the Helicobacter pylori infection and tobacco smoking are warranted.

In conclusion, our results further suggest a significant association between the rs2910164 polymorphism and increased risk of gastric cancer in a large two-stage case–control study in the Chinese population. Although the associations appeared to be statistically significant in our population, the initial findings should be independently verified in diverse ethnic populations since genetic polymorphisms often vary between ethnic groups.

Footnotes

Acknowledgments

This work was supported in part by “the Six Talent Summit” Project of Jiangsu Province (SWYY-71-028), the National Natural Science Foundation of Jiangsu (BK2011194), and the National Natural Science Foundation of China (30872084, 30972444, and 81001274).

Disclosure Statement

We declare that we have no conflict of interest.

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