Pri-miR-34b/c rs4938723 Polymorphism Is Associated with a Decreased Risk of Gastric Cancer

Abstract

Previous studies have demonstrated that miR-34 family members are abnormally expressed in gastric cancer. Overexpression of the miR-34 family suppresses gastric carcinogenesis, whereas downregulation of the miR-34 family promotes tumorigenesis. p53 can bind to the promoter region of miR-34b/c, leading to an increase of miR-34b/c expression. Recently, a variant in the promoter region of pri-miR-34b/c (rs4938723) has been discovered, with the function of altering the binding efficiency of transcription factor GATA. The purpose of this study was to examine the role of the miR-34b/c rs4938723 and TP53 Arg72Pro polymorphisms in the susceptibility of gastric cancer. We analyzed the distribution of the two polymorphisms in 197 patients with gastric cancer and 289 age-, gender-, ethnicity-, and living area-matched controls using polymerase chain reaction-restriction fragment length polymorphism and DNA direct sequencing. We found that the CT and CT/CC genotypes of the miR-34b/c rs4938723 were associated with a significantly decreased risk of gastric cancer compared with the TT genotype (CT vs. TT: odds ratio [OR]=0.66; 95% confidence interval [95% CI], 0.45-0.97; and CT/CC vs. TT: OR=0.67; 95% CI, 0.47-0.97, respectively). Combined analysis showed that subjects carrying the miR-34b/c rs4938723 CT/CC and TP53 CG/CC genotypes had a 0.62-fold decreased risk to develop gastric cancer compared with subjects carrying the miR-34b/c rs4938723 TT and TP53 CG/CC genotypes (OR=0.62; 95% CI, 0.40-0.96). These findings suggest that the miR-34b/c rs4938723 may individually and jointly have a protective effect on the risk of gastric risk.

Introduction

Gastric cancer is a common malignancy developing from the lining of the stomach. It is estimated that there were 22,220 new cases and 10,990 deaths in 2014 (Siegel et al., 2014). The highest rates are observed in developing countries, and 42% of the cases occur in China (Parkin et al., 2005). The geographical distribution provides strong evidence that environmental components are risk factors for the development of gastric cancer, such as Helicobacter pylori infection, salted food intake, and tobacco smoking (Tsugane, 2005; Dikshit et al., 2011; Wang et al., 2012; D'Elia et al., 2014). In addition to the environmental factors, genetic factors have been determined to be responsible for gastric carcinogenesis (Gao et al., 2009; Li et al., 2013b; He et al., 2014). Among the genetic factors, tumor suppressor TP53 genetic alterations are fundamental events in the early stage and advanced tumors of gastric cancer (Bellini et al., 2012).

The TP53 gene located on 17p13.1 encodes a nuclear p53 protein of 393 amino acids, which plays a crucial role in the maintenance of genomic stability (Benchimol et al., 1985; Belyi et al., 2010). As a transcription factor, the p53 protein has been reported to be implicated in the apoptotic pathway through targeting multiple elements (Zhao et al., 2012). Recent work has demonstrated that p53 participates in carcinogenesis through another pathway of regulating miRNAs (He et al., 2007; Ji et al., 2008; Soutto et al., 2014).

Corney et al. (2007) reported that miR-34b and miR-34c cooperate in suppressing cell proliferation and adhesion-independent growth by targeting p53. Furthermore, p53 can bind to the promoter region of miR-34b/c, which led to an increase of miR-34b/c expression (Corney et al., 2007). In humans, a nonsynonymous variant in a proline-rich domain of the p53 protein results in a substitution of an arginine for a proline at codon 72 (Arg72Pro, rs1042522) (Matlashewski et al., 1987). The Arg72Pro polymorphism exhibits differences in biological properties, with the 72Pro variant having a lower apoptotic potential than the 72Arg variant (Dumont et al., 2003). Based on this background, varieties of reports have investigated the role of the Arg72Pro polymorphism in several kinds of cancers, including gastric cancer (Francisco et al., 2011; Tang et al., 2012; Cheng et al., 2014).

Recently, an rs4938723T/C polymorphism in the promoter region of pri-miR-34b/c has been identified (Xu et al., 2011). The T to C change of the rs4938723 polymorphism has been reported to affect the binding of GATA-X, which eventually altered transcription activities of the promoter (Hasegawa et al., 2003; van Rietschoten et al., 2004; Xu et al., 2011). To date, several studies reported the association of the miR-34b/c rs4938723 polymorphism with several types of cancer, such as hepatocellular carcinoma (Xu et al., 2011; Han et al., 2013; Son et al., 2013), colorectal cancer (Gao et al., 2013; Oh et al., 2014), nasopharyngeal carcinoma (Li et al., 2013a), esophageal squamous cell carcinoma (Yin et al., 2013; Zhang et al., 2014a), osteosarcoma (Tian et al., 2014), and renal cell cancer (Zhang et al., 2014b). Therefore, we hypothesized that the polymorphism might be related to the risk of gastric cancer. In this study, we performed a case-control study to examine the prevalence of the miR-34b/c rs4938723 in a Chinese population. Furthermore, interaction analysis of the miR-34b/c rs4938723 and TP53 Arg72Pro polymorphisms was also tested.

Materials and Methods

Study population

The study protocol was approved by the institutional review boards of the hospital. A total of 486 subjects were enrolled in this study, including 197 patients with gastric cancer and 289 healthy controls of equivalent age, gender, ethnicity, and living area. All participants were of the Chinese Han population and recruited from the Luoyang Central Hospital Affiliated to Zhengzhou University between July 2010 and December 2012. The study population has been described previously (Li et al., 2013b). Briefly, the patients were mostly men (63.5%) with a mean age of 57.7±12.2 years. In addition to clinical presentation, the diagnosis of gastric cancer was confirmed by histological examination. The patients were limited to cases without a family history of gastric cancer. The controls were recruited from a health checkup center at the hospital. The vast majority of the controls were males (60.2%) as well, and the mean age was 56.3±11.3 years. We excluded the controls if they had evidence of family history of cancer or inflammatory diseases, such as gastritis. Demographic and clinical data, including age, gender, differentiation status, clinical stage, and metastasis status, were obtained based on medical records.

Genotyping

DNA was extracted from peripheral blood leukocytes using an extraction kit (Bioteke, Beijing, China). Genotyping for the miR-34b/c rs4938723 and TP53 Arg72Pro polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism analysis. The amplification primers and restriction enzymes were selected as described previously (Xu et al., 2011; Li et al., 2012; Gao et al., 2013). For the purpose of quality assurance, ∼10% samples were genotyped in duplicate by two researchers independently, and discordant results were resolved by direct sequencing.

Statistical analyses

Demographic information between cases and controls was compared using the Student's t-test or χ² test. The Hardy-Weinberg equilibrium (HWE) was determined using standard χ² statistics. The genotype and allelic frequencies were analyzed by means of the χ² test. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were used to assess the effect of the miR-34b/c rs4938723 and TP53 Arg72Pro polymorphisms on the risk of gastric cancer. A p-value of <0.05 was considered statistically significant. All analyses were done using SPSS 13.0 (SPSS, Inc., Chicago, IL).

Results

The characteristics of controls and patients with gastric cancer are presented in Table 1. The age of controls was slightly younger than patients, and the male subjects in the control group were slightly less than those in the case group. However, there is no significant difference between cases and controls based on age and gender (p>0.05).

Table 1.

Characteristics of Controls and Patients with Gastric Cancer

	Controls, n=289	Patients, n=197	p-Value
Age (years, mean±SD)	56.3 (±11.3)	57.7±12.2	NS
Gender
Male	174 (60.2)	125 (63.5)	NS
Female	115 (39.8)	72 (36.5)
Differentiated status
Well to moderately		77 (39.1)
Poorly to undifferentiated		120 (60.9)
Clinical stage
I-II		88 (44.7)
III-IV		109 (55.3)
Metastasis
Yes		84 (42.6)
No		113 (57.4)

NS, not significant; SD, standard deviation.

The genotype and allele frequencies of the miR-34b/c rs4938723 and TP53 Arg72Pro polymorphisms are listed in Table 2. We tested the genotypes for HWE, and the genotype distributions of both polymorphisms fit the HWE in cases and controls. The CT and CT/CC genotypes of the miR-34b/c rs4938723 were associated with significantly decreased risks of gastric cancer compared with the TT genotype (CT vs. TT: OR=0.66; 95% CI, 0.45-0.97, and CT/CC vs. TT: OR=0.67; 95% CI, 0.47-0.97, respectively). Stratification analysis showed no statistically significant correlation between the miR-34b/c rs4938723 polymorphism and clinicopathological parameters, including differentiated status, clinical stage, and metastasis status. Moreover, the genotype distribution of the TP-53 Arg72Pro is not significantly different between cases and controls.

Table 2.

Association Between miR-34b/c rs4938723 and TP53 Arg72Pro Polymorphisms and Susceptibility of Gastric Cancer

Polymorphisms	Controls (n=289) (%)	GC (n=197) (%)	OR (95% CI)	p-Value
rs4938723
TT	121 (41.9)	102 (51.8)	1.0 (ref)
CT	137 (47.4)	76 (38.6)	0.66 (0.45-0.97)	0.03
CC	31 (10.7)	19 (9.6)	0.73 (0.39-1.36)	0.32
CT/CC	168 (58.1)	95 (48.2)	0.67 (0.47-0.97)	0.03
T allele	379 (65.6)	280 (71.1)	1.0 (ref)
C allele	199 (34.4)	114 (28.9)	0.78 (0.59-1.02)	0.07
TP53 Arg72Pro
GG	93 (32.2)	65 (33.0)	1.0 (ref)
CG	154 (53.3)	104 (52.8)	0.97 (0.65-1.45)	0.87
CC	42 (14.5)	28 (14.2)	0.95 (0.54-1.69)	0.87
G allele	340 (58.8)	234 (59.4)	1.0 (ref)
C allele	238 (41.2)	160 (40.6)	0.98 (0.75-1.27)	0.86

Bold values indicate P<0.05.

95% CI, 95% confidence interval; GC, gastric cancer; OR, odds ratio.

The combined effects of the miR-34b/c rs4938723 and TP53 Arg72Pro polymorphisms on the risk of gastric cancer were also detected. As shown in Table 3, subjects carrying the miR-34b/c rs4938723 CT/CC and TP53 CG/CC genotypes had a 0.62-fold decreased risk to develop gastric cancer compared with subjects carrying the miR-34b/c rs4938723 TT and TP53 CG/CC genotypes (OR=0.62; 95% CI, 0.40-0.96).

Table 3.

Combined Effects of miR-34b/c rs4938723 and TP53 Arg72Pro Polymorphisms on the Risk of Gastric Cancer

Genotypes	Controls, n (%)	GC, n (%)	OR (95% CI)	p-Value
rs4938723 TT+TP53 CG/CC	79 (27.3)	69 (35.0)	1.0 (ref)
rs4938723 CT/CC+TP53 CG/CC	117 (40.5)	63 (32.0)	0.62 (0.40-0.96)	0.03
rs4938723 TT+TP53 GG	42 (14.5)	33 (16.8)	0.90 (0.52-1.57)	0.71
rs4938723 CT/CC+TP53 GG	51 (17.6)	32 (16.2)	0.72 (0.42-1.24)	0.24

Bold values indicate P<0.05.

Discussion

We analyzed the role of the miR-34b/c rs4938723 and TP53 Arg72Pro polymorphisms in the susceptibility of gastric cancer and found evidence of miR-34b/c rs4938723 being associated with the risk of gastric cancer. Specifically, individuals who carried the miR-34b/c rs4938723 CT and CT/CC genotypes had a 0.66- and 0.67-fold reduced risk for developing gastric cancer, respectively. Moreover, subjects carrying the miR-34b/c rs4938723 CT/CC and TP53 CG/CC genotypes had a 0.62-fold decreased risk to develop gastric cancer with a 95% CI of 0.40-0.96. These results suggest that rs4938723 in the promoter of pri-miR-34b/c may be related to the etiology of gastric cancer.

Human miR-34 consists of miR-34a, miR-34b, and miR-34c, which express abnormally in various types of cancer, including gastric cancer (Corney et al., 2010; Wang et al., 2013, 2014). Overexpression of the miR-34 family can suppress gastric carcinogenesis, whereas downregulation of the miR-34 family can promote tumorigenesis (Wang et al., 2014). Restoration of miR-34 can inhibit cell proliferation and apoptosis that is mediated by p53 (Corney et al., 2007; Ji et al., 2008; Hermeking, 2010). Inversely, p53 can bind to the promoter region of miR-34b/c, which induces expression of miR-34b/c, indicating a positive feedback loop between p53 and miR-34 (Corney et al., 2007; Yamakuchi and Lowenstein, 2009).

Recently, a variant in the promoter region of pri-miR-34b/c (rs4938723) has been discovered, with the function of altering the binding efficiency of transcription factor GATA (Xu et al., 2011). Because of the potential role of the miR-34b/c rs4938723 polymorphism, several studies have been conducted to examine the effect of the miR-34b/c rs4938723 polymorphism on cancer susceptibility. However, no report evaluated the role of the polymorphism in gastric cancer risk. In this study, we provided evidence that miR-34b/c rs4938723 is a protective factor for the development of gastric cancer.

In agreement with our results, Gao et al. (2013) reported that the miR-34b/c rs4938723CC genotype had a 0.56-fold decreased risk of colorectal cancer in a Chinese population. The TP53 and miR-34b/c interaction analysis showed that the combined genotypes of TT-GC and CC-GG were associated with a 0.63- and 0.38-fold reduced risk of colorectal cancer in a Korean population (Oh et al., 2014). Similarly, the decreased risks were also observed in esophageal squamous cell carcinoma (Yin et al., 2013; Zhang et al., 2014a).

Inconsistent with our results, some authors reported that the miR-34b/c rs4938723 TC/CC genotypes were associated with a significantly increased risk of hepatocellular carcinoma (Xu et al., 2011; Han et al., 2013; Son et al., 2013). The risk effect of the miR-34b/c rs4938723 TC/CC genotypes was more evident in subjects carrying the TP53 CC genotype, drinkers, male subjects, and individuals aged <55 years old (Xu et al., 2011). The increased risk was also found in nasopharyngeal carcinoma (Li et al., 2013a), osteosarcoma (Tian et al., 2014), and renal cell cancer (Zhang et al., 2014b). Nevertheless, the increased risk was not confirmed in hepatocellular carcinoma by subsequent meta-analysis (Liang et al., 2013). The possibilities for the difference may be that the miR-34b/c rs4938723 polymorphism has different roles according to the types of cancer. Additionally, the potential role of gene-environment cannot be ruled out. Thus, further association studies are of great value in diverse types of cancer.

In the last decades, several studies have focused on the relationship between the TP53 Arg72Pro polymorphism and the risk of gastric cancer, but conflicting results have been obtained. In an attempt to clarify the issue, meta-analysis was used by pooling the data together. Conflicting results, however, still occurred. Some meta-analyses showed that the TP53 Arg72Pro polymorphism may contribute to the susceptibility of gastric cancer (Francisco et al., 2011; Tang et al., 2012; Cheng et al., 2014). In contrast, a meta-analysis reported by Zhou et al. (2007) showed no significant difference in the TP53 Arg72Pro genotype distribution between cases and controls. The result in the current study is in accordance with the previous negative finding. After combined analysis with the miR-34b/c rs4938723, a statistical significance was found, indicating that the TP53 polymorphism together with other genetic polymorphisms may modulate the individual risk of gastric cancer.

Taken together, this study suggests that the miR-34b/c rs4938723 may individually and jointly have a protective effect on the risk of gastric risk. There are some limitations in the study, such as hospital-based design, limited sample size, and the absence of gene-environmental interaction analysis. Therefore, population-based studies with larger samples should be considered in future investigations of the miR-34b/c rs4938723 in relation to gastric cancer risk.

Footnotes

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 81302149), the Science & Technology Pillar Program of Sichuan Province (14ZC1838), Distinguished Young Scientist of Sichuan University (No. 2013SCU04A38), the PhD Programs Foundation of Ministry of Education of China (No. 20130181120011), the Science & Technology Project of Henan Province (No. 132300410105), and the PhD Scientific Research Foundation of Henan University of Science and Technology (No. 09001492).

Author Disclosure Statement

No competing financial interests exist.

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