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A miR-29c binding site genetic variant in the 3′-untranslated region of LAMTOR3 gene is associated with gastric cancer risk
Biomedicine & Pharmacotherapy 69 (2015) 70–75
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Original article
A miR-29c binding site genetic variant in the 30 -untranslated region of LAMTOR3 gene is associated with gastric cancer risk Peng Song a,1, Weizhi Wang b,1, Guoquan Tao c,1, Haiyan Chu d,e, Meilin Wang d,e, Dongmei Wu d,e, Na Tong d,e, Weida Gong f, Jianwei Zhou g, Zhengdong Zhang d,e, Baolin Wang a,*, Haixia Zhu h,**, Qinghong Zhao a,* a
Department of General Surgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China c Department of General Surgery, Huai-An First People’s Hospital Affiliated to Nanjing Medical University, Huai-an,, China d Department of Environmental Genomics, Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, Nanjing Medical University, Nanjing, China e Department of Genetic Toxicology, The Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, China f Department of General Surgery, Yixing Cancer Hospital, Yixing, China g Department of Molecular Cell Biology & Toxicology, School of Public Health, Cancer Center, Nanjing Medical University, Nanjing, China h Core Laboratory of Nantong Tumor Hospital, Nantong, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 October 2014 Accepted 5 November 2014
Single nucleotide polymorphisms (SNPs) in the 30 -untranslated regions (UTRs) targeted by putative mircoRNAs (miRNAs) could influence the susceptibility of cancer. Recently, miR-29c has been reported to be down-regulated in gastric cancer (GC) and serve as a tumor suppressor that regulated tumor progression. The present study was aimed at investigating whether the miR-29c binding site SNPs within the 30 -UTRs of target genes affected the gastric cancer risk. Using bioinformatics tools, we chose three SNPs (IGHMBP2 rs3750980, LAMTOR3 rs11944405 and WWOX rs2288035) located in miR-29c binding sites. We genotyped these three SNPs to assess their associations with GC risk in a case-control study comprising 753 GC cases and 950 controls. Among these three SNPs, we found a significantly decreased risk of GC associated with the LAMTOR3 rs11944405 T > C polymorphism [TC vs. TT, adjusted odds ratio (OR) = 0.79, 95% confidence interval (CI) = 0.63–0.99; TC/CC vs. TT, adjusted OR = 0.81, 95% CI = 0.65–1.00]. The significant association was also presented in the subgroup analysis by age ( 65), sex (female), depth of invasion (T3/T4), lymph node metastasis (N1-3), distant metastasis (M0) and TNM stage (III/IV). However, no significant association was detected for IGHMBP2 rs3750980 and WWOX rs2288035. Our results suggested that the LAMTOR3 rs11944405 polymorphism may be a potential biomarker for genetic susceptibility to GC. ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Single nucleotide polymorphism LAMTOR3 miR-29c Gastric cancer Genetic susceptibility
1. Introduction Gastric cancer (GC) remains one of the most highly aggressive and lethal neoplasms worldwide, with highest incidence rates in Eastern Asia, Eastern Europe, and South America. Although * Corresponding authors. Department of General Surgery, The Second Affiliated Hospital of Nanjing Medical University, 121, Jiangjiayuan Road, Nanjing 210011, China. Tel.: +86 25 58509832; fax: +86 25 58509994. ** Corresponding author. Core Laboratory of Nantong Tumor Hospital, 48, West Qingnian Road, Nantong 226006, China. E-mail addresses: [email protected] (B. Wang), [email protected] (H. Zhu), [email protected] (Q. Zhao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biopha.2014.11.008 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
stomach cancer rates have decreased substantially in most parts of the world, it is estimated that nearly one million new cases and over 700,000 deaths have happened each year [1]. Accumulating evidence has indicated that multiple risk factor exposures (i.e., Helicobacter pylori infection, alcohol consumption, tobacco smoking, diet poor in fresh fruits and vegetables or rich in salt and nitrate) and genetic variations play important roles in the process of GC [2–4]. With the development of molecular genetics in recent decades, single nucleotide polymorphisms (SNPs) in apoptosis, inflammatory, immune response, metabolism, tumor-related genes are reported to associate with different susceptibility to this malignancy [5]. Identifying new susceptibility loci for GC advances our understanding of the gastric carcinogenesis.
P. Song et al. / Biomedicine & Pharmacotherapy 69 (2015) 70–75
MicroRNAs (miRNAs), a class of small non-coding singlestranded RNA molecules (1822 nucleotides), acting as gene regulators suppress protein expression by pairing to complementary sequences in the 30 -untranslated regions (UTRs) of target mRNA [6]. As important potential regulators, miRNAs are thought to be involved in diverse biological functions as well as cancer. However, naturally occurring SNPs in the miRNA target-binding site can disturb its regulation. Thus, it gives us a clue that SNPs occurring in the miRNA binding site can be candidate biomarkers, which may be a valuable indication and provide accurate prediction of cancer progression. Up to now, a number of studies have investigated the role of miR-29c and found it differentially expressed in a variety of human cancer, such as breast cancer [7], lung cancer [8,9], esophageal cancer [10], hepatocellular carcinoma [11], and gliomas [12]. As for GC, the expression of miR-29c is commonly down-regulated in tumor relative to adjacent normal tissues [13,14]. Additionally, previous studies have demonstrated that miR-29c acting as a tumor suppressor has a critical impact on the aggressive and progressive phenotypes of GC [14,15]. In the present study, we selected three SNPs within miR-29c binding sites (IGHMBP2 rs3750980, LAMTOR3 rs11944405 and WWOX rs2288035) to evaluate their association with GC risk. 2. Materials and methods
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absorbance at 260 and 280 nm. Genotyping was conducted by the TaqMan methodology in 384-well plates, and the outputs were read with the Sequence Detection Software on an ABI 7900HT instrument (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. To ensure accuracy of the genotyping, each plate included four negative controls, and two persons completed the genotype analysis independently in a blind fashion. Besides, about 10% of the samples were randomly selected to verify the first genotyping, and the results were 100% concordant. Due to DNA quality, several samples failed in genotyping, and were excluded in the further analysis. The structure of primers and probes for each SNP were listed in Table S1 (supplemental material). 2.4. Statistical analysis Hardy-Weinberg equilibrium was conducted by a goodness-offit Chi2 test to evaluate the genotype frequencies among the cancerfree controls. The Chi2 test was performed to examine the frequency distributions of demographic characteristics. The odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the association between the genotypes of the selected SNPs with GC risk from both unconditional univariate and multivariate logistic regression models. All of the statistical analyses were performed with SAS software version 9.1 (SAS Institute Inc., Cary, NC, USA) and two-sided P < 0.05 was the criterion for statistical significance.
2.1. Ethics statement 3. Results This research was approved by the institutional review board of Nanjing Medical University. All participants were voluntary and signed informed consent, and then donated approximately 5 mL venous blood and possible tissues. 2.2. Study population A total of 753 histologically confirmed gastric adenocarcinoma patients and 950 cancer-free controls participated in case-control study. They were recruited from The Second Affiliated Hospital of Nanjing Medical University (Nanjing, China), Cancer Hospital of Nantong (Nantong, China) and Yixing Cancer Hospital (Yixing, China) between March 2006 and January 2010. For GC patients, surgical-pathologic staging was according to the TNM classification by the American Joint Committee on Cancer (AJCC, 6th). Histopathology of tumors was classified into diffuse or intestinal types based on Lauren’s criteria [16]. 2.3. SNPs selection and genotyping The online software of miRNASNP (http://www.bioguo.org/ miRNASNP/) and PolymiRTS (http://compbio.uthsc.edu/miRSNP/) were used to identify the putative SNPs within miR-29c binding sites. The following criteria were included: targets gain by SNPs that located in the seed region of the miR29c binding sites; the predicted SNPs were identified by both the bioinformatics tools simultaneously; minor allelic frequency (MAF) was 5% or more in Han Chinese in Beijing (CHB).
Finally, IGHMBP2 rs3750980, LAMTOR3 rs11944405 and WWOX rs2288035 were selected as the candidate SNPs. Genomic DNA of case-control study was extracted from peripheral blood. DNA purity and concentrations in the sample were calculated by spectrophotometric measurement of
3.1. Characteristics and clinical features of subjects Characteristics and clinical features of included subjects are summarized in Table 1. There was no difference in the distribution of age and sex between cases and controls (P = 0.486 for age, P = 0.437 for sex), because of frequency matching used in the study design. Of the 753 cases, 295 (39.2%) patients had cardia GC and 458 (60.8%) patients had non-cardia GC. Among the cases, 437 (58.0%) patients were diffuse type and 316 (42.0%) were intestinal type. Besides, 26.8%, 21.9%, 35.3%, and 15.9% of patients were classified as TNM stage I, II, III, and IV, respectively. 3.2. Association of the selected SNPs with gastric cancer risk The genotype frequencies of the selected SNPs (rs3750980, rs11944405 and rs2288035) are presented in Table 2. All the three polymorphisms were in Hardy-Weinberg equilibrium among controls (P = 0.577 for rs3750980, P = 0.981 for rs11944405, and P = 0.244 for rs2288035). Two-sided Chi2 test showed that the genotype distributions of these three SNPs among cases and controls were not significantly different. We then tested the association of each SNP with GC risk using logistic regression. For rs11944405, when using the TT genotype as the reference, the TC genotype had a significantly decreased risk of GC (adjusted OR = 0.79, 95% CI = 0.63– 0.99), and the CC genotype had no significant association (adjusted OR = 0.97, 95% CI = 0.52–1.83). To improve the power for detecting the effect of variant genotypes, we combined the TC and CC genotypes and found it was also associated with a decreased risk of GC, compared to the TT genotype (adjusted OR = 0.81, 95% CI = 0.65– 1.00). However, no association with GC risk was observed for rs3750980 and rs2288035. 3.3. Stratified analysis of the associations between rs11944405 polymorphism and clinical variables of gastric cancer We analyzed the correlations between the SNP rs11944405 and GC risk stratifying by clinicopathological features. As shown in
P. Song et al. / Biomedicine & Pharmacotherapy 69 (2015) 70–75
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4. Discussion
Table 1 Demographic and clinical features of including subjects. Variables
Cases n
Age (years) 65 432 > 65 321 Sex Male 512 Female 241 Tumor site Cardia 295 Nocardia 458 Histological types Intestinal 316 Diffuse 437 b Depth of invasion T1 130 T2 130 381 T3 T4 112 Lymph node metastasis N0 297 N1/N2/N3 456 Distant metastasis M0 655 M1 98 Clinical stage I 202 II 165 III 266 IV 120
Pa
Controls %
n
%
57.4 42.6
529 421
55.7 44.3
0.486
68.0 32.0
629 321
66.2 33.8
0.437
39.2 60.8 42.0 58.0 17.3 17.3 50.6 14.8 39.4 60.6 87.0 13.0 26.8 21.9 35.3 16.0
a Two-sided Chi2 test for the frequency distributions of selected variables between cases and controls.
Table 3, the risk of GC for the TC/CC genotypes was decreased compared to the TT genotypes in the subgroups of age 65, females, T3/T4, N1-3, M0, and TNM stage III/IV (adjusted OR = 0.69, 95% CI = 0.51–0.93 for age 65; adjusted OR = 0.66, 95% CI = 0.45– 0.98 for females; adjusted OR = 0.72, 95% CI = 0.56–0.92 for T3/T4; adjusted OR = 0.73, 95% CI = 0.56–0.94 for N1-3; adjusted OR = 0.78, 95% CI = 0.63–0.99 for M0 and adjusted OR = 0.66, 95% CI = 0.50–0.88 for stage III/IV). Additionally, strong association between the rs11944405 variant genotypes and tumor TNM stage was found. Logistic regression revealed that patients carried the variant genotypes with stage III/IV had a 0.68-fold reduced risk of GC compared to patients with stage I/II (Table 4). Furthermore, the median ages of GC onset based on the genotypes of rs11944405 were calculated, and no significant differences were detected (supplemental material, Table S2).
Accumulated evidence has suggested that mutations at the miRNAs binding sites altered the interactions of miRNAs and its targets, ultimately contributing to the individual difference in susceptibility and severity of cancer [17,18]. Matsuo et al. identified miR-29c as the significantly down-regulated miRNA in gastric carcinoma relative to normal gastric epithelium by a miRNA microarray, which covers 470 human miRNAs [13]. This phenomenon that miR-29c was down-regulated in GC was proved by several studies, and the possible role of miR-29c as a tumor suppressor was proposed. Besides, a series of its target genes were reported, such as RRC2, MCL-1, B7-H3, MMP2, SIRT1, and CCND2 [7,9,11,13–15,19]. Together with the data reported previously, miR-29c played important role in the tumor development. Therefore we hypothesized that genetic variants within miR-29c binding sites can modify the risk of GC. In this study, we chose three SNPs IGHMBP2 rs3750980, LAMTOR3 rs11944405 and WWOX rs2288035, which located in miR-29c binding sties to investigate their association with the risk of GC. Among these three SNPs, our results showed that LAMTOR3 rs11944405C allele was associated with a decreased risk of GC. The stratified analysis exhibited that the protective effect of the C allele was more pronounced among subgroups of age 65, females, T3/ T4, N1-3, M0, and TNM stage III/IV. Moreover, this SNP was significantly correlated with the TNM stage, suggesting it might be related to the progression of GC. Taken together, these data indicated that the rs11944405 might be a promising susceptibility biomarker for GC. LAMTOR3 (MP1) is a scaffolding protein that functions in the extracellular signal-regulated kinase (ERK) cascade [20]. The protein is localized to late endosomes by the adaptor protein LAMTOR2, and both of them comprise a stable heterodimeric complex to active ERK [21]. Activated ERKs can translocate to the nucleus, where they regulate the gene expression. In addition to its function in MEK/ERK pathway, LAMTOR3 has been reported to be a regulator of mammalian target of rapamycin (mTOR) signaling cascade, which promotes cell growth, proliferation, migration, and apoptosis in response to growth factors, energy levels, and amino acids [21,22]. However, due to ERK and mTOR activation is downstream of many signaling pathways, the aberrant activation of ERK or mTOR may lead to human disorders including cancer [23,24]. A recent publication reported that LAMTOR3 participated in gastrin-induced cellular responses linked to proliferation and homeostasis of the gastric mucosa mainly by facilitating ERK pathway [25]. In brief, LAMTOR3 creates a core platform for both the ERK and mTOR pathways involving in tumorigenesis and acts
Table 2 Associations between the selected SNPs and gastric cancer risk. SNPs
rs3750980
rs11944405
rs2288035
a b
Genotypes
CC CT TT CT + TT TT TC CC TC + CC CC CG GG CG + GG
Cases
Controls
Call rate
Pa
(95% CI)b
n
%
n
%
%
391 314 48 362 569 166 18 184 440 281 32 313
51.9 41.7 6.4 48.1 75.6 22.0 2.4 24.4 58.4 37.3 4.3 41.6
474 402 72 474 676 250 22 272 550 357 42 399
50.0 42.4 7.6 50.0 71.3 26.4 2.3 28.7 58.0 37.6 4.4 42.0
99.8
0.538
99.8
0.430 0.119
Two-sided Chi2 test for either genotype distributions between cases and controls. Adjusted for age and sex in logistic regression model.
99.9
Adjusted OR
0.049 0.972
0.843
1.00 0.95 0.81 0.93 1.00 0.79 0.97 0.81 1.00 0.98 0.95 0.93
(reference) (0.78–1.16) (0.55–1.20) (0.77–1.12) (0.63–0.99) (0.52–1.83) (0.65–1.00) (0.81–1.20) (0.59–1.52) (0.58–1.49)
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Table 3 Stratified analysis between the rs11944405 polymorphism and gastric cancer risk. rs11944405 T > C genotypes Variables
Age (years) 65 > 65 Sex Male Female Tumor site Cardia Non-cardia Histological types Diffuse Intestinal Depth of invasion T1/T2 T3/T4 Lymph node metastasis N0 N1/N2/N3 Distant metastasis M0 M1 Clinical stage I/II III/IV a b
TT (n, %)
Pa
Adjusted OR (95% CI)b
TC/CC (n, %)
Cases
Controls
Cases
Controls
334 (77.3) 235 (73.2)
369 (69.8) 307 (73.3)
98 (22.7) 86 (26.8)
160 (30.2) 112 (26.7)
0.009 0.985
0.69 (0.51–0.93) 0.99 (0.71–1.38)
386 (75.4) 183 (75.9)
458 (73.0) 218 (67.9)
126 (24.6) 58 (24.1)
169 (27.0) 103 (32.1)
0.369 0.037
0.89 (0.68–1.17) 0.66 (0.45–0.98)
227 (76.9) 342 (74.7)
676 (71.3) 676 (71.3)
68 (23.1) 116 (25.3)
272 (28.7) 272 (28.7)
0.057 0.186
0.75 (0.55–1.01) 0.85 (0.66–1.09)
327 (74.8) 242 (76.6)
676 (71.3) 676 (71.3)
110 (25.2) 74 (23.4)
272 (28.7) 272 (28.7)
0.173 0.069
0.83 (0.64–1.08) 0.77 (0.57–1.03)
186 (71.5) 383 (77.7)
676 (71.3) 676 (71.3)
74 (28.5) 110 (22.3)
272 (28.7) 272 (28.7)
0.942 0.009
0.99 (0.73–1.34) 0.72 (0.56–0.92)
216 (72.7) 353 (77.4)
676 (71.3) 676 (71.3)
81 (27.3) 103 (22.6)
272 (28.7) 272 (28.7)
0.614 0.016
0.93 (0.70–1.25) 0.73 (0.56–0.94)
498 (76.0) 71 (72.4)
676 (71.3) 676 (71.3)
157 (24.0) 27 (27.6)
272 (28.7) 272 (28.7)
0.036 0.812
0.78 (0.63–0.99) 0.96 (0.60–1.52)
264 (71.9) 305 (79.0)
676 (71.3) 676 (71.3)
103 (28.1) 81 (21.0)
272 (28.7) 272 (28.7)
0.821 0.004
0.97 (0.74–1.27) 0.66 (0.50–0.88)
Two-sided x2 test for either genotype distributions between cases and controls. Adjusted for age and sex in logistic regression model.
as a convergence point for them. Therefore, variants in LAMTOR3 gene might modify individuals’ susceptibility to GC. Recently, two GWAS unveiled 4q23, which LAMTOR3 located in as a cancerrelated chromosomal region [26,27]. De Araujo et al. observed functional correlation between LAMTOR3 rs148972953 and the status of estrogen receptor (ER) and progesterone receptor (PR) in 296 breast cancer samples (P = 0.035 for ER, P = 0.006 for PR) [28]. Our results provided the first evidence of a genetic association between the SNP rs11944405 in LAMTOR3 and susceptibility of GC. As mention above, 30 -UTR variation affected miRNA targeting, which result in cancer susceptibility. The SNP rs11944405 T > C of LAMTOR3 gene was exactly located in the miR-29c binding site. The variant rs11944405C allele gained a better binding for miR-29c to target the 30 -UTR of LAMTOR3,
and thus suppressed its expression. This might partly explain the observation that the C allele decreased the risk of GC because of the important role of LAMTOR3 in ERK and mTOR signaling cascades. Besides, Jun et al. reported that marked up-regulation of LAMTOR3 was observed in pancreatic adenocarcinoma cells by immunestaining of a tissue microarray [21]. However, we should acknowledge that genetic mutation was not the only reason affecting the GC occurrence. As we know, many factors may affect the LAMTOR3 expression level and activity, including transcription, post-transcriptional regulation, translation, and post-translation process. When stratified by age and sex, the SNP rs11944405 presented a protective factor for GC among subgroup of younger subjects (age 65 years) and females. Younger subjects with strong immune system and better health condition seemed to more be
Table 4 Association between the SNP rs11944405 and clinical characteristics of gastric cancer. Variables
Histological types Intestinal Diffuse Depth of invasion T1/T2 T3/T4 Lymph node metastasis N0 N1/N2/N3 Distant metastasis M0 M1 TNM Stages I/II III/IV a b
Adjusted OR (95% CI)a
Genotypes
Pb
TT
TC/CC
TC/CC vs.TT
242 327
74 110
1.00 (reference) 1.14 (0.81–1.60)
0.580
186 383
74 110
1.00 (reference) 0.72 (0.51–1.02)
0.062
216 353
81 103
1.00 (reference) 0.78 (0.56–1.09)
0.144
498 71
157 27
1.00 (reference) 1.21 (0.75–1.94)
0.442
264 305
103 81
1.00 (reference) 0.68 (0.49–0.95)
0.024
Adjusted for age and sex in logistic regression model. Two-sided Chi2 test for the frequency distributions of clinical variables between TT genotype and TC/CC genotypes among gastric cancer patients.
74
P. Song et al. / Biomedicine & Pharmacotherapy 69 (2015) 70–75
sensitive to genetic effects rather than multiple environmental exposures [29,30]. We further analyzed whether the effect of rs11944405 was more prominent in early-onset gastric cancer. However, no significant difference was observed between the GC onset age and the genotypes of LAMTOR3 rs11944405. The genderspecific difference could be partially owing to steroid sex hormones, such as estrogen. Estrogen receptors were widely detected in cancer cells, and most tumor tissues expressed the estrogen receptors depend on ERK or mTOR pathway for proliferation [31–33]. Marina et al. found that LAMTOR3 was essential for the survival of estrogen receptor positive breast cancer cell lines [34]. Thus, the LAMTOR3 polymorphism might affect carcinogenesis of GC in a gender-specific manner. In all, these results needed more confirmations. Some possible limitations in this study should be acknowledged. First, the selection bias was inevitable for this hospitalbased case-control study. However, we matched the controls to case by age, sex and putative confounding factors to minimize the bias as much as possible. Second, the number of subjects in our study was moderate, especially for stratified analysis, resulting in insufficient statistical power. Third, lacking some risk factors information, such as Helicobacter pylori infection, alcohol consumption, and tobacco smoking, limited us to further analysis the potential interaction, since gene-environment can modulate cancer risk. Therefore, large sample studies with detailed risk factors information are warranted to validate our results. Fourth, the SNP rs11944405 located in the miR-29c binding sites was identified based on bioinformatics software. Further functional assay is needed to reveal their relevance. In conclusion, we provided the first evidence that the SNP rs11944405 at miR-29c binding site within the 30 -UTR of LAMTOR3 gene was associated with gastric cancer susceptibility. Our finding should be verified by larger prospective studies. Author contributions Z.Z., H.Z., Q.Z. and B.W. designed the study and applied for Research Ethics Board approval. Z.Z., H.Z, Q.Z., B.W., G.T., J.Z. and W.G. recruited the patients and collected the data. P.S. and W.Z. prepared the manuscript draft. P.S., W.Z., H.C., M.W., D.W. and N.T. carried out the data analysis. All authors approved the final article. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This study was partly supported by National Natural Science Foundation of China (81230068, 81302490, 81373091 and 81001274), Natural Science Foundation of Jiangsu Province (BK2011194, BK2012842), the Key Program for Basic Research of Jiangsu Provincial Department of Education (12KJA330002), Jiangsu Provincial Science and Technology Innovation Team, and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biopha.2014.11.008.
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Original article
A miR-29c binding site genetic variant in the 30 -untranslated region of LAMTOR3 gene is associated with gastric cancer risk Peng Song a,1, Weizhi Wang b,1, Guoquan Tao c,1, Haiyan Chu d,e, Meilin Wang d,e, Dongmei Wu d,e, Na Tong d,e, Weida Gong f, Jianwei Zhou g, Zhengdong Zhang d,e, Baolin Wang a,*, Haixia Zhu h,**, Qinghong Zhao a,* a
Department of General Surgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China c Department of General Surgery, Huai-An First People’s Hospital Affiliated to Nanjing Medical University, Huai-an,, China d Department of Environmental Genomics, Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, Nanjing Medical University, Nanjing, China e Department of Genetic Toxicology, The Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, China f Department of General Surgery, Yixing Cancer Hospital, Yixing, China g Department of Molecular Cell Biology & Toxicology, School of Public Health, Cancer Center, Nanjing Medical University, Nanjing, China h Core Laboratory of Nantong Tumor Hospital, Nantong, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 October 2014 Accepted 5 November 2014
Single nucleotide polymorphisms (SNPs) in the 30 -untranslated regions (UTRs) targeted by putative mircoRNAs (miRNAs) could influence the susceptibility of cancer. Recently, miR-29c has been reported to be down-regulated in gastric cancer (GC) and serve as a tumor suppressor that regulated tumor progression. The present study was aimed at investigating whether the miR-29c binding site SNPs within the 30 -UTRs of target genes affected the gastric cancer risk. Using bioinformatics tools, we chose three SNPs (IGHMBP2 rs3750980, LAMTOR3 rs11944405 and WWOX rs2288035) located in miR-29c binding sites. We genotyped these three SNPs to assess their associations with GC risk in a case-control study comprising 753 GC cases and 950 controls. Among these three SNPs, we found a significantly decreased risk of GC associated with the LAMTOR3 rs11944405 T > C polymorphism [TC vs. TT, adjusted odds ratio (OR) = 0.79, 95% confidence interval (CI) = 0.63–0.99; TC/CC vs. TT, adjusted OR = 0.81, 95% CI = 0.65–1.00]. The significant association was also presented in the subgroup analysis by age ( 65), sex (female), depth of invasion (T3/T4), lymph node metastasis (N1-3), distant metastasis (M0) and TNM stage (III/IV). However, no significant association was detected for IGHMBP2 rs3750980 and WWOX rs2288035. Our results suggested that the LAMTOR3 rs11944405 polymorphism may be a potential biomarker for genetic susceptibility to GC. ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Single nucleotide polymorphism LAMTOR3 miR-29c Gastric cancer Genetic susceptibility
1. Introduction Gastric cancer (GC) remains one of the most highly aggressive and lethal neoplasms worldwide, with highest incidence rates in Eastern Asia, Eastern Europe, and South America. Although * Corresponding authors. Department of General Surgery, The Second Affiliated Hospital of Nanjing Medical University, 121, Jiangjiayuan Road, Nanjing 210011, China. Tel.: +86 25 58509832; fax: +86 25 58509994. ** Corresponding author. Core Laboratory of Nantong Tumor Hospital, 48, West Qingnian Road, Nantong 226006, China. E-mail addresses: [email protected] (B. Wang), [email protected] (H. Zhu), [email protected] (Q. Zhao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biopha.2014.11.008 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
stomach cancer rates have decreased substantially in most parts of the world, it is estimated that nearly one million new cases and over 700,000 deaths have happened each year [1]. Accumulating evidence has indicated that multiple risk factor exposures (i.e., Helicobacter pylori infection, alcohol consumption, tobacco smoking, diet poor in fresh fruits and vegetables or rich in salt and nitrate) and genetic variations play important roles in the process of GC [2–4]. With the development of molecular genetics in recent decades, single nucleotide polymorphisms (SNPs) in apoptosis, inflammatory, immune response, metabolism, tumor-related genes are reported to associate with different susceptibility to this malignancy [5]. Identifying new susceptibility loci for GC advances our understanding of the gastric carcinogenesis.
P. Song et al. / Biomedicine & Pharmacotherapy 69 (2015) 70–75
MicroRNAs (miRNAs), a class of small non-coding singlestranded RNA molecules (1822 nucleotides), acting as gene regulators suppress protein expression by pairing to complementary sequences in the 30 -untranslated regions (UTRs) of target mRNA [6]. As important potential regulators, miRNAs are thought to be involved in diverse biological functions as well as cancer. However, naturally occurring SNPs in the miRNA target-binding site can disturb its regulation. Thus, it gives us a clue that SNPs occurring in the miRNA binding site can be candidate biomarkers, which may be a valuable indication and provide accurate prediction of cancer progression. Up to now, a number of studies have investigated the role of miR-29c and found it differentially expressed in a variety of human cancer, such as breast cancer [7], lung cancer [8,9], esophageal cancer [10], hepatocellular carcinoma [11], and gliomas [12]. As for GC, the expression of miR-29c is commonly down-regulated in tumor relative to adjacent normal tissues [13,14]. Additionally, previous studies have demonstrated that miR-29c acting as a tumor suppressor has a critical impact on the aggressive and progressive phenotypes of GC [14,15]. In the present study, we selected three SNPs within miR-29c binding sites (IGHMBP2 rs3750980, LAMTOR3 rs11944405 and WWOX rs2288035) to evaluate their association with GC risk. 2. Materials and methods
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absorbance at 260 and 280 nm. Genotyping was conducted by the TaqMan methodology in 384-well plates, and the outputs were read with the Sequence Detection Software on an ABI 7900HT instrument (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. To ensure accuracy of the genotyping, each plate included four negative controls, and two persons completed the genotype analysis independently in a blind fashion. Besides, about 10% of the samples were randomly selected to verify the first genotyping, and the results were 100% concordant. Due to DNA quality, several samples failed in genotyping, and were excluded in the further analysis. The structure of primers and probes for each SNP were listed in Table S1 (supplemental material). 2.4. Statistical analysis Hardy-Weinberg equilibrium was conducted by a goodness-offit Chi2 test to evaluate the genotype frequencies among the cancerfree controls. The Chi2 test was performed to examine the frequency distributions of demographic characteristics. The odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the association between the genotypes of the selected SNPs with GC risk from both unconditional univariate and multivariate logistic regression models. All of the statistical analyses were performed with SAS software version 9.1 (SAS Institute Inc., Cary, NC, USA) and two-sided P < 0.05 was the criterion for statistical significance.
2.1. Ethics statement 3. Results This research was approved by the institutional review board of Nanjing Medical University. All participants were voluntary and signed informed consent, and then donated approximately 5 mL venous blood and possible tissues. 2.2. Study population A total of 753 histologically confirmed gastric adenocarcinoma patients and 950 cancer-free controls participated in case-control study. They were recruited from The Second Affiliated Hospital of Nanjing Medical University (Nanjing, China), Cancer Hospital of Nantong (Nantong, China) and Yixing Cancer Hospital (Yixing, China) between March 2006 and January 2010. For GC patients, surgical-pathologic staging was according to the TNM classification by the American Joint Committee on Cancer (AJCC, 6th). Histopathology of tumors was classified into diffuse or intestinal types based on Lauren’s criteria [16]. 2.3. SNPs selection and genotyping The online software of miRNASNP (http://www.bioguo.org/ miRNASNP/) and PolymiRTS (http://compbio.uthsc.edu/miRSNP/) were used to identify the putative SNPs within miR-29c binding sites. The following criteria were included: targets gain by SNPs that located in the seed region of the miR29c binding sites; the predicted SNPs were identified by both the bioinformatics tools simultaneously; minor allelic frequency (MAF) was 5% or more in Han Chinese in Beijing (CHB).
Finally, IGHMBP2 rs3750980, LAMTOR3 rs11944405 and WWOX rs2288035 were selected as the candidate SNPs. Genomic DNA of case-control study was extracted from peripheral blood. DNA purity and concentrations in the sample were calculated by spectrophotometric measurement of
3.1. Characteristics and clinical features of subjects Characteristics and clinical features of included subjects are summarized in Table 1. There was no difference in the distribution of age and sex between cases and controls (P = 0.486 for age, P = 0.437 for sex), because of frequency matching used in the study design. Of the 753 cases, 295 (39.2%) patients had cardia GC and 458 (60.8%) patients had non-cardia GC. Among the cases, 437 (58.0%) patients were diffuse type and 316 (42.0%) were intestinal type. Besides, 26.8%, 21.9%, 35.3%, and 15.9% of patients were classified as TNM stage I, II, III, and IV, respectively. 3.2. Association of the selected SNPs with gastric cancer risk The genotype frequencies of the selected SNPs (rs3750980, rs11944405 and rs2288035) are presented in Table 2. All the three polymorphisms were in Hardy-Weinberg equilibrium among controls (P = 0.577 for rs3750980, P = 0.981 for rs11944405, and P = 0.244 for rs2288035). Two-sided Chi2 test showed that the genotype distributions of these three SNPs among cases and controls were not significantly different. We then tested the association of each SNP with GC risk using logistic regression. For rs11944405, when using the TT genotype as the reference, the TC genotype had a significantly decreased risk of GC (adjusted OR = 0.79, 95% CI = 0.63– 0.99), and the CC genotype had no significant association (adjusted OR = 0.97, 95% CI = 0.52–1.83). To improve the power for detecting the effect of variant genotypes, we combined the TC and CC genotypes and found it was also associated with a decreased risk of GC, compared to the TT genotype (adjusted OR = 0.81, 95% CI = 0.65– 1.00). However, no association with GC risk was observed for rs3750980 and rs2288035. 3.3. Stratified analysis of the associations between rs11944405 polymorphism and clinical variables of gastric cancer We analyzed the correlations between the SNP rs11944405 and GC risk stratifying by clinicopathological features. As shown in
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4. Discussion
Table 1 Demographic and clinical features of including subjects. Variables
Cases n
Age (years) 65 432 > 65 321 Sex Male 512 Female 241 Tumor site Cardia 295 Nocardia 458 Histological types Intestinal 316 Diffuse 437 b Depth of invasion T1 130 T2 130 381 T3 T4 112 Lymph node metastasis N0 297 N1/N2/N3 456 Distant metastasis M0 655 M1 98 Clinical stage I 202 II 165 III 266 IV 120
Pa
Controls %
n
%
57.4 42.6
529 421
55.7 44.3
0.486
68.0 32.0
629 321
66.2 33.8
0.437
39.2 60.8 42.0 58.0 17.3 17.3 50.6 14.8 39.4 60.6 87.0 13.0 26.8 21.9 35.3 16.0
a Two-sided Chi2 test for the frequency distributions of selected variables between cases and controls.
Table 3, the risk of GC for the TC/CC genotypes was decreased compared to the TT genotypes in the subgroups of age 65, females, T3/T4, N1-3, M0, and TNM stage III/IV (adjusted OR = 0.69, 95% CI = 0.51–0.93 for age 65; adjusted OR = 0.66, 95% CI = 0.45– 0.98 for females; adjusted OR = 0.72, 95% CI = 0.56–0.92 for T3/T4; adjusted OR = 0.73, 95% CI = 0.56–0.94 for N1-3; adjusted OR = 0.78, 95% CI = 0.63–0.99 for M0 and adjusted OR = 0.66, 95% CI = 0.50–0.88 for stage III/IV). Additionally, strong association between the rs11944405 variant genotypes and tumor TNM stage was found. Logistic regression revealed that patients carried the variant genotypes with stage III/IV had a 0.68-fold reduced risk of GC compared to patients with stage I/II (Table 4). Furthermore, the median ages of GC onset based on the genotypes of rs11944405 were calculated, and no significant differences were detected (supplemental material, Table S2).
Accumulated evidence has suggested that mutations at the miRNAs binding sites altered the interactions of miRNAs and its targets, ultimately contributing to the individual difference in susceptibility and severity of cancer [17,18]. Matsuo et al. identified miR-29c as the significantly down-regulated miRNA in gastric carcinoma relative to normal gastric epithelium by a miRNA microarray, which covers 470 human miRNAs [13]. This phenomenon that miR-29c was down-regulated in GC was proved by several studies, and the possible role of miR-29c as a tumor suppressor was proposed. Besides, a series of its target genes were reported, such as RRC2, MCL-1, B7-H3, MMP2, SIRT1, and CCND2 [7,9,11,13–15,19]. Together with the data reported previously, miR-29c played important role in the tumor development. Therefore we hypothesized that genetic variants within miR-29c binding sites can modify the risk of GC. In this study, we chose three SNPs IGHMBP2 rs3750980, LAMTOR3 rs11944405 and WWOX rs2288035, which located in miR-29c binding sties to investigate their association with the risk of GC. Among these three SNPs, our results showed that LAMTOR3 rs11944405C allele was associated with a decreased risk of GC. The stratified analysis exhibited that the protective effect of the C allele was more pronounced among subgroups of age 65, females, T3/ T4, N1-3, M0, and TNM stage III/IV. Moreover, this SNP was significantly correlated with the TNM stage, suggesting it might be related to the progression of GC. Taken together, these data indicated that the rs11944405 might be a promising susceptibility biomarker for GC. LAMTOR3 (MP1) is a scaffolding protein that functions in the extracellular signal-regulated kinase (ERK) cascade [20]. The protein is localized to late endosomes by the adaptor protein LAMTOR2, and both of them comprise a stable heterodimeric complex to active ERK [21]. Activated ERKs can translocate to the nucleus, where they regulate the gene expression. In addition to its function in MEK/ERK pathway, LAMTOR3 has been reported to be a regulator of mammalian target of rapamycin (mTOR) signaling cascade, which promotes cell growth, proliferation, migration, and apoptosis in response to growth factors, energy levels, and amino acids [21,22]. However, due to ERK and mTOR activation is downstream of many signaling pathways, the aberrant activation of ERK or mTOR may lead to human disorders including cancer [23,24]. A recent publication reported that LAMTOR3 participated in gastrin-induced cellular responses linked to proliferation and homeostasis of the gastric mucosa mainly by facilitating ERK pathway [25]. In brief, LAMTOR3 creates a core platform for both the ERK and mTOR pathways involving in tumorigenesis and acts
Table 2 Associations between the selected SNPs and gastric cancer risk. SNPs
rs3750980
rs11944405
rs2288035
a b
Genotypes
CC CT TT CT + TT TT TC CC TC + CC CC CG GG CG + GG
Cases
Controls
Call rate
Pa
(95% CI)b
n
%
n
%
%
391 314 48 362 569 166 18 184 440 281 32 313
51.9 41.7 6.4 48.1 75.6 22.0 2.4 24.4 58.4 37.3 4.3 41.6
474 402 72 474 676 250 22 272 550 357 42 399
50.0 42.4 7.6 50.0 71.3 26.4 2.3 28.7 58.0 37.6 4.4 42.0
99.8
0.538
99.8
0.430 0.119
Two-sided Chi2 test for either genotype distributions between cases and controls. Adjusted for age and sex in logistic regression model.
99.9
Adjusted OR
0.049 0.972
0.843
1.00 0.95 0.81 0.93 1.00 0.79 0.97 0.81 1.00 0.98 0.95 0.93
(reference) (0.78–1.16) (0.55–1.20) (0.77–1.12) (0.63–0.99) (0.52–1.83) (0.65–1.00) (0.81–1.20) (0.59–1.52) (0.58–1.49)
P. Song et al. / Biomedicine & Pharmacotherapy 69 (2015) 70–75
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Table 3 Stratified analysis between the rs11944405 polymorphism and gastric cancer risk. rs11944405 T > C genotypes Variables
Age (years) 65 > 65 Sex Male Female Tumor site Cardia Non-cardia Histological types Diffuse Intestinal Depth of invasion T1/T2 T3/T4 Lymph node metastasis N0 N1/N2/N3 Distant metastasis M0 M1 Clinical stage I/II III/IV a b
TT (n, %)
Pa
Adjusted OR (95% CI)b
TC/CC (n, %)
Cases
Controls
Cases
Controls
334 (77.3) 235 (73.2)
369 (69.8) 307 (73.3)
98 (22.7) 86 (26.8)
160 (30.2) 112 (26.7)
0.009 0.985
0.69 (0.51–0.93) 0.99 (0.71–1.38)
386 (75.4) 183 (75.9)
458 (73.0) 218 (67.9)
126 (24.6) 58 (24.1)
169 (27.0) 103 (32.1)
0.369 0.037
0.89 (0.68–1.17) 0.66 (0.45–0.98)
227 (76.9) 342 (74.7)
676 (71.3) 676 (71.3)
68 (23.1) 116 (25.3)
272 (28.7) 272 (28.7)
0.057 0.186
0.75 (0.55–1.01) 0.85 (0.66–1.09)
327 (74.8) 242 (76.6)
676 (71.3) 676 (71.3)
110 (25.2) 74 (23.4)
272 (28.7) 272 (28.7)
0.173 0.069
0.83 (0.64–1.08) 0.77 (0.57–1.03)
186 (71.5) 383 (77.7)
676 (71.3) 676 (71.3)
74 (28.5) 110 (22.3)
272 (28.7) 272 (28.7)
0.942 0.009
0.99 (0.73–1.34) 0.72 (0.56–0.92)
216 (72.7) 353 (77.4)
676 (71.3) 676 (71.3)
81 (27.3) 103 (22.6)
272 (28.7) 272 (28.7)
0.614 0.016
0.93 (0.70–1.25) 0.73 (0.56–0.94)
498 (76.0) 71 (72.4)
676 (71.3) 676 (71.3)
157 (24.0) 27 (27.6)
272 (28.7) 272 (28.7)
0.036 0.812
0.78 (0.63–0.99) 0.96 (0.60–1.52)
264 (71.9) 305 (79.0)
676 (71.3) 676 (71.3)
103 (28.1) 81 (21.0)
272 (28.7) 272 (28.7)
0.821 0.004
0.97 (0.74–1.27) 0.66 (0.50–0.88)
Two-sided x2 test for either genotype distributions between cases and controls. Adjusted for age and sex in logistic regression model.
as a convergence point for them. Therefore, variants in LAMTOR3 gene might modify individuals’ susceptibility to GC. Recently, two GWAS unveiled 4q23, which LAMTOR3 located in as a cancerrelated chromosomal region [26,27]. De Araujo et al. observed functional correlation between LAMTOR3 rs148972953 and the status of estrogen receptor (ER) and progesterone receptor (PR) in 296 breast cancer samples (P = 0.035 for ER, P = 0.006 for PR) [28]. Our results provided the first evidence of a genetic association between the SNP rs11944405 in LAMTOR3 and susceptibility of GC. As mention above, 30 -UTR variation affected miRNA targeting, which result in cancer susceptibility. The SNP rs11944405 T > C of LAMTOR3 gene was exactly located in the miR-29c binding site. The variant rs11944405C allele gained a better binding for miR-29c to target the 30 -UTR of LAMTOR3,
and thus suppressed its expression. This might partly explain the observation that the C allele decreased the risk of GC because of the important role of LAMTOR3 in ERK and mTOR signaling cascades. Besides, Jun et al. reported that marked up-regulation of LAMTOR3 was observed in pancreatic adenocarcinoma cells by immunestaining of a tissue microarray [21]. However, we should acknowledge that genetic mutation was not the only reason affecting the GC occurrence. As we know, many factors may affect the LAMTOR3 expression level and activity, including transcription, post-transcriptional regulation, translation, and post-translation process. When stratified by age and sex, the SNP rs11944405 presented a protective factor for GC among subgroup of younger subjects (age 65 years) and females. Younger subjects with strong immune system and better health condition seemed to more be
Table 4 Association between the SNP rs11944405 and clinical characteristics of gastric cancer. Variables
Histological types Intestinal Diffuse Depth of invasion T1/T2 T3/T4 Lymph node metastasis N0 N1/N2/N3 Distant metastasis M0 M1 TNM Stages I/II III/IV a b
Adjusted OR (95% CI)a
Genotypes
Pb
TT
TC/CC
TC/CC vs.TT
242 327
74 110
1.00 (reference) 1.14 (0.81–1.60)
0.580
186 383
74 110
1.00 (reference) 0.72 (0.51–1.02)
0.062
216 353
81 103
1.00 (reference) 0.78 (0.56–1.09)
0.144
498 71
157 27
1.00 (reference) 1.21 (0.75–1.94)
0.442
264 305
103 81
1.00 (reference) 0.68 (0.49–0.95)
0.024
Adjusted for age and sex in logistic regression model. Two-sided Chi2 test for the frequency distributions of clinical variables between TT genotype and TC/CC genotypes among gastric cancer patients.
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P. Song et al. / Biomedicine & Pharmacotherapy 69 (2015) 70–75
sensitive to genetic effects rather than multiple environmental exposures [29,30]. We further analyzed whether the effect of rs11944405 was more prominent in early-onset gastric cancer. However, no significant difference was observed between the GC onset age and the genotypes of LAMTOR3 rs11944405. The genderspecific difference could be partially owing to steroid sex hormones, such as estrogen. Estrogen receptors were widely detected in cancer cells, and most tumor tissues expressed the estrogen receptors depend on ERK or mTOR pathway for proliferation [31–33]. Marina et al. found that LAMTOR3 was essential for the survival of estrogen receptor positive breast cancer cell lines [34]. Thus, the LAMTOR3 polymorphism might affect carcinogenesis of GC in a gender-specific manner. In all, these results needed more confirmations. Some possible limitations in this study should be acknowledged. First, the selection bias was inevitable for this hospitalbased case-control study. However, we matched the controls to case by age, sex and putative confounding factors to minimize the bias as much as possible. Second, the number of subjects in our study was moderate, especially for stratified analysis, resulting in insufficient statistical power. Third, lacking some risk factors information, such as Helicobacter pylori infection, alcohol consumption, and tobacco smoking, limited us to further analysis the potential interaction, since gene-environment can modulate cancer risk. Therefore, large sample studies with detailed risk factors information are warranted to validate our results. Fourth, the SNP rs11944405 located in the miR-29c binding sites was identified based on bioinformatics software. Further functional assay is needed to reveal their relevance. In conclusion, we provided the first evidence that the SNP rs11944405 at miR-29c binding site within the 30 -UTR of LAMTOR3 gene was associated with gastric cancer susceptibility. Our finding should be verified by larger prospective studies. Author contributions Z.Z., H.Z., Q.Z. and B.W. designed the study and applied for Research Ethics Board approval. Z.Z., H.Z, Q.Z., B.W., G.T., J.Z. and W.G. recruited the patients and collected the data. P.S. and W.Z. prepared the manuscript draft. P.S., W.Z., H.C., M.W., D.W. and N.T. carried out the data analysis. All authors approved the final article. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This study was partly supported by National Natural Science Foundation of China (81230068, 81302490, 81373091 and 81001274), Natural Science Foundation of Jiangsu Province (BK2011194, BK2012842), the Key Program for Basic Research of Jiangsu Provincial Department of Education (12KJA330002), Jiangsu Provincial Science and Technology Innovation Team, and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biopha.2014.11.008.
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