Genetic polymorphisms of IL17A and pri-microRNA-938, targeting IL17A 3′-UTR, influence susceptibility to gastric cancer

Human Immunology 73 (2012) 747–752

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Genetic polymorphisms of IL17A and pri-microRNA-938, targeting IL17A 30 -UTR, influence susceptibility to gastric cancer Tomiyasu Arisawa a,⇑, Tomomitsu Tahara b, Hisakazu Shiroeda a, Yasuhito Matsue a, Takahiro Minato a, Tomoe Nomura a, Hideto Yamada a, Ranji Hayashi a, Takashi Saito a, Kazuhiro Matsunaga a, Tomoki Fukuyama a, Nobuhiko Hayashi a, Toshimi Otsuka a, Atsushi Fukumura a, Masakatsu Nakamura a, Tomoyuki Shibata b a b

Department of Gastroenterology, Kanazawa Medical University, 1-1, Daigaku, Uchinada-machi, Ishikawa 920-0293, Japan Department of Gastroenterology, Fujita Health University, 1-98, Dengakugakubo, Kutsukake-cho, Toyoake 470-1192, Japan

a r t i c l e

i n f o

Article history: Received 5 January 2012 Accepted 16 April 2012 Available online 23 April 2012

a b s t r a c t We report an association between gastric cancer (GC) and polymorphisms in IL17A, rs2275913 (197 G > A), rs3748067 (⁄1249 C > T), and pri-miR-938, rs2505901 (T > C). We employed the multiplex PCR-SSCP method to detect gene polymorphisms in 337 GC cases and 587 controls. The minor allele frequency of rs2275913 was significantly higher, and those of rs3748067 and rs2505901 significantly lower, in GC cases than controls. The rs2275913 AA homozygote was associated with an increased risk (OR, 2.38; 95%CI, 1.63–3.46; p < 0.0001) for the development of both intestinal and diffuse types of GC. The rs3748067 T polymorphism was associated with a decreased risk for intestinal GC (OR, 0.511; 95%CI, 0.272–0.962; p = 0.037), whereas rs2505901 C locus carried a decreased risk overall for GC (OR, 0.733; 95%CI, 0.545–0.985; p = 0.039). In addition, rs3748067 T allele was inversely correlated with lymph node metastasis. Our results suggest that polymorphisms in both IL17A and pri-miR-938 contribute to cancer risk susceptibility and therefore can affect the development of gastric cancer. Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

1. Introduction Gastric cancer is one of the most common cancers worldwide, but the etiology of this tumor remains unclear. Helicobacter pylori (H. pylori) infection is now accepted as a crucial event in the development of atrophic gastritis and is implicated in the development of gastric carcinoma, especially those not located in cardia [1–5]. However, there is marked variation in the extent of gastric inflammation among H. pylori-infected patients, and only a small percentage of them actually develop gastric cancer. Although bacterial virulence factors are associated with enhanced inflammation and cancer development [6,7], it is unclear why a considerable proportion of patients infected with cagA+ H. pylori strains do not develop severe pathologies. This suggests that genetic factors play an important role in the long-term outcome of H. pylori infection [8–12]. Interleukin-17 (IL-17) is a relatively recently described cytokine that bridges the adaptive and innate immune systems. IL-17A is a member of the IL-17 cytokine family responsible for the pathogenic activity of Th17 cells [13], a distinct lineage of CD4+ effector cells ⇑ Corresponding author. Fax: +81 76 286 0892. E-mail address: [email protected] (T. Arisawa).

[14], and induces multiple proinflammatory mediators, including chemokines, cytokines, and metalloproteinases, from epithelial and fibroblast cells [15]. It has been demonstrated that both IL-17 RNA transcripts and protein are expressed at a higher level in whole gastric mucosal and lamina propria mononuclear cell samples from H. pylori-infected patients than in those from uninfected subjects [16]. Thus, an important role of IL-17 in the inflammatory response to H. pylori colonization has been indicated [16,17]. We have already reported that the IL-17A gene (IL17A) polymorphism (rs2275913 G > A) and IL-17F gene (IL17F) polymorphism (rs763780 T > C) are closely associated with susceptibility to gastric carcinogenesis [18] and ulcerative colitis [19]. Several studies on the association between rs2275913 and rheumatoid arthritis, gastric carcinogenesis and asthma were subsequently instigated [20–22]. However, microRNAs are now also considered important in disease susceptibility due to their ability to regulate mRNA expression post-transcriptionally by binding to the 30 -UTR of targeted genes. In the IL17A 30 -UTR, there is a polymorphism rs3748067 (⁄1249 C > T) and many, potentially strong, binding sites for microRNAs including microRNA938 (miR-938). In addition, polymorphism (rs2505901 T > C) in pri-miR-938 coding region is in linkage disequilibrium with rs12416605, which changes the seeding sequence of miR-938 (UGU[G/A]CCC).

0198-8859/$36.00 - see front matter Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humimm.2012.04.011

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We report herein an investigation into the association of IL17A and pri-miR938 polymorphism with susceptibility to gastric carcinogenesis. 2. Subjects and methods 2.1. Clinical samples Our gastric cancer group included 337 patients (GC cases), enrolled at the Endoscopy Center of Fujita Health University Hospital or Kanazawa Medical University Hospital between July 2006 and December 2009. One hundred and ninety six patients, who consented to further genetic analysis, were recruited from our previous study [18]. As a control group, 587 subjects without malignant neoplasm, confirmed endoscopically and histologically, were selected at random from our DNA biobank, collected over the same period as that defined above (controls). Our final study cohort comprised 924 subjects for whom at least one polymorphism could be clearly analyzed. All subjects underwent upper gastrointestinal endoscopy and patients with severe systemic diseases, malignancies in other organs, and who had received nonsteroidal anti-inflammatory drugs, antibiotics, and H. pylori eradication treatment were excluded. H. pylori infection status was assessed by serology, histological examination, or the urea breath test. Patients were diagnosed as having infection when at least one of the diagnostic tests was positive. The Ethics Committees of Fujita Health University and Kanazawa Medical University approved the protocol, and written informed consent was obtained from all of the participating subjects. 2.2. Genotyping of polymorphisms DNA was isolated from biopsy specimens or peripheral blood and genotyped using the PCR-SSCP method as reported previously [18,19]. The following primers were used to detect rs3748067 C > T and rs2505901 T > C, (1249F: 50 -cccctcagagatcaacagaccaaca-30 and 1249R: 50 -gcgaaaatggttacgatgtgaaacttg-30 , and miR938F: 50 -gtgtatg taatacctctgagcctttgggg-30 and miR938R: 50 -aacatgccgtacctgcttaacctgcctt-30 , respectively). A one-tube multiplex PCR was carried out in a volume of 20 lL containing 0.1 lg of genomic DNA. The DNA was denatured at 95 °C for 3 min, followed by 35 cycles at 95 °C for 30 s, 52 °C for 40 s, and 72 °C for 45 s, with final extension

at 72 °C for 5 min. Thereafter, 2 lL of the PCR product was denatured with 10 lL of formamide (Sigma–Aldrich Co., St. Louis, USA) at 90 °C for 5 min. SSCP was carried out at 18 °C using a GenePhor DNA separation system with GeneGel Excel 12.5/24 (Amersham Biosciences Corp., USA), after which the denatured single strand DNA bands were detected using a DNA Silver Staining Kit (Amersham Biosciences Corp.). The following primers were used to detect rs2275913 G > A, (IL17AF: 50 -aacaagtaagaatgaaaagaggacatggt-30 and IL17AR: 50 -ccccc aatgaggtcatagaagaatc-30 ). PCR was carried out in a volume of 20 lL containing 0.1 lg of genomic DNA. The DNA was denatured at 96 °C for 90 s, followed by 35 cycles at 96 °C for 15 s, 58 °C for 30 s, and 72 °C for 45 s, with final extension at 72 °C for 3 min. Thereafter, SSCP was carried out at 6 °C in the same manner as described above. 2.3. Statistical analysis Data is expressed as mean ± SD. Mean ages between GC cases and controls were compared using Student’s t-test. The ratios of male/female and H. pylori infection were compared between 2 groups using a 2  2 table and the Fisher’s exact test. Allele and genotype frequencies were calculated by direct counting. The allele counts and genotype distribution were also compared by Fisher’s exact test. Furthermore, the strength of association between genotype frequencies and the disease was assessed by calculating the odds ratio (OR) at 95% confidence intervals (CI). Adjusted ORs were calculated with the use of logistical regression analysis after adjustment for age, gender and H. pylori infection status. The association of genotypes with the progression of gastric cancer was assessed by ANCOVA using the number of allele as a co-variate. When setting a = 0.05, the b value was calculated by post-hoc analysis. For all analyses, the level of significance was set at p < 0.05. 3. Results 3.1. Subject characteristics and genotype frequencies As shown in Fig. 1, single strand DNAs was clearly separated by SSCP. In controls, rs2275913 and rs2505901 were in Hardy– Weinberg equilibrium (p = 0.093 and 1.0, respectively), whereas rs3748067 was not. The mean age of controls was significantly lower than that in GC cases (Table 1).

Fig. 1. PCR-SSCP using clinical samples. Single strand DNAs were clearly separated by SSCP.

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T. Arisawa et al. / Human Immunology 73 (2012) 747–752 Table 1 Subject characteristics and genotype frequencies.

a b c d

Controls

GC cases

p value <0.0001a <0.0001b <0.0001b

The number of subjects Mean years ± SD Male:female HP positive rate IL17A rs2275913 G > A GG GA AA (Unknown) A allele frequency

587 61.4 ± 13.7 314: 273 57.2%

337 65.3 ± 11.4 234: 103 85.8%

218 293 72 4 37.5%

112 137 84 4 45.8%

IL17A rs3748067 C > T CC CT TT (Unknown) T allele frequency

494 56 23 14 9.77%

298 22 7 10 5.50%

0.033c

pri-miR938 rs2505901 T > C TT TC CC (Unknown) C allele frequency

324 215 35 13 24.8%

211 112 10 4 19.8%

0.042c

<0.0001c 0.0005d

0.0097d

0.039c 0.015d

Student’s t-test. Fisher’s exact test. The deviation of genotype distribution, GC cases vs. controls. The frequency of minor allele, GC cases vs. controls.

The minor allele frequency of rs2275913 was significantly higher, but those of rs3748067 and rs2505901 were significantly lower, in GC cases than in controls (p = 0.0005, 1-bpower = 0.928; p = 0.0097, 1-bpower = 0.896; p = 0.015, 1-bpower = 0.679, respectively). The genotype frequency of the 197 AA homozygote was significantly higher in GC cases than controls (p < 0.0001, 1-bpower = 0.997). The wild type homozygote frequencies of rs3748067 and rs2505901 were significantly higher in GC cases than controls (p = 0.033, 1-bpower = 0.581 and p = 0.042, 1-bpower = 0.52, respectively), whereas the frequency of the rs2505901 minor allele homozygote was significantly lower in GC cases than controls (p = 0.033, 1-bpower = 0.544, Table 1). 3.2. The association between gene polymorphisms and gastric carcinogenesis The rs2275913 AA homozygote was strongly associated with susceptibility to gastric carcinogenesis (OR, 2.38; 95%CI, 1.63–3.46; p < 0.0001), both intestinal and diffuse types of cancer (OR, 2.48; 95%CI, 1.60–3.84; p < 0.0001 and OR, 2.12; 95%CI, 1.30–3.44;

p = 0.026, respectively, Table 2). The rs3748067 T polymorphism carried a decreased risk for the development of intestinal gastric cancer (OR, 0.511; 95%CI, 0.272–0.962; p = 0.037), although this genotype was also weakly associated with gastric cancer overall (p = 0.096, Table 2). Conversely, the rs2505901 C genotype was associated with a decreased risk for the development of gastric cancer overall (OR, 0.733; 95%CI, 0.545–0.985; p = 0.039), although this genotype was weakly associated with both intestinal and diffuse types of gastric cancer (p = 0.078 and 0.092, respectively). In subjects younger than 60 years old, the rs2275913 AA homozygote conferred an increased risk for the development of gastric cancer (OR, 3.20; 95%CI, 1.56–6.57; p = 0.0016), whereas rs3748067 and rs2505901 were not associated with gastric carcinogenesis at all (Table 3). In subjects older than 60 years of age, the rs2275913 AA homozygote was associated with an increased risk for gastric carcinogenesis (OR, 2.20; 95%CI, 1.40–3.44; p = 0.0006). The rs3748067 T genotype carried a decreased risk for gastric carcinogenesis (OR, 0.499; 95%CI, 0.269–0.926; p = 0.027), although the rs2505901 C genotype was weakly associated with gastric carcinogenesis (p = 0.067). 3.3. The association between genetic polymorphisms and the progression of gastric cancer We further investigated the influence of genetic polymorphism on the progression of gastric cancer. According to the UICC (Unio Internationalis Contra Cancrum) classification ver.6, gastric cancer cases at stage 0–I were classified as early GC cases and those at stage II–IV classified as advanced GC cases. Thus, in our study, early GC and advanced GC cases consisted of 197 and 132 cases, respectively (unknown: 8 cases). The minor allele rs2275913 was significantly correlated, and those of rs3748067 and rs2505901 were inversely correlated, to the progression of gastric cancer by ANCOVA (p < 0.0001, p = 0.0070 and 0.019, respectively, Fig. 2). However, the genotype distribution of rs3748067 was different in early and advanced GC cases. Hence, the association of this polymorphism with staging factors was investigated. The minor allele rs3748067 was inversely correlated to both depth of cancer and lymph node metastasis (p = 0.012 and 0.0055 by ANCOVA, respectively, Fig. 3). When compared among the patients with gastric cancers at early and advanced stages, it was inversely correlated to lymph node metastasis, but not to the depth of cancer. 4. Discussion We have previously reported that the rs2275913 AA homozygote is associated with an increased risk for the development of

Table 2 Association between genetic polymorphisms and gastric cancer. IL17A rs2275913 G > A

GG

GA

AA

AA vs. G carrier; OR (95%CI)

Controls (583) Overall GC cases (333) Intestinal (199) Diffuse (134) IL17A rs3748067 C > T Controls (573) Overall GC cases (327) Intestinal (193) Diffuse (131) (Unknown) miR938 rs2505901 T > C Controls (574) Overall GC cases (333) Intestinal (196) Diffuse (134) (Unknown)

218 112 64 48 CC 494 298 179 117 2 TT 324 211 124 85 2

293 137 82 55 CT 56 22 10 11 1 TC 215 112 65 46 1

72 84 53 31 TT 23 7 4 3 0 CC 35 10 7 3 0

Reference 2.38 (1.63–3.46) 2.48 (1.60–3.84) 2.12 (1.30–3.44) T carrier vs. CC; OR (95%CI) Reference 0.666 (0.413–1.08) 0.511 (0.272–0.962) 0.829 (0.446–1.54) – C carrier vs. TT; OR (95%CI) Reference 0.733 (0.545–0.985) 0.723 (0.505–1.04) 0.710 (0.476–1.06) –

By logistical regression analysis after adjustment for age, gender and H. pylori infection status (): number of subjects.

p Value <0.0001 <0.0001 0.0026 p Value 0.096 0.037 0.55 – p Value 0.039 0.078 0.092 –

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Table 3 The risk of gastric carcinogenesis with genotype in young and old subjects. 660 years old IL17A rs2275913 G > A Controls (235) GC cases (114) IL17A rs3748067 C > T Controls (233) GC cases (113) pri-miR-938 rs2505901 T > C Controls (233) GC cases (114)

GG 98 46 CC 204 100 TT 128 70

GA 113 42 CT 21 10 TC 89 42

AA 24 26 TT 8 3 CC 16 2

AA vs. G carrier; OR (95%CI) Reference 3.20 (1.56–6.57) T carrier vs. CC; OR (95%CI) Reference 1.05 (0.474–2.31) C carrier vs. TT; OR (95%CI) Reference 0.817 (0.491–1.36)

p Value

<60 years old IL17A rs2275913 G > A Controls (347) GC cases (219) IL17A rs3748067 C > T Controls (339) GC cases (214) pri-miR-938 rs2505901 T > C Controls (340) GC cases (219)

GG 120 66 CC 289 198 TT 195 141

GA 180 95 CT 35 12 TC 126 70

AA 47 58 TT 15 4 CC 19 8

AA vs. G carrier; OR (95%CI) Reference 2.20 (1.40–3.44) T carrier Vs. CC; OR (95%CI) Reference 0.499 (0.269–0.926) C carrier vs. TT; OR (95%CI) Reference 0.709 (0.491–1.03)

p Value

0.0016 p Value 0.91 p Value 0.44

0.0006 p Value 0.027 p Value 0.067

By logistical regression analysis after adjustment for gender and H. pylori infection status (): number of subjects.

Fig. 2. The relationship between genotype frequency and clinical stage of gastric cancer. When considering that controls, the early GC, and the advanced GC progress continuously, the correlation of genotype frequency to clinical stage was estimated by ANCOVA. Early GC: stage 0–I; advanced GC: stage II–IV.

both intestinal and diffuse types of gastric cancer [18]. In our current study, we report similar findings in a larger sample size. In addition, we find that the rs3748067 T genotype carries a decreased risk for intestinal cancer and the rs2505901 C genotype confers a decreased risk for gastric cancer overall. Although our study for the allelic association has adequate bpower, a Type II error cannot be excluded in the assessment of the association between genotype and disease due to a relatively small sample size. The role of Th17 in the pathogenesis of inflammation is still well unknown. Firstly, Luzza et al. reported that increased production of active IL-17A during H. pylori infection may play an important role in the inflammatory response to H. pylori colonization [16]. Thereafter, several studies revealed that IL-17, a key regulator of neutro-

phil chemotaxis, is produced in excess in H pylori-infected stomachs [17,23]. These results suggest that IL-17A may contribute to H. pylori-induced gastric inflammation. Although Th17 cytokines are considered crucial for enhancing the effector phase of the T-cell responses that cause tissue inflammation and damage in the gut, recent studies have shown that Th17 cytokines can also exert protective effects in the intestine. O’Connor et al. showed that the greater severity of colitis induced by the transfer of IL-17Adeficient T cells is associated with an enhanced Th1 cell response, raising the possibility that the anti-inflammatory effect of IL-17A in the gut can rely on the negative regulation of Th1 cell responses [24]. Thus, IL-17A seems to have dual actions, inflammatory or protective, depending of the co-expression of other cytokines [25].

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Fig. 3. The relationship between rs3748067 genotype frequency and depth of gastric cancer or lymph node metastasis. The minor allele rs3748067 was inversely correlated to both depth of cancer and lymph node metastasis. When compared among patients with gastric cancers at early and advanced stages of GC, it was inversely correlated to lymph node metastasis, not to the depth of cancer. LN(): without lymph node metastasis; LN(+): with lymph node metastasis.

Our results indicate that the rs2275913 AA homozygote is significantly associated with susceptibility to gastric cancer. We have not obtained the data how the rs2275913 affects the gastric carcinogenesis. There have been few studies aimed at determining the influences of rs2275913 on IL17A gene expression. Chen et al. demonstrated that rs2275913 was not related to phytohemagglutinin induced IL-17 production in peripheral blood mononuclear cells (PBMCs) [22]. However, this experimental design is not pertinent to the inflammatory conditions observed in H. pylori infection. In addition, many kinds and subtypes of cells existed in PBMCs and the networks of regulating immune response and cytokine production are complicated. Therefore, the mechanism by which rs2275913 could influence the pathogenesis of gastric carcinogenesis remains unclear. To the best of our knowledge, there have been no reports of an association between rs3748067 and human disorders. In general, microRNAs post-transcriptionally regulate the expression of mRNA through binding to 30 -UTRs of target genes. There are many binding sites for microRNAs such as miR-4656, -578 and -1587, including miR-938, in the 30 -UTR of IL17A. Altered conformation of 30 -UTR caused by polymorphism may thus affect binding between microRNAs and mRNA. In our current study, we show that rs3748067 was negatively associated with susceptibility to gastric cancer. That is, the minor 30 -UTR polymorphism inhibited development of intestinal cancer. These facts suggest that both polymorphisms in the 50 -promoter and the 30 -UTR affect gastric carcinogenesis. In our current study, the minor allele rs3748067 was inversely correlated with GC clinical stage, especially with lymph node metastasis. Several studies have reported the important roles of Th17 in the development and progression of gastric cancer [26–28]. Reports that an increased proportion of Th17 cells in peripheral blood was associated with clinical stage and that the expression level of IL-17 mRNA in gastric tumors was associated with lymph node involvement may

support our results. However, why only rs3748067 was associated with the progression of gastric cancer is unclear. Recently, Mi et al. investigated the association between type 1 diabetes and rs12416605 [29]. The rs12416605 genotype changes the seeding sequence of miR-938 and is in linkage disequilibrium with rs2505901, which is located 45 bp upstream of MIR938 encoding miR-938, indicating that the minor allele rs2505901 may affect the function of miR-938 as well as rs12416605 mutant. Thus, we investigated the association of rs2505901 with susceptibility to gastric cancer. In our present study, the rs2505901 minor allele was associated with a decreased risk for gastric cancer. Our epidemiological study does not reveal, however, whether the effect of rs2505901 on the gastric carcinogenesis is attributable to altered binding to the IL17A 30 -UTR, which includes a strong binding site for miR-938. Actually, miR938 has many target genes, such as transcription factor 7-like2 (TCF7L2) and interferon regulatory factor 8 (IRF8). Further studies will be needed to clarify precisely what genes miR-938 targets in gastric carcinogenesis. In addition, the binding site of miR-938 is located from ⁄246 to ⁄252, approximately 1kbp upstream of rs3748067 in the IL17A 30 -UTR (http:// mirdb.org/miRDB). Further experimentation is also required to define more precisely the interaction of rs3748067 with miR-938 and its influence on the gastric carcinogenesis. In conclusion, the rs2275913 AA homozygote was associated with an increased risk for both intestinal and diffuse types of gastric carcinogenesis, as we reported in our smaller previous study. In addition, the minor allele rs3748067 was associated with a decreased risk for intestinal cancer and rs2505901 was negatively associated with susceptibility to gastric cancer overall. These results suggest that polymorphisms in both the IL17A 30 -UTR and pri-miR-938, as well as IL17A promoter, contribute to cancer risk susceptibility and therefore can affect the development of gastric cancer.

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