Tumor Biol. DOI 10.1007/s13277-014-2255-8

RESEARCH ARTICLE

IL-17 gene polymorphism is associated with susceptibility to gastric cancer Nan Wang & Jingyue Yang & Jianguo Lu & Qing Qiao & Guoqiang Bao & Tao Wu & Xianli He

Received: 11 March 2014 / Accepted: 18 June 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract We conducted a case–control study to investigate the role of three common IL-17A and IL-17F single nucleotide polymorphisms (SNPs) on the susceptibility to gastric cancer. A case–control study was conducted using a Chinese study population of 462 gastric cancer subjects and 462 controls. Polymerase chain reaction restriction fragment length of polymorphism (PCR-RFLP) was taken to genotype rs2275913, rs763780, and rs3748067 within the IL-17 gene. When comparing demographic characteristics of gastric cancer between gastric cancer cases and control groups, cancer cases were more likely to be cigarette smokers and alcohol drinkers, have cancer history in the first relatives, and have higher infection rate of Helicobacter pylori. By conditional regression analysis, individuals carrying IL-17 rs2275913 GA, AA genotype, and A allele were associated with an increased gastric cancer risk. Those carrying rs3748067 CC genotype and C allele had a significantly increased risk for the development of gastric cancer. Moreover, rs2275913 and rs3748067 variations had association with cigarette smoking, alcohol drinking, and H. pylori infection on the risk of gastric cancer. These results suggest that rs2275913 and rs3748067 variations significantly increase gastric cancer risk in a Chinese population.

Keywords IL-17 . Single nucleotide polymorphisms . Gastric cancer . Susceptibility Nan Wang and Jingyue Yang contribute equally to this study. N. Wang : J. Lu : Q. Qiao : G. Bao : T. Wu : X. He (*) Department of General Surgery, Tangdu Hospital, Fourth Military Medical University, No.1 Xin’yi Road, Xi’an 710038, China e-mail: [email protected] J. Yang Department of Oncology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China

Introduction Gastric cancer is the fourth most common malignancy worldwide. It is estimated that there were approximately 989,600 new gastric cancer cases in 2008, and these cases were mainly occurred in Eastern Asian countries, particularly in China [1, 2]. It is well known that the development of gastric cancer is a multifactorial process, which includes many genetic and environmental factors [3]. Although Helicobacter pylori infections have been confirmed to have a role in the susceptibility to gastric cancer [4–6], the real etiology of gastric cancer is still unclear. Previous studies have shown that a group of host immune factors, when aberrantly expressed, is associated with susceptibility to gastric cancer, including interleukin-1 (IL-1), IL-8, IL-10, and tumor necrosis factors-α genes [7–10]. It is hypothesized that polymorphisms of these interleukin genes can cause a proinflammatory gastric environmental, and thus promote tissue for carcinogenesis. Interleukin-17 (IL-17) is a cytokine secreted exclusively by activated T cells that bridges the adaptive and innate immune systems [11, 12]. IL-17 plays an important role in both innate and adaptive immunities and can function on a variety of cell types [11, 13]. IL-17A and IL-17F are two key cytokines of the IL-17 cytokine family preferentially produced by helper T 17 (Th17) cells, and the two cytokines are a hallmark cytokine which contribute to the pathogenic activity of the lineage of CD4+ effector cells and multiple proinflammatory mediators [14, 15]. Previous studies have shown that IL-17A and IL-17F have a role in both innate and adaptive immune responses during the pathological process of cancer [16]. Several previous studies have reported that IL-17A and IL-17F single nucleotide polymorphisms (SNPs) are associated with the risk of gastric cancer [8, 12, 17–19], but the results of these studies are controversial. The discrepancy of these results may be caused by the different genetic distributions of IL-17 in different populations, different study designs, and sample sizes.

Tumor Biol.

Therefore, we conducted a case–control study to investigate the role of three common IL-17A and IL-17F single nucleotide polymorphisms (SNPs) on the susceptibility to gastric cancer in a Chinese population and their association with environmental factors.

Materials and methods Study population A case–control study was conducted using a Chinese study population of 462 gastric cancer subjects and 462 controls. All gastric cancer cases were newly diagnosed and histopathologically confirmed to be primary gastric cancer, and they were selected from Tangdu Hospital of Fourth Military Medical University. Patients who had secondary or recurrent tumors or a history of other malignant neoplasm and had inadequate organ function were excluded from the case group. For the frequency-matched controls on age and sex, 462 control subjects were collected from the heath checkup center of Tangdu Hospital of Fourth Military Medical University, who came to our hospital for health checkup. Controls who had a history of cancer were excluded from the control group. Informed consent was obtained from all cases and control subjects when they agreed to participate into our study. The study protocol was approved by the ethics committee of Tangdu Hospital of Fourth Military Medical University. Demographic and related lifestyle factors such as sex, age, dietary habits, alcohol consumption, tobacco smoking, and family history of cancer were collected by a face-to-face questionnaire. The questionnaires were finished by trained interviewers who were not aware of the study hypothesis. Smoking status was divided into nonsmokers and smokers. The smokers were defined as those who smoked more than one cigarette/pipe per day for at least half a year. Alcohol drinking status was divided into nondrinkers and drinkers. Alcohol drinkers were defined as those who consumed 50 g alcohol (200 ml beers, 100 wine ml, and 50 ml white spirit) per week for at least half a year. Family history was referred to first or second degree relatives, such as parents, grandparents, siblings, and offspring. H. pylori infection of all cases and controls were determined using H. pylori-specific IgG by ELISA (Diagnostic Automation, CA, USA) and a rapid urea breath test. Subjects who tested positive by either of the two examinations were defined as positive for H. pylori. Genotyping A 5-ml sample of venous blood was collected from each subject following the interview. The blood samples were

kept at −20 °C until use, with EDTA 0.5 mg/ml used as anticoagulant. Genomic DNA was extracted from the whole blood using a TIANamp blood DNA kit (Tiangen Biotech, Beijing, China) according to manufacturer’s instructions. Polymerase chain reaction restriction fragment length of polymorphism (PCR-RFLP) was taken to genotype rs2275913 (-197G > A), rs763780(7488T > C), and rs3748067(*1249C>T) within the IL-17 gene. The PCR primers of rs2275913 (-197G>A), rs3748067(*1249C>T), and rs763780(7488T>C) were designed using primer 5.0 software according to the manufacturer’s instructions. For rs2275913, the primer sequences were 5′-GCCCTTCCCA TTTTCCTTCAGA-3′ (forward) and 5′-CCAATCAACTGG GGATGGATGA-3′(reverse), which generated the 210-bp fragment. For rs3748067, the primers sequences were 5′CTGTTTCCATCCGTGCAGGTC-3′(forward) and 5′TGGTGACTGTTGGCTGCACCT-3′(reverse), which generated the 188-bp fragment. For rs763780, the primers sequences were 5′-AAGCAGGGAGCCTGCAGAGTG-3′ (forward) and 5′-GGCACCACACAACCCAGAAAG-3′(reverse), which generated the 217-bp fragment. Amplification was conducted under the cycling program: an initial denaturation for 5 min at 95 °C, followed by 35 step cycles of denaturation at 95 °C for 30 s, annealing at 62 °C for 30 s, and extension at 72 °C for 30 s, followed by a final extension at 72 °C for 10 min. PCR products were verified 2 % agarose gel stained with ethidium bromide and ultraviolet light. The results were confirmed by sequencing the PCR products using an automated sequencing system. Reproducibility was verified by repeat analysis of a randomly chosen subgroup of 5 % of the subjects. Statistical analysis A chi-square (χ2) was taken to evaluate the Hardy–Weinberg equilibriums in control groups. The demographic and lifestyle characteristics between groups were compared using the χ2 test or student t test. The genetic and allele frequencies between groups were evaluated by χ2 test. The association between genetic polymorphisms and the risk of gastric cancer was estimated by conditional multiple logistical regression model, and the results were expressed as odds ratios (ORs) and 95 % confidence intervals (CIs). The OR (95 % CI) was adjusted for potential risk factors, such as sex, age, smoking and drinking status, and family history of cancer as well as H. pylori infection. The homozygous genotypes of the three SNPs were taken as reference group. We evaluated the associations between genetic polymorphisms and environmental factors on the risk of gastric cancer. All P values were two-sided, and a P value 0.05). When comparing demographic characteristics of gastric cancer between gastric cancer cases and control groups, cancer cases were more likely to be cigarette smokers (OR=2.15, 95 % CI=1.64–2.82) and alcohol drinkers (OR=1.97, 95 % CI=1.50–2.58), have cancer history in the first relatives (OR=24.82, 95 % CI=6.40– 212.01), and have higher infection rate of H. pylori (OR= 1.89, 95 % CI=1.44–2.49). In our study population, the genotype and allele distributions of IL-17 rs2275913, rs763780, and rs3748067 in controls were in line with the Hardy–Weinberg equilibrium. The genotype and allele distributions of the three SNPs in the gastric cancer cases and control subjects are shown in Table 2. There was a significant difference in the genotype distributions of rs2275913 and rs3748067 (P values

IL-17 gene polymorphism is associated with susceptibility to gastric cancer.

We conducted a case-control study to investigate the role of three common IL-17A and IL-17F single nucleotide polymorphisms (SNPs) on the susceptibili...
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