DNA AND CELL BIOLOGY Volume 34, Number 1, 2014 ª Mary Ann Liebert, Inc. Pp. 1–6 DOI: 10.1089/dna.2014.2628
ORIGINAL RESEARCH ARTICLE
Interleukin 17A Polymorphism Elevates Gene Expression and Is Associated with Increased Risk of Nonsmall Cell Lung Cancer Sensen Cheng,1,* Zhulin Shao,1,* Xiuchun Liu,2,* Liangjun Guo,2 Xia Zhang,2 Qinyun Na,3 Xiaofeng Chen,3 Yuan Ma,1 Jinsong Zheng,2 Bao Song,2 and Jie Liu 2
Interleukin 17 (IL-17), also known as IL-17A, is a proinflammatory cytokine and plays critical roles in tumor immunity. Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancers. The aim of this study was to investigate the correlation between IL-17A genetic polymorphisms and susceptibility to NSCLC. Two single nucleotide polymorphisms (SNPs) in IL-17A gene, rs3819024A/G and rs8193037G/A, were detected in 322 NSCLC patients and 366 healthy donors. Data revealed that prevalence of IL-17A rs8193037GA and AA genotypes were significantly higher in the patients than in controls (odds ratio [OR]: 2.20, 95% confidence interval [CI]: 1.53–3.16, p < 0.001; and OR: 3.19, 95% CI: 1.42–7.15, p = 0.003). Stratification analyses showed that rs8193037A allele had significantly higher percentage in adenocarcinoma than in squamous cell carcinoma (OR: 1.72, 95% CI: 1.12–2.64, p = 0.013). When examining the possible function of the SNPs, we found that in vitro stimulated peripheral blood mononuclear cells from subjects possessing rs8193037A allele produced significantly more IL-17 than those with the GG genotype, and this phenomenon could be observed in both controls and the NSCLC patients. These data indicate IL-17A polymorphism is associated with increased risk of NSCLC probably by elevating gene expression.
(Spriggs, 1997; Smith and Colbert, 2014). IL-17A plays critical roles in both adaptive immunity and innate immunity (Martin et al., 2014). This cytokine is one of the major factors protecting human bodies against certain pathogens at mucosal and epithelial levels (Harvey et al., 2014). IL-17A is also involved in controlling fungal infection (Bar et al., 2014). Dysregulation of IL-17A causes chronic inflammation and excessive expression of other proinflammatory cytokines (Mok et al., 2010). The role of IL-17 in NSCLC remains controversial (Duan et al., 2014). An induction of IL 17A mRNA and protein expression was noted in lung CD4 + T cells in patients with NSCLC compared with healthy controls, suggesting that IL17 is involved in lung cancer (Xu et al., 2014). Also, study showed that overexpression of IL-17 in vitro could promote tumor growth and angiogenesis in immune-compromised mice (Li et al., 2012a; Neurath and Finotto, 2012). Intranasal treatment of a neutralizing IL-17A antibody could cause a significant reduction of tumor growth in mice model with experimental lung adenocarcinoma compared with control treated mice (Chen et al., 2010). Further, IL-17 may directly promote the invasion of NSCLC cells both in vitro and in vivo
Introduction
L
ung cancer is the leading cause of death worldwide; more than a million people die from this disease each year (Lee and Forey, 2013). Nonsmall cell lung cancer (NSCLC) accounts for up to 80% of all lung cancer cases. Despite the development of antitumor therapy, the prognosis for patients with lung cancer remains poor, with a 5-year survival rate of less than 20% (Mok, 2011). Pathogenesis of the malignancy remains unclear. However, recent studies have provided evidence that genetic factors may influence the development of NSCLC (Li et al., 2012b; Shi et al., 2012; Bai et al., 2013). Interleukin (IL)-17, also known as IL-17A, was first cloned in 1993 and was identified as cytotoxic T lymphocyteassociated antigen (CTLA)-8 (Rouvier et al., 1993; Yao et al., 1995). Early studies reported that IL-17A was mainly produced by activated memory CD4 + T cells but could be induced in CD8 + T cells, natural killer (NK) T cells, dendritic cells (DCs), and possibly other cells (Rouvier et al., 1993; Yao et al., 1995). Later, specialized T cells, called Th17 cells, were identified to be the major source of IL-17A 1
School of Medicine and Life Sciences, University of Jinan, Shandong Academy of Medical Sciences, Jinan, China. Department of Oncology, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, Jinan, China. Department of Surgery, Huadong Hospital, Shanxi, China. *These authors contributed equally to the work.
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(Chen et al., 2010; Kirshberg et al., 2011; Li et al., 2012a). In contrast, recent reports indicated that tumor growth in subcutaneous tissue and lung tumor metastasis were enhanced in IL-17 - / - mice and that the mechanism was associated with reducing IFN gamma-producing tumor-infiltrating NK and T cells (Kryczek et al., 2009). It implicates that IL-17-mediated responses are protective against tumor development. Single nucleotide polymorphisms (SNPs) may have different functions and therefore may affect the pathogenesis of diseases. In this study, we investigated IL-17A rs3819024A/ G and rs8193037G/A polymorphisms in NSCLC, and examined their effects on IL-17 expression. Materials and Methods Patients and controls
The study group included 322 NSCLC cases. The histological types of lung cancers were identified according to the World Health Organization classifications. The pathologic stages were determined according to the International System for Staging Lung Cancer. Samples from the healthy control population were collected from individuals residing in the same geographic areas without histories of malignancy or other major diseases such as chronic obstructive pulmonary disease and cardiovascular diseases. The healthy control group included 239 males and 127 females. To exclude the possible effects of ethnicity, only Han Chinese were included in this study. Informed consent was obtained from all study participants according to the Helsinki Declaration. This study was approved by the review boards of Shandong Academy of Medical Sciences and Fudan University. DNA extraction and genotyping
Genomic DNA was extracted from 5 mL frozen whole blood using the DNA Extraction Kit (Qiagen, Inc., Hilden, Germany) according to the manufacturer’s protocol. The rs3819024A/G and rs8193037G/A polymorphisms were genotyped by a primer-introduced restriction analysis–polymerase chain reaction (PIRA-PCR) assay. For rs8193037G/A, mismatched G and C were introduced to the antisense primer to replace A at + 3 and + 1 bp from the polymorphic site to create a BsmBI restriction site (sense 5¢-TCACCTTT GTCCAGTCT CTA-3¢ and antisense 5¢-GTACCTTGATTTT CCATTTGAT CTTTCGTC-3¢). Similarly, for rs8193037G/A, a mismatched G was introduced to the sense primer to replace C at - 3 bp from the polymorphic site to create a BsaHI restriction site (sense 5¢-ATGGTGTCACCCCTGAACCCACT GCGAGAC-3¢ and antisense 5¢-CCTATCCTACATATACA GTA-3¢). The restriction enzymes of the two loci (rs3819024A/G and rs8193037G/A) were BsmBI and BsaHI (NEB, Ipswich, MA), respectively. Genotyping was performed in a double-blind manner. Different genotype patterns by the PIRA-PCR assay were randomly selected for direct sequencing to determine the genotypes by using an automated sequencer (Perkin-Elmer ABI model 377 genetic analysis; Applied Biosystems, Foster City, CA), and the results were 100% concordant. Cell preparation, cell culture, and measurement of IL-17
Heparinized blood samples were collected. Peripheral blood mononuclear cells (PBMCs) were isolated using a Fi-
CHENG ET AL.
coll-Hypaque gradient (Pharmacia Biotech, Uppsala, Sweden) and were induced to secrete IL-17 by culturing the PBMCs (106/well) in 24-well plates for 48 h in RPMI 1640 supplemented with 10% fetal bovine serum in the presence of 5 mg/mL phytohemagglutinin (PHA; Pharmacia Biotech) at 37C in 5% CO2. The concentrations of IL-17 in collected supernatants were measured by an enzyme-linked immunosorbent assay (ELISA; Mabtech, Nacka Strand, Sweden). Statistical analysis
The SPSS statistical software package version 19.0 was used for all of the statistical analyses. Genotype and allele frequencies of IL-17A polymorphisms were compared between NSCLC cases and controls using the Chi-square test and odds ratios (OR), and 95% confidence intervals (CIs) were calculated to assess the relative risk conferred by a particular allele and genotype. The linkage disequilibrium between these polymorphisms and the haplotypes were conducted using the SHEsis software, from the website http:// analysis.bio-x.cn/ (Bio-X, Inc., Shanghai, China). Expression of IL-17A was compared by Student’s t-test. Statistical significance was assumed at p < 0.05. Results IL-17A polymorphisms among the patients and controls
The selected characteristics of the cases and controls are presented in Table 1. No significant differences were observed between the NSCLC cases and controls in regard to age and gender, whereas proportion of smokers was clearly higher in patients than in controls. The distribution of IL-17A SNPs in NSCLC cases and controls are shown in Table 2. Polymorphic rs3819024AG and GG genotypes did not present significant variations between patients and controls. For the
Table 1. Characteristics of Nonsmall Cell Lung Cancer Patients and Controls Characteristics Age £ 50 > 50 Gender Male Female Smoking status Nonsmokers Smokers Clinical stage I II III IV Histological type Squamous cell carcinoma Adenocarcinoma Other types
NSCLC (n = 322) (%)
Controls (n = 366) (%)
129 (40.1) 193 (59.9)
151 (41.3) 211 (58.7)
> 0.05
217 (67.4) 105 (32.6)
239 (65.3) 127 (34.7)
> 0.05
101 (31.4) 221 (68.6)
251 (68.6) 115 (31.4)
< 0.001
48 52 121 101
(14.9) (16.1) (37.6) (31.4)
113 (35.1) 171 (53.1) 38 (11.8)
NSCLC, nonsmall cell lung cancer.
p-Value
IL-17A POLYMORPHISMS AND NSCLC
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Table 2. The Distribution of IL-17A Single Nucleotide Polymorphisms in Nonsmall Cell Lung Cancer Cases and Controls Polymorphisms rs3819024A/G Genotype AA AG GG Allele A G rs8193037G/A Genotype GG GA AA Allele G A
Cases (n = 322) (%)
Controls (n = 366) (%)
OR (95% CI)
p-Value
111 (34.5) 146 (45.3) 65 (20.2)
119 (32.5) 170 (46.4) 77 (21.1)
Ref. 0.92 (0.66–1.29) 0.91 (0.59–1.38)
0.634 0.641
368 (57.1) 276 (42.9)
408 (55.7) 324 (44.3)
Ref. 0.94 (0.76–1.17)
0.600
204 (63.4) 98 (30.4) 20 (6.2)
293 (80.1) 64 (17.5) 9 (2.4)
Ref. 2.20 (1.53–3.16) 3.19 (1.42–7.15)
< 0.001a 0.003a
506 (78.6) 138 (21.4)
650 (88.8) 82 (11.2)
Ref. 2.16 (1.61–2.91)
< 0.001a
a p-Value < 0.05. CI, confidence interval; IL, interleukin; OR, odds ratio.
rs8193037G/A SNP, 98 (30.4%) patients and 64 (17.5%) controls were detected carrying GA genotype; 20 (6.2%) patients and 9 (2.4%) controls were detected carrying AA genotype. Distribution of GA and AA genotypes was significantly higher in patients than in controls (OR: 2.20, 95% CI: 1.53– 3.16, p < 0.001; OR: 3.19, 95% CI: 1.42–7.15, p = 0.003). In addition, we analyzed the linkage status of the two SNPs and no linkage disequilibrium was found (data not shown). These data suggest that the IL-17A rs8193037G/A SNP is associated with increased susceptibility to NSCLC in the Chinese population.
rs3819024A/G and rs8193037G/A SNPs were associated with specific histological type of NSCLC (Table 3). For the rs3819024A/G polymorphism, no significant differences in genotypes were observed between adenocarcinoma and squamous cell carcinoma. As for the rs8193037G/A polymorphism, proportion of GA and AA showed nonsignificant increases in adenocarcinoma than in squamous cell carcinoma ( p = 0.053 and p = 0.089, Table 3), whereas percentage of A allele revealed significantly higher value in adenocarcinoma than in squamous cell carcinoma ( p = 0.013, Table 3). These data suggest that the rs8193037G/A polymorphism is more correlated with the risk of adenocarcinoma than squamous cell carcinoma.
IL-17A SNPs in different histological types of NSCLC cases
IL-17A SNP and histological specificity of NSCLC
Adenocarcinoma and squamous cell carcinoma are the two major histological types of NSCLC. We evaluated whether the
Due to the observation that rs8193037G/A polymorphism appeared to be more associated with increased risk of
Table 3. IL-17A Single Nucleotide Polymorphisms in Different Histological Types of Nonsmall Cell Lung Cancer Cases Polymorphisms
Adenocarcinoma (n = 171) (%)
SCC (n = 113) (%)
OR (95% CI)
p-Value
58 (33.9) 77 (45.0) 36 (21.1)
39 (34.5) 51 (45.1) 23 (20.4)
Ref. 1.02 (0.59–1.74) 1.05 (0.54–2.04)
0.956 0.880
193 (56.4) 149 (43.6)
129 (57.1) 97 (42.9)
Ref. 1.03 (0.73–1.44)
0.879
98 (57.3) 60 (35.1) 13 (7.6)
80 (70.8) 29 (25.7) 4 (3.5)
Ref. 1.69 (0.99–2.88) 2.65 (0.83–8.46)
0.053 0.089
256 (74.9) 86 (25.1)
189 (83.6) 37 (16.4)
Ref. 1.72 (1.12–2.64)
0.013a
rs3819024A/G Genotype AA AG GG Allele A G rs8193037G/A Genotype GG GA AA Allele G A a p-Value < 0.05. SCC, squamous cell carcinoma.
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CHENG ET AL.
Table 4. rs8193037 Single Nucleotide Polymorphism in Major Histological Types of Nonsmall Cell Lung Cancer Cases and Controls Polymorphism
Adenocarcinoma (n = 171) (%)
Controls (n = 366) (%)
OR (95% CI)
p-Value
98 (57.3) 60 (35.1) 13 (7.6)
293 (80.1) 64 (17.5) 9 (2.4)
Ref. 2.80 (1.84–4.27) 4.32 (1.79–10.42)
< 0.001 < 0.001
256 (74.9) 86 (25.1)
650 (88.8) 82 (11.2)
Ref. 2.66 (1.90–3.72)
< 0.001
rs8193037G/A Genotype AA AG GG Allele A G Polymorphism rs8193037G/A Genotype GG GA AA Allele G A a
SCC (n = 113) (%)
Controls (n = 366) (%)
OR (95% CI)
p-Value
80 (70.8) 29 (25.7) 4 (3.5)
293 (80.1) 64 (17.5) 9 (2.4)
Ref. 1.66 (1.00–2.75) 1.65 (0.49–5.50)
0.029a 0.411
189 (83.6) 37 (16.4)
650 (88.8) 82 (11.2)
Ref. 1.55 (1.02–2.36)
0.039a
p-Value < 0.05.
adenocarcinoma, we investigated whether the SNP was only correlated with adenocarcinoma in NSCLC. Data showed that percentages of AG and AA genotypes were significantly higher in adenocarcinoma cases than in controls (Table 4). However, prevalence of GA genotype and A allele were also significantly increased in squamous cell carcinoma than in controls ( p = 0.029 and p = 0.039, Table 4). These results indicate that the association between IL-17A rs8193037G/A polymorphism and risk of NSCLC was not limited to adenocarcinoma.
FIG. 1. Peripheral blood mononuclear cells from patients or controls were stimulated in vitro with PHA and levels of secreted IL-17 were determined by ELISA. Levels of IL-17 among different genotypes of rs3819024 in healthy controls (A) and NSCLC patients (B) are shown. Levels of IL-17 between rs8193037GG genotype and A carrier in healthy controls (C) and NSCLC patients (D) are shown. ELISA, enzyme-linked immunosorbent assay; IL, interleukin; NS, not significant; NSCLC, nonsmall cell lung cancer; PHA, phytohemagglutinin.
Impact of the SNPs on the secretion of IL-17
To substantiate the biological significances of rs3819024A/ G and rs8193037G/A polymorphisms, we examined whether different genotypes could affect IL-17 secretion. PBMCs from patients or controls were stimulated in vitro with PHA and levels of secreted IL-17 were determined by ELISA. For the rs3819024A/G SNP, a total of 62 healthy controls and 58 patients were included. As shown in Figure 1A and B, levels of IL-17 among different genotypes of rs3819024 remained
IL-17A POLYMORPHISMS AND NSCLC
FIG. 2. Histological types of NSCLC and the effect of rs8193037G/A SNP on IL-17 expression. When considering the GG genotype, the excretion of IL-17 remained similar between adenocarcinoma (AD) and squamous cell carcinoma (SCC) patients. However, when considering the A allele, level of IL-17 was moderately elevated in adenocarcinoma cases than in squamous cell carcinoma cases. Forty-five subjects were included in figure, in which 12 AD patients and 11 SCC patients were GG genotype, and 10 AD patients and 12 SCC patients were GA or AA genotype. SNP, single nucleotide polymorphism.
similar. For the rs8193037G/A SNP, a total of 65 healthy controls and 60 patients were included. PBMCs from healthy controls with rs8193037A allele produced significantly higher levels of IL-17 than those with GG genotype ( p = 0.012, Fig. 1C). This phenomenon was also observed in the NSCLC patients ( p = 0.023, Fig. 1D). We further evaluated whether histological types of NSCLC could affect the function of rs8193037G/A SNP. When considering the GG genotype, we found that the excretion of IL-17 remained similar between adenocarcinoma and squamous cell carcinoma patients. However, when considering the A allele, level of IL-17 was moderately elevated in adenocarcinoma cases than in squamous cell carcinoma cases (Fig. 2). Discussion
Genetic variants could act as risk factors for NSCLC. A genome-wide association study (GWAS) has identified that rs7086803, rs9387478, and rs2395185 greatly contribute to lung cancer susceptibility in never-smoking women in Asia (Lan et al., 2012). Another research group has reported that rs2853677, rs10937405, rs7216064, and rs3817963 are associated with susceptibility to lung adenocarcinoma in the Japanese population (Shiraishi et al., 2012). In this study, we identified that IL-17A rs8193037G/A SNP was associated with increased susceptibility to NSCLC, especially to adenocarcinoma in the Han Chinese population. However, recent literature investigated another polymorphism in IL17A gene, rs2275913, and did not found any correlation with lung cancer risk in the Tunisian population (Kaabachi et al.,
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2014). It would be interesting to conduct a similar research in the Chinese population for comparison. The rs3819024A/G SNP is located at the 5¢ untranslated region of IL-17A gene. It has been reported that it is associated with an increased risk of gastric cardia adenocarcinoma in a Chinese population (Ren et al., 2014). In addition, Wang et al. (2012) have shown that the polymorphism may be involved in the development of allergic rhinitis with asthma. However, we did not find any association between this SNP and NSCLC in our study population (Table 2). Function of the polymorphism remains unclear. We evaluated whether rs3819024 could affect gene expression but did not observe any differences in IL-17 excretion from subjects with different genotypes (Fig. 1A, B), suggesting that the SNP may not affect the promoter activity of IL-17A gene. Studies have shown that rs8193037 may be involved in various diseases. Zhang et al. (2013) have reported that the polymorphism is associated with increased risk of inflammatory bowel disease in a Chinese Han population. Also, it has been shown that frequencies of IL-17A rs8193037 GG homozygote and G allele are significantly higher in the patients with coronary artery disease than those in the controls (Zhang et al., 2011). Our data revealed that rs8193037 GA and AA genotypes were associated with NSCLC (Table 2), indicating a functional significance of the polymorphism. rs8193037 is located at 122-bp upstream from the translation start site of IL-17A, which is close to the activator protein 1 (AP-1) region (Liu et al., 2004). It is possible that the SNP may affect the gene expression of IL-17A by interfering with AP-1. Our data showed that subjects with rs8193037A allele produced significantly higher levels of IL-17 than those with GG genotype ( p < 0.05, Fig. 1C, D), suggesting a potential mechanism of rs8193037 on the pathogenesis of human diseases. Our data identified that rs8193037G/A polymorphism was more correlated with risk of adenocarcinoma than squamous cell carcinoma (Table 3), but was not limited to adenocarcinoma (Table 4). Since the polymorphism may affect IL-17 expression, these results indicate that different histological types of NSCLC may have different responses to IL-17. In addition, our data showed that adenocarcinoma patients carrying rs8193037A allele had IL-17 production than squamous cell carcinoma patients with rs8193037A (Fig. 2), indicating a special correlation between IL-17 and adenocarcinoma. Further study on this field in needed. In summary, we identified that IL-17A rs8193037G/A SNP was associated with increased susceptibility to NSCLC, especially to adenocarcinoma. Functional analyses revealed that in vitro stimulated PBMCs carrying rs8193037A allele produced significantly more IL-17 than those with the GG genotype. These data indicate IL-17A polymorphism is associated with increased risk of NSCLC probably by elevating gene expression. Acknowledgments
This research was supported by Jinan Science Research Project Foundation (201102051) and Shandong Provincial Natural Science Foundation (ZR2013HM079). Disclosure Statement
No competing financial interests exist.
6 References
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Address correspondence to: Jie Liu, MD, PhD Department of Oncology Shandong Cancer Hospital and Institute Shandong Academy of Medical Sciences 440 Jiyan Road Jinan Shandong 250117 China E-mail: [email protected] Received for publication July 22, 2014; received in revised form September 1, 2014; accepted September 7, 2014.
ORIGINAL RESEARCH ARTICLE
Interleukin 17A Polymorphism Elevates Gene Expression and Is Associated with Increased Risk of Nonsmall Cell Lung Cancer Sensen Cheng,1,* Zhulin Shao,1,* Xiuchun Liu,2,* Liangjun Guo,2 Xia Zhang,2 Qinyun Na,3 Xiaofeng Chen,3 Yuan Ma,1 Jinsong Zheng,2 Bao Song,2 and Jie Liu 2
Interleukin 17 (IL-17), also known as IL-17A, is a proinflammatory cytokine and plays critical roles in tumor immunity. Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancers. The aim of this study was to investigate the correlation between IL-17A genetic polymorphisms and susceptibility to NSCLC. Two single nucleotide polymorphisms (SNPs) in IL-17A gene, rs3819024A/G and rs8193037G/A, were detected in 322 NSCLC patients and 366 healthy donors. Data revealed that prevalence of IL-17A rs8193037GA and AA genotypes were significantly higher in the patients than in controls (odds ratio [OR]: 2.20, 95% confidence interval [CI]: 1.53–3.16, p < 0.001; and OR: 3.19, 95% CI: 1.42–7.15, p = 0.003). Stratification analyses showed that rs8193037A allele had significantly higher percentage in adenocarcinoma than in squamous cell carcinoma (OR: 1.72, 95% CI: 1.12–2.64, p = 0.013). When examining the possible function of the SNPs, we found that in vitro stimulated peripheral blood mononuclear cells from subjects possessing rs8193037A allele produced significantly more IL-17 than those with the GG genotype, and this phenomenon could be observed in both controls and the NSCLC patients. These data indicate IL-17A polymorphism is associated with increased risk of NSCLC probably by elevating gene expression.
(Spriggs, 1997; Smith and Colbert, 2014). IL-17A plays critical roles in both adaptive immunity and innate immunity (Martin et al., 2014). This cytokine is one of the major factors protecting human bodies against certain pathogens at mucosal and epithelial levels (Harvey et al., 2014). IL-17A is also involved in controlling fungal infection (Bar et al., 2014). Dysregulation of IL-17A causes chronic inflammation and excessive expression of other proinflammatory cytokines (Mok et al., 2010). The role of IL-17 in NSCLC remains controversial (Duan et al., 2014). An induction of IL 17A mRNA and protein expression was noted in lung CD4 + T cells in patients with NSCLC compared with healthy controls, suggesting that IL17 is involved in lung cancer (Xu et al., 2014). Also, study showed that overexpression of IL-17 in vitro could promote tumor growth and angiogenesis in immune-compromised mice (Li et al., 2012a; Neurath and Finotto, 2012). Intranasal treatment of a neutralizing IL-17A antibody could cause a significant reduction of tumor growth in mice model with experimental lung adenocarcinoma compared with control treated mice (Chen et al., 2010). Further, IL-17 may directly promote the invasion of NSCLC cells both in vitro and in vivo
Introduction
L
ung cancer is the leading cause of death worldwide; more than a million people die from this disease each year (Lee and Forey, 2013). Nonsmall cell lung cancer (NSCLC) accounts for up to 80% of all lung cancer cases. Despite the development of antitumor therapy, the prognosis for patients with lung cancer remains poor, with a 5-year survival rate of less than 20% (Mok, 2011). Pathogenesis of the malignancy remains unclear. However, recent studies have provided evidence that genetic factors may influence the development of NSCLC (Li et al., 2012b; Shi et al., 2012; Bai et al., 2013). Interleukin (IL)-17, also known as IL-17A, was first cloned in 1993 and was identified as cytotoxic T lymphocyteassociated antigen (CTLA)-8 (Rouvier et al., 1993; Yao et al., 1995). Early studies reported that IL-17A was mainly produced by activated memory CD4 + T cells but could be induced in CD8 + T cells, natural killer (NK) T cells, dendritic cells (DCs), and possibly other cells (Rouvier et al., 1993; Yao et al., 1995). Later, specialized T cells, called Th17 cells, were identified to be the major source of IL-17A 1
School of Medicine and Life Sciences, University of Jinan, Shandong Academy of Medical Sciences, Jinan, China. Department of Oncology, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, Jinan, China. Department of Surgery, Huadong Hospital, Shanxi, China. *These authors contributed equally to the work.
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(Chen et al., 2010; Kirshberg et al., 2011; Li et al., 2012a). In contrast, recent reports indicated that tumor growth in subcutaneous tissue and lung tumor metastasis were enhanced in IL-17 - / - mice and that the mechanism was associated with reducing IFN gamma-producing tumor-infiltrating NK and T cells (Kryczek et al., 2009). It implicates that IL-17-mediated responses are protective against tumor development. Single nucleotide polymorphisms (SNPs) may have different functions and therefore may affect the pathogenesis of diseases. In this study, we investigated IL-17A rs3819024A/ G and rs8193037G/A polymorphisms in NSCLC, and examined their effects on IL-17 expression. Materials and Methods Patients and controls
The study group included 322 NSCLC cases. The histological types of lung cancers were identified according to the World Health Organization classifications. The pathologic stages were determined according to the International System for Staging Lung Cancer. Samples from the healthy control population were collected from individuals residing in the same geographic areas without histories of malignancy or other major diseases such as chronic obstructive pulmonary disease and cardiovascular diseases. The healthy control group included 239 males and 127 females. To exclude the possible effects of ethnicity, only Han Chinese were included in this study. Informed consent was obtained from all study participants according to the Helsinki Declaration. This study was approved by the review boards of Shandong Academy of Medical Sciences and Fudan University. DNA extraction and genotyping
Genomic DNA was extracted from 5 mL frozen whole blood using the DNA Extraction Kit (Qiagen, Inc., Hilden, Germany) according to the manufacturer’s protocol. The rs3819024A/G and rs8193037G/A polymorphisms were genotyped by a primer-introduced restriction analysis–polymerase chain reaction (PIRA-PCR) assay. For rs8193037G/A, mismatched G and C were introduced to the antisense primer to replace A at + 3 and + 1 bp from the polymorphic site to create a BsmBI restriction site (sense 5¢-TCACCTTT GTCCAGTCT CTA-3¢ and antisense 5¢-GTACCTTGATTTT CCATTTGAT CTTTCGTC-3¢). Similarly, for rs8193037G/A, a mismatched G was introduced to the sense primer to replace C at - 3 bp from the polymorphic site to create a BsaHI restriction site (sense 5¢-ATGGTGTCACCCCTGAACCCACT GCGAGAC-3¢ and antisense 5¢-CCTATCCTACATATACA GTA-3¢). The restriction enzymes of the two loci (rs3819024A/G and rs8193037G/A) were BsmBI and BsaHI (NEB, Ipswich, MA), respectively. Genotyping was performed in a double-blind manner. Different genotype patterns by the PIRA-PCR assay were randomly selected for direct sequencing to determine the genotypes by using an automated sequencer (Perkin-Elmer ABI model 377 genetic analysis; Applied Biosystems, Foster City, CA), and the results were 100% concordant. Cell preparation, cell culture, and measurement of IL-17
Heparinized blood samples were collected. Peripheral blood mononuclear cells (PBMCs) were isolated using a Fi-
CHENG ET AL.
coll-Hypaque gradient (Pharmacia Biotech, Uppsala, Sweden) and were induced to secrete IL-17 by culturing the PBMCs (106/well) in 24-well plates for 48 h in RPMI 1640 supplemented with 10% fetal bovine serum in the presence of 5 mg/mL phytohemagglutinin (PHA; Pharmacia Biotech) at 37C in 5% CO2. The concentrations of IL-17 in collected supernatants were measured by an enzyme-linked immunosorbent assay (ELISA; Mabtech, Nacka Strand, Sweden). Statistical analysis
The SPSS statistical software package version 19.0 was used for all of the statistical analyses. Genotype and allele frequencies of IL-17A polymorphisms were compared between NSCLC cases and controls using the Chi-square test and odds ratios (OR), and 95% confidence intervals (CIs) were calculated to assess the relative risk conferred by a particular allele and genotype. The linkage disequilibrium between these polymorphisms and the haplotypes were conducted using the SHEsis software, from the website http:// analysis.bio-x.cn/ (Bio-X, Inc., Shanghai, China). Expression of IL-17A was compared by Student’s t-test. Statistical significance was assumed at p < 0.05. Results IL-17A polymorphisms among the patients and controls
The selected characteristics of the cases and controls are presented in Table 1. No significant differences were observed between the NSCLC cases and controls in regard to age and gender, whereas proportion of smokers was clearly higher in patients than in controls. The distribution of IL-17A SNPs in NSCLC cases and controls are shown in Table 2. Polymorphic rs3819024AG and GG genotypes did not present significant variations between patients and controls. For the
Table 1. Characteristics of Nonsmall Cell Lung Cancer Patients and Controls Characteristics Age £ 50 > 50 Gender Male Female Smoking status Nonsmokers Smokers Clinical stage I II III IV Histological type Squamous cell carcinoma Adenocarcinoma Other types
NSCLC (n = 322) (%)
Controls (n = 366) (%)
129 (40.1) 193 (59.9)
151 (41.3) 211 (58.7)
> 0.05
217 (67.4) 105 (32.6)
239 (65.3) 127 (34.7)
> 0.05
101 (31.4) 221 (68.6)
251 (68.6) 115 (31.4)
< 0.001
48 52 121 101
(14.9) (16.1) (37.6) (31.4)
113 (35.1) 171 (53.1) 38 (11.8)
NSCLC, nonsmall cell lung cancer.
p-Value
IL-17A POLYMORPHISMS AND NSCLC
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Table 2. The Distribution of IL-17A Single Nucleotide Polymorphisms in Nonsmall Cell Lung Cancer Cases and Controls Polymorphisms rs3819024A/G Genotype AA AG GG Allele A G rs8193037G/A Genotype GG GA AA Allele G A
Cases (n = 322) (%)
Controls (n = 366) (%)
OR (95% CI)
p-Value
111 (34.5) 146 (45.3) 65 (20.2)
119 (32.5) 170 (46.4) 77 (21.1)
Ref. 0.92 (0.66–1.29) 0.91 (0.59–1.38)
0.634 0.641
368 (57.1) 276 (42.9)
408 (55.7) 324 (44.3)
Ref. 0.94 (0.76–1.17)
0.600
204 (63.4) 98 (30.4) 20 (6.2)
293 (80.1) 64 (17.5) 9 (2.4)
Ref. 2.20 (1.53–3.16) 3.19 (1.42–7.15)
< 0.001a 0.003a
506 (78.6) 138 (21.4)
650 (88.8) 82 (11.2)
Ref. 2.16 (1.61–2.91)
< 0.001a
a p-Value < 0.05. CI, confidence interval; IL, interleukin; OR, odds ratio.
rs8193037G/A SNP, 98 (30.4%) patients and 64 (17.5%) controls were detected carrying GA genotype; 20 (6.2%) patients and 9 (2.4%) controls were detected carrying AA genotype. Distribution of GA and AA genotypes was significantly higher in patients than in controls (OR: 2.20, 95% CI: 1.53– 3.16, p < 0.001; OR: 3.19, 95% CI: 1.42–7.15, p = 0.003). In addition, we analyzed the linkage status of the two SNPs and no linkage disequilibrium was found (data not shown). These data suggest that the IL-17A rs8193037G/A SNP is associated with increased susceptibility to NSCLC in the Chinese population.
rs3819024A/G and rs8193037G/A SNPs were associated with specific histological type of NSCLC (Table 3). For the rs3819024A/G polymorphism, no significant differences in genotypes were observed between adenocarcinoma and squamous cell carcinoma. As for the rs8193037G/A polymorphism, proportion of GA and AA showed nonsignificant increases in adenocarcinoma than in squamous cell carcinoma ( p = 0.053 and p = 0.089, Table 3), whereas percentage of A allele revealed significantly higher value in adenocarcinoma than in squamous cell carcinoma ( p = 0.013, Table 3). These data suggest that the rs8193037G/A polymorphism is more correlated with the risk of adenocarcinoma than squamous cell carcinoma.
IL-17A SNPs in different histological types of NSCLC cases
IL-17A SNP and histological specificity of NSCLC
Adenocarcinoma and squamous cell carcinoma are the two major histological types of NSCLC. We evaluated whether the
Due to the observation that rs8193037G/A polymorphism appeared to be more associated with increased risk of
Table 3. IL-17A Single Nucleotide Polymorphisms in Different Histological Types of Nonsmall Cell Lung Cancer Cases Polymorphisms
Adenocarcinoma (n = 171) (%)
SCC (n = 113) (%)
OR (95% CI)
p-Value
58 (33.9) 77 (45.0) 36 (21.1)
39 (34.5) 51 (45.1) 23 (20.4)
Ref. 1.02 (0.59–1.74) 1.05 (0.54–2.04)
0.956 0.880
193 (56.4) 149 (43.6)
129 (57.1) 97 (42.9)
Ref. 1.03 (0.73–1.44)
0.879
98 (57.3) 60 (35.1) 13 (7.6)
80 (70.8) 29 (25.7) 4 (3.5)
Ref. 1.69 (0.99–2.88) 2.65 (0.83–8.46)
0.053 0.089
256 (74.9) 86 (25.1)
189 (83.6) 37 (16.4)
Ref. 1.72 (1.12–2.64)
0.013a
rs3819024A/G Genotype AA AG GG Allele A G rs8193037G/A Genotype GG GA AA Allele G A a p-Value < 0.05. SCC, squamous cell carcinoma.
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Table 4. rs8193037 Single Nucleotide Polymorphism in Major Histological Types of Nonsmall Cell Lung Cancer Cases and Controls Polymorphism
Adenocarcinoma (n = 171) (%)
Controls (n = 366) (%)
OR (95% CI)
p-Value
98 (57.3) 60 (35.1) 13 (7.6)
293 (80.1) 64 (17.5) 9 (2.4)
Ref. 2.80 (1.84–4.27) 4.32 (1.79–10.42)
< 0.001 < 0.001
256 (74.9) 86 (25.1)
650 (88.8) 82 (11.2)
Ref. 2.66 (1.90–3.72)
< 0.001
rs8193037G/A Genotype AA AG GG Allele A G Polymorphism rs8193037G/A Genotype GG GA AA Allele G A a
SCC (n = 113) (%)
Controls (n = 366) (%)
OR (95% CI)
p-Value
80 (70.8) 29 (25.7) 4 (3.5)
293 (80.1) 64 (17.5) 9 (2.4)
Ref. 1.66 (1.00–2.75) 1.65 (0.49–5.50)
0.029a 0.411
189 (83.6) 37 (16.4)
650 (88.8) 82 (11.2)
Ref. 1.55 (1.02–2.36)
0.039a
p-Value < 0.05.
adenocarcinoma, we investigated whether the SNP was only correlated with adenocarcinoma in NSCLC. Data showed that percentages of AG and AA genotypes were significantly higher in adenocarcinoma cases than in controls (Table 4). However, prevalence of GA genotype and A allele were also significantly increased in squamous cell carcinoma than in controls ( p = 0.029 and p = 0.039, Table 4). These results indicate that the association between IL-17A rs8193037G/A polymorphism and risk of NSCLC was not limited to adenocarcinoma.
FIG. 1. Peripheral blood mononuclear cells from patients or controls were stimulated in vitro with PHA and levels of secreted IL-17 were determined by ELISA. Levels of IL-17 among different genotypes of rs3819024 in healthy controls (A) and NSCLC patients (B) are shown. Levels of IL-17 between rs8193037GG genotype and A carrier in healthy controls (C) and NSCLC patients (D) are shown. ELISA, enzyme-linked immunosorbent assay; IL, interleukin; NS, not significant; NSCLC, nonsmall cell lung cancer; PHA, phytohemagglutinin.
Impact of the SNPs on the secretion of IL-17
To substantiate the biological significances of rs3819024A/ G and rs8193037G/A polymorphisms, we examined whether different genotypes could affect IL-17 secretion. PBMCs from patients or controls were stimulated in vitro with PHA and levels of secreted IL-17 were determined by ELISA. For the rs3819024A/G SNP, a total of 62 healthy controls and 58 patients were included. As shown in Figure 1A and B, levels of IL-17 among different genotypes of rs3819024 remained
IL-17A POLYMORPHISMS AND NSCLC
FIG. 2. Histological types of NSCLC and the effect of rs8193037G/A SNP on IL-17 expression. When considering the GG genotype, the excretion of IL-17 remained similar between adenocarcinoma (AD) and squamous cell carcinoma (SCC) patients. However, when considering the A allele, level of IL-17 was moderately elevated in adenocarcinoma cases than in squamous cell carcinoma cases. Forty-five subjects were included in figure, in which 12 AD patients and 11 SCC patients were GG genotype, and 10 AD patients and 12 SCC patients were GA or AA genotype. SNP, single nucleotide polymorphism.
similar. For the rs8193037G/A SNP, a total of 65 healthy controls and 60 patients were included. PBMCs from healthy controls with rs8193037A allele produced significantly higher levels of IL-17 than those with GG genotype ( p = 0.012, Fig. 1C). This phenomenon was also observed in the NSCLC patients ( p = 0.023, Fig. 1D). We further evaluated whether histological types of NSCLC could affect the function of rs8193037G/A SNP. When considering the GG genotype, we found that the excretion of IL-17 remained similar between adenocarcinoma and squamous cell carcinoma patients. However, when considering the A allele, level of IL-17 was moderately elevated in adenocarcinoma cases than in squamous cell carcinoma cases (Fig. 2). Discussion
Genetic variants could act as risk factors for NSCLC. A genome-wide association study (GWAS) has identified that rs7086803, rs9387478, and rs2395185 greatly contribute to lung cancer susceptibility in never-smoking women in Asia (Lan et al., 2012). Another research group has reported that rs2853677, rs10937405, rs7216064, and rs3817963 are associated with susceptibility to lung adenocarcinoma in the Japanese population (Shiraishi et al., 2012). In this study, we identified that IL-17A rs8193037G/A SNP was associated with increased susceptibility to NSCLC, especially to adenocarcinoma in the Han Chinese population. However, recent literature investigated another polymorphism in IL17A gene, rs2275913, and did not found any correlation with lung cancer risk in the Tunisian population (Kaabachi et al.,
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2014). It would be interesting to conduct a similar research in the Chinese population for comparison. The rs3819024A/G SNP is located at the 5¢ untranslated region of IL-17A gene. It has been reported that it is associated with an increased risk of gastric cardia adenocarcinoma in a Chinese population (Ren et al., 2014). In addition, Wang et al. (2012) have shown that the polymorphism may be involved in the development of allergic rhinitis with asthma. However, we did not find any association between this SNP and NSCLC in our study population (Table 2). Function of the polymorphism remains unclear. We evaluated whether rs3819024 could affect gene expression but did not observe any differences in IL-17 excretion from subjects with different genotypes (Fig. 1A, B), suggesting that the SNP may not affect the promoter activity of IL-17A gene. Studies have shown that rs8193037 may be involved in various diseases. Zhang et al. (2013) have reported that the polymorphism is associated with increased risk of inflammatory bowel disease in a Chinese Han population. Also, it has been shown that frequencies of IL-17A rs8193037 GG homozygote and G allele are significantly higher in the patients with coronary artery disease than those in the controls (Zhang et al., 2011). Our data revealed that rs8193037 GA and AA genotypes were associated with NSCLC (Table 2), indicating a functional significance of the polymorphism. rs8193037 is located at 122-bp upstream from the translation start site of IL-17A, which is close to the activator protein 1 (AP-1) region (Liu et al., 2004). It is possible that the SNP may affect the gene expression of IL-17A by interfering with AP-1. Our data showed that subjects with rs8193037A allele produced significantly higher levels of IL-17 than those with GG genotype ( p < 0.05, Fig. 1C, D), suggesting a potential mechanism of rs8193037 on the pathogenesis of human diseases. Our data identified that rs8193037G/A polymorphism was more correlated with risk of adenocarcinoma than squamous cell carcinoma (Table 3), but was not limited to adenocarcinoma (Table 4). Since the polymorphism may affect IL-17 expression, these results indicate that different histological types of NSCLC may have different responses to IL-17. In addition, our data showed that adenocarcinoma patients carrying rs8193037A allele had IL-17 production than squamous cell carcinoma patients with rs8193037A (Fig. 2), indicating a special correlation between IL-17 and adenocarcinoma. Further study on this field in needed. In summary, we identified that IL-17A rs8193037G/A SNP was associated with increased susceptibility to NSCLC, especially to adenocarcinoma. Functional analyses revealed that in vitro stimulated PBMCs carrying rs8193037A allele produced significantly more IL-17 than those with the GG genotype. These data indicate IL-17A polymorphism is associated with increased risk of NSCLC probably by elevating gene expression. Acknowledgments
This research was supported by Jinan Science Research Project Foundation (201102051) and Shandong Provincial Natural Science Foundation (ZR2013HM079). Disclosure Statement
No competing financial interests exist.
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Address correspondence to: Jie Liu, MD, PhD Department of Oncology Shandong Cancer Hospital and Institute Shandong Academy of Medical Sciences 440 Jiyan Road Jinan Shandong 250117 China E-mail: [email protected] Received for publication July 22, 2014; received in revised form September 1, 2014; accepted September 7, 2014.
