Single nucleotide polymorphism and haplotype association of the interleukin-8 gene with nasopharyngeal carcinoma

Clinical Immunology (2007) 125, 309–317

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y c l i m

Single nucleotide polymorphism and haplotype association of the interleukin-8 gene with nasopharyngeal carcinoma Ye-Sheng Wei a,b,⁎, Yan Lan a , Ren-Guang Tang a , Qun-Qing Xu c , Yan Huang a , Hong-Bing Nong d , Wei-Tong Huang a a

Center of Clinical Laboratory, Affiliated Hospital of Youjiang Medical College for Nationalities, Baise 533000, Guangxi, China b Institute of Medical Laboratory, Youjiang Medical College for Nationalities, Baise 533000, Guangxi, China c Center of Scientific Laboratory, Youjiang Medical College for Nationalities, Baise 533000, Guangxi, China d Department of Otolaryngology, Affiliated Hospital of Youjiang Medical College for Nationalities, Baise 533000, Guangxi, China Received 9 April 2007; accepted with revision 26 July 2007 Available online 27 August 2007

KEYWORDS Interleukin-8; Single nucleotide polymorphism; Haplotype; NPC

Abstract The cytokine interleukin-8 (IL-8) may play a role in the pathogenesis of nasopharyngeal carcinoma (NPC) through the modulation of tumor immune response or enhanced angiogenesis. Polymorphism of IL-8 gene, which may affect the production level of cytokine, has been inversely associated with a number of cancers. To test this hypothesis, we investigated the relationship of IL-8 gene polymorphisms and NPC in a Chinese population. We analyzed single nucleotide polymorphisms (SNPs) of IL-8 gene −845 T/C, −738 T/A, −353 A/T, −251 A/Tand + 678 T/C in 280 patients with NPC and 290 age and sex matched controls, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and polymerase chain reactionsequence specific primers method (PCR-SSP). There were significant differences in the genotype and allele distribution of −251 A/T polymorphism of the IL-8 gene among cases and controls. The −251 AA and AT genotypes were associated with a significantly increased risk of NPC as compared with the −251 TT genotypes (OR = 1.820, 95% CI, 1.120–2.959, P = 0.015 and OR = 1.590, 95% CI, 1.104–2.290, P = 0.013, respectively). Haplotype analysis revealed that the homozygosity of the AAT haplotype (defined by SNPs at positions −353, −251 and + 678) of IL-8 gene conveys the highest risk for NPC compared with the homozygosity for the TTC haplotype (OR = 1.396; 95% CI, 1.064– 1.831; P = 0.016). The −251 A/T polymorphism of IL-8 and its haplotype are associated with NPC in a Chinese population. Our data suggests that IL-8 gene may play a role in the development of NPC. © 2007 Elsevier Inc. All rights reserved.

⁎ Corresponding author. Center of Clinical Laboratory, Affiliated Hospital of Youjiang Medical College for Nationalities, Baise 533000, Guangxi, China. Fax: +86 776 2848653. E-mail address: [email protected] (Y.-S. Wei). 1521-6616/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2007.07.010

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Introduction Nasopharyngeal carcinoma (NPC) is a malignancy arising from the epithelial cells lining the nasopharynx. It has a marked geographic and ethnic distribution. It is rare among Caucasians in Western Europe and North America, with the incidence rate usually less than 1/100,000 [1]. The disease occurs at high frequency in Southern China. The highest aged-adjusted incidence has been reported in South China to be 30–50/100,000 [1], and it has caused very serious health problem in these areas. Etiological factors include tobacco smoking, alcohol consumption, Epstein–Barr virus infection, consumption of salted fish and other environmental factors [2–5]. In addition to genetic factors such as chromosomal aberrations, single nucleotide polymorphisms (SNPs) of XRCC1, HLA-E, and Cyclin D1 also play a role in the development of NPC [6–8]. Only a few people develop the disease in areas where NPC is endemic even though everyone is exposed to the same environment, suggesting that genetic differences such as single nucleotide polymorphisms may contribute to NPC carcinogenesis. However, the molecular mechanism of NPC induced by these factors is still unknown. Interleukin-8 (IL-8; CXCL8) belongs to the superfamily of CXC chemokines attracting neutrophils and macrophages and manifests a wide range of proinflammatory effects [9,10]. In addition, IL-8 has been implicated in a wide variety of other processes, including angiogenesis, tumor growth, invasion, and potential metastasis in cancer [11]. IL-8 is produced by various malignant cells including NPC [12–15], and increased blood levels of IL-8 have been demonstrated in several malignant cancer [16–18]. These observations suggested that the IL-8 gene might be a candidate for cancer. The gene encoding IL-8 is located on chromosome 4q13– q21 in humans. The IL-8 gene consists of four exons, three introns, and a proximal promoter region [19]. Polymorphisms at positions −251 of the IL-8 promoter region are correlated with IL-8 production or protein expression [20,21]. Recently, genetic polymorphisms of the IL-8 gene have been implicated in the susceptibility to a range of cancers, including oral cancer [22], breast carcinoma [23], and gastric cancer [24,25]. No studies, to date, have examined the association

Table 1

between genetic polymorphisms in IL-8 genes and NPC. In this study, we evaluated whether IL-8 gene −845 T/C, −738 T/A, − 353 A/T, − 251 A/T and + 678 T/C polymorphisms are associated with NPC in a Chinese population.

Materials and methods Study population The case-control population contained 570 adult unrelated Chinese who were selected from the same population living in China between December 2005 and November 2006 (Table 1). A total of 280 NPC patients were recruited from Department of Otolaryngology, West China hospital, Sichuan University. The only selection criterion for patients was that their NPC diagnosis had been pathologically confirmed. The patients (204 males; 76 females) had a mean (S.D.) age of 49.4 (9.5) years. The control group comprised 290 healthy volunteers who visited the general health check-up division at the West China hospital, Sichuan University. Selection criteria for controls were no evidence of any personal or family history of cancer or other serious illness. The mean age of the control group (192 males and 98 females) was 48.2 (10.3) years. There was no significant difference between patients and control subjects in terms of gender and age distribution. Written informed consent was obtained from all the subjects, and the study was performed with the approval of the ethics committee of Chinese Human Genome.

DNA extraction Genomic DNA was extracted from EDTA-anticoagulated peripheral blood leukocytes by the salting-out method [26]. Briefly, 5 ml of blood was mixed with Triton lysis buffer (0.32 M sucrose, 1% Triton X100, 5 mM MgCl2, H2O, 10 mM Tris–HCl, pH 7.5). Leukocytes were spun down and washed with H2O. The pellet was incubated with proteinase K at 56 °C and subsequently salted out at 4 °C using a saturated NaCl solution. Precipitated proteins were removed by centrifugation. The DNA in the supernatant fluid was dissolved in 500 ul H2O.

Primer sequences and reaction conditions for genotyping IL-8 polymorphisms

Gene

Method

Primer sequence

Annealing temperature (°C)

Restriction enzyme

Reference

−845 T/C

PCR-RFLP

61

Vsp I

Lee et al. [27]

−738T/A

PCR-RFLP

61

Xba I

Lee et al. [27]

−353 A/T

PCR-RFLP

59

Mfe I

Lee et al. [27]

−251 A/T

PCR-RFLP

57

Ase I

Heinzmann et al. [20]

+ 678 T/C

PCR-SSP

F: 5′-AACCCAGCAGCTCCAGTG-3′ R: 5′-AGATAAGCCAGCCAATCATT-3′ F: 5′-AACCCAGCAGCTCCAGTG-3′ R: 5′-AGATAAGCCAGCCAATCATT-3′ F: 5′-GAATTCAGTAACCCAGGCAT-3′ R: 5′-AAGCTTGTGTGCTCTGCTGTCTCT-3′ F: 5′-CCATCATGATAGCATCTGTA-3′ R: 5′-CCACAATTTGGTGAATTATTAA-3′ F: 5′-AGTTGAGCAAAAGGTAACTCAGA-3′ R: 5′-GTCATAACTGACAACATTGAACA-3′ R: 5′-GTCATAACTGACAACATTGAACG-3′

60

Lee et al. [27]

SNP and haplotype association of the IL-8 gene with nasopharyngeal carcinoma

311

Determination of IL-8 genotype

fragments of 341 and 193 bp for wild-type allele (allele T”), or 534 bp for mutation-type allele (allele C”), the fragments were separated by electrophoresis in 8% polyacrylamide gel and stained with ethidium bromide for visualization under UV light. The amplified products (− 738 T/A) were digested with XbaI (New England Biolabs) for 3 h at 37 °C, producing fragments of 302 and 232 bp for wild-type allele (allele T”), or 534 bp for mutation-type allele (allele A”), the fragments were separated by electrophoresis in 8% polyacrylamide gel ”

”

”

”

”

”

”

Figure 1 PCR-RFLP assay for analyzing the −353 A/T polymorphism of IL-8. PCR product was digested by MfeI and separated on 2.0% agarose gel electrophoresis. Lanes 1, 2 homozygous TT genotype showed only one fragment of 1527 bp, lane 3, 6 heterozygous AT genotype showed three fragments of 1527, 1230 and 297 bp, lanes 4, 5 homozygous AA genotype showed two fragments of 1230 and 297 bp, lane M was loaded with appropriate molecular markers.

Figure 2 PCR-RFLP assay for analyzing the −251 A/T polymorphism of IL-8. PCR product was digested by AseI and separated on 8% polyacrylamide gels electrophoresis. Lanes 1, 2 and 7 heterozygous AT genotype showed three fragments of 173, 152 and 21 (not shown) bp, lane 3, 6 homozygous TT genotype showed only one fragment of 173 bp, lanes 4, 5 homozygous AA genotype showed two fragments of 152 and 21 (not shown) bp, lane M was loaded with appropriate molecular markers.

”

The IL-8 promoter −845 T/C, −738T/A, −353 A/T, and −251 A/T genotypes were determined by using a polymerase chain reaction-restriction fragment length polymorphism method, IL-8 + 678 T/C genotypes were determined by using a polymerase chain reaction-sequence specific primers method (PCR-SSP), and the PCR primers were designed based on described previously [20,27] (see Table 1 for primer sequences and reaction conditions). The PCR reactions were performed in a total volume of 25 ul containing 100 ng genomic DNA, 0.5 μmol/l of each primer, 200 μmol/l of each dNTP, 10 × PCR buffer supplied by Invitrogen Corp (10 mmol/l Tris–HCl, pH 8.8, 50 mmol/l KCl), 2.0 mmol/l MgCl2, and 2.5 U of DNA Taq polymerase. The PCR cycle conditions consisted of an initial denaturation step at 94 °C for 5 min followed by 35 cycles of 30 s at 94 °C; 55 s at 61 °C for −845 T/C and −738 T/ A, 59 °C for −353 A/T, 57 °C for − 251 A/T and 60 °C for +678 T/C; 1 min at 72 °C; and a final elongation at 72 °C for 8 min. The amplified products (− 353 A/T) were digested with MfeI (New England Biolabs) for 3 h at 37 °C, producing fragments of 1230 and 297 bp for wild-type allele (allele A”), or 1527 bp for mutation-type allele (allele T”), the fragments were separated by electrophoresis in 2.0% agarose gel and stained with ethidium bromide for visualization under UV light (Fig. 1). The amplified products (−251 A/T) were digested with AseI (New England Biolabs) for 3 h at 37 °C, producing fragments of 152 and 21 bp for wild-type allele (allele A”), or 173 bp for mutation-type allele (allele T”), the fragments were separated by electrophoresis in 8% polyacrylamide gel and stained with ethidium bromide for visualization under UV light (Fig. 2). The amplified products (+ 678 T/C) were separated by electrophoresis in 8% polyacrylamide gel and stained with ethidium bromide for visualization under UV light (Fig. 3). The amplified products (−845 T/C) were digested with VspI (New England Biolabs) for 3 h at 37 °C, producing

Figure 3 PCR-SSP assay for analyzing the + 678 T/C polymorphism of IL-8. PCR product was separated on 8% polyacrylamide gels electrophoresis. PCR product was 321 bp, Lanes 1–3 showed homozygous TT, heterozygous TC and homozygous CC genotype, respectively, lane M was loaded with appropriate molecular markers.

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Y.-S. Wei et al. genotype frequencies among the subjects with the expected genotype frequencies. The linkage disequilibrium (LD) between the polymorphisms was quantified using the Shi’s standardized coefficient D′ (|D′|) [28]. The haplotypes and their frequencies were estimated based on a Bayesian algorithm using the Phase program [29]. Statistical significance was assumed at the P b 0.05 level. The SPSS statistical software package version 11.5 was used for all of the statistical analysis.

Results Characteristics of the study population

Figure 4 PCR-RFLP assay for analyzing the −845 T/C polymorphism of IL-8. PCR product was digested by VspI and separated on 8% polyacrylamide gels electrophoresis. Lanes 1– 7 homozygous TT genotype showed two fragments of 341 and 193 bp, lane M was loaded with appropriate molecular markers.

and stained with ethidium bromide for visualization under UV light (IL-8 − 845 T/C and − 738 T/A did not show any polymorphism in either patients or controls, Figs. 4 and 5).

The demographics of the cases and controls enrolled in this study are shown in Table 2. There were no significant differences between the cases and controls for the mean age or gender distribution, smoking and alcohol consumption status, and this suggested that the matching based on these two variables was adequate. In the 280 NPC patients, the diagnosis was poorly differentiated squamous cell carcinoma (n = 229), others include poorly differentiated adenocarcinoma (n = 2), moderately differentiated squamous cell carcinoma (n = 7), and undifferentiated cancer (n = 42).

The genotype and allele frequencies of IL-8 Statistical analysis Genotype and allele frequencies of IL-8 were compared between NPC cases and controls using the χ2 test and Fisher’s exact test when appropriate, and odds ratios (OR) and 95% confidence intervals (CIs) were calculated to assess the relative risk conferred by a particular allele and genotype. Demographic and clinical data between groups were compared by χ2 test and by Student’s t-test. Hardy– Weinberg equilibrium was tested with a goodness of fit χ2test with one degree of freedom to compare the observed

Among the five SNPs investigated in this study, IL-8 − 845 T/C and −738 T/A did not show any polymorphism in either patients or controls. Therefore, these two sites were excluded from the following analysis. The genotype and allele frequencies of the IL-8 gene −353 A/T, − 251 A/T and +678 T/C polymorphisms among the controls and the cases are shown in Table 3. The genotype distributions of the three polymorphisms among the controls and the cases were in Hardy–Weinberg equilibrium. The frequencies of the AA, AT, and TT genotypes of −251 A/Twere 14.5%, 42.1%, and 43.4% in controls, and were 19.3%, 48.9%, and 31.8% in cases, respectively. There were significant differences in the genotype and allele frequencies of the IL-8 gene −251 A/T polymorphism between NPC and control

Table 2

Figure 5 PCR-RFLP assay for analyzing the −738 T/A polymorphism of IL-8. PCR product was digested by XbaI and separated on 8% polyacrylamide gels electrophoresis. Lanes 1– 6 heterozygous TT genotype showed two fragments of 302 and 232 bp, lane M was loaded with appropriate molecular markers.

Characteristics of the study population

Variable

NPC patients n = 280 (%)

Controls n = 290 (%)

P

Age (mean ± S.D.) Sex Male Female Cigarette smoking Non-smokers Smokers Alcohol consumption Non-drinkers Drinkers Clinical stages Stages I and II Stages III and IV

49.4 ± 9.5

48.2 ± 10.3

0.573

204 (72.9) 76 (27.1)

192 (66.2) 98 (33.8)

0.085

71 (25.4) 209 (74.6)

84 (29.0) 206 (71.0)

0.333

82 (29.3) 198 (70.7)

101(34.8) 189 (65.2)

0.157

90 (32.1) 190 (67.9)

SNP and haplotype association of the IL-8 gene with nasopharyngeal carcinoma

+678 [|D′| = 0.883]. Major TTC haplotype accounted for 51.1% and 60.2% of these eight haplotypes in both the cases and the controls, respectively. By haplotype analyses, we found that AAT haplotype was associated with a significantly increased risk of NPC as compared with the TTC haplotype (OR = 1.396; 95% CI, 1.064–1.831; P = 0.016).

Table 3 The genotype and allele frequencies of IL-8 polymorphism in NPC patients and controls Polymorphism

NPC patients n = 280 (%)

IL-8 −353 A/T genotypes AA 55 (19.6) AT 139 (49.6) TT alleles 86 (30.7) A 249 (44.5) T 311 (55.5) IL-8 −251 A/T genotypes AA 54 (19.3) AT 137 (48.9) TT alleles 89 (31.8) A 245 (43.8) T 315 (56.3) IL-8 +678 T/C genotypes TT 38 (13.6) TC 104 (37.1) CC alleles 138 (49.3) T 180 (32.1) C 380 (67.9)

Controls n = 290 (%)

χ2

P

47 (16.2) 131 (45.2) 112 (38.6) 225 (38.8) 355 (61.2)

4.104

0.128

42 (14.5) 122 (42.1) 126 (43.4) 206 (35.5) 374 (64.5)

3.772

The genotype and allele frequencies of IL-8 polymorphisms in relation to pathological indices of NPC severity

0.052

8.563

0.014

8.076

0.004

No association was found between IL-8 gene -353A/T, -251 A/T and +678 T/C polymorphisms and different clinical stafe as shown in Table 5. Genotype and allele frequencies of the IL-8 gene -353 A/T, - 251 A/T and +6787 T/C polymorphisms in Stage I and II were not significantly different than that in Stages III and IV of PNC patients (P N 0.05).

Discussion 35 (12.1) 111 (38.3) 144 (49.7) 181 (31.2) 399 (68.8)

0.304

0.859

0.115

0.734

groups. The −251 AA and AT genotypes were associated with a significantly increased risk of NPC as compared with the −251 TT genotypes (OR = 1.820, 95% CI, 1.120–2.959, P = 0.015 and OR = 1.590, 95% CI, 1.104–2.290, P = 0.013, respectively). The −251 A allele was associated with a significantly increased risk of NPC as compared with the − 251 T allele (OR = 1.412, 95% CI, 1.113–1.792, P = 0.004). However, genotype and allele frequencies of the IL-8 −353 A/T and +678 T/C polymorphisms in NPC patients were not significantly different than that in healthy controls (P N 0.05).

Haplotype analysis of the IL-8 gene Haplotype analyses were performed and the possible eight haplotype frequencies are shown in Table 4. Linkage disequilibrium was observed between allele A at locus −353 and allele A at locus −251 [|D′| = 0.902] and allele A at locus −353 and allele T at locus +678 [|D′| = 0.876], and linkage disequilibrium was observed between locus −251 and locus Table 4

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NPC remains as a public health problem in many countries in endemic areas including China. Although it is treatable, most of NPC patients are diagnosed at a late stage of the disease and too late to be treated. Hence, the potential risk factors for prediction of which patients without clinical symptoms are likely to have NPC would be valuable to physicians and NPC patients. Because it will provide more informed planning for an early and efficient treatment. There have been many attempts to detect primary tumor characteristics with the potential tumor markers. Although some serological assays including viral genome detection for NPC correlate with early diagnosis, prognosis, recurrence and survival, molecular markers for the cancer predisposition and susceptibility are awaited. In this study, we investigated the IL-8 gene polymorphisms and determined whether these genetic factors are related to the occurrence of NPC in a Chinese population. Our results showed that the IL-8 gene − 251 A/T polymorphism was significantly associated with the risk of NPC. The − 251 AA and AT genotypes were associated with a significantly increased risk of NPC as compared with the − 251 TT genotypes (OR = 1.820, 95% CI, 1.120–2.959, P = 0.015 and OR = 1.590, 95% CI, 1.104–2.290, P = 0.013, respectively). Moreover, their haplotype AAT haplotype was associated with a significantly increased risk of NPC as compared with the TTC haplotype (OR = 1.396; 95% CI, 1.064–1.831; P = 0.016). This finding

Haplotype distribution in the patients with NPC and in controls

IL-8 gene (−353/−251/+ 678) haplotypes

Cases 2n = 560 (%)

Controls 2n = 580 (%)

OR (95% CI)

P

TTC AAT TTT TAC TAT ATC ATT AAC

286 (51.1) 167 (29.8) 11 (2.0) 8 (1.4) 5 (0.9) 6 (1.1) 9 (1.6) 68 (12.1)

349 146 7 3 1 2 5 67

1.000 1.396 1.918 3.254 6.101 3.661 2.197 1.238

0.016 0.177 0.068 0.061 0.091 0.153 0.259

(60.2) (25.2) (1.2) (0.5) (0.2) (0.3) (0.9) (11.6)

(Ref) (1.064–1.831) (0.734–5.010) (0.855–12.379) (0.709–52.522) (0.733–18.276) (0.728–6.627) (0.854–1.796)

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Table 5 The genotype and allele frequencies of IL-8 polymorphism in relation to pathological indices of NPC severity Variable

χ2

NPC patients

P

Clinical stages Stages I and Stages III and II n = 90 (%) IV n = 190 (%) IL-8 −353 A/T genotypes AA 16 (17.8) AT 46 (51.1) TT alleles 28 (31.1) A 78 (43.3) T 102 (56.7)

39 (20.5) 93 (48.9) 58 (30.5) 171 (45.0) 209 (55.0)

0.299 0.861

IL-8 −251 A/T genotypes AA 15 (16.7) AT 45 (50.0) TT alleles 30 (33.3) A 75 (41.7) T 105 (58.3)

39 (20.5) 92 (48.4) 59 (31.1) 170 (44.7) 210 (55.3)

0.603 0.740

IL-8 + 678 T/C genotypes TT 13 (14.4) TC 31 (34.4) CC alleles 46 (51.1) T 57 (31.7) C 123 (68.3)

25 (13.2) 73 (38.4) 92 (48.4) 123 (32.4) 257 (67.6)

0.424 0.809

0.137 0.711

0.468 0.494

0.028 0.868

suggests that IL-8 gene −251 A/T polymorphism could be used as genetic susceptibility markers of the NPC. There were other epidemiological studies in diverse ethnic populations found inconsistent results with ours, and majority of them have focused on the A/T polymorphism at −251 upstream from the transcriptional start site. In the current study, the frequencies of the −251 A alleles among the healthy controls was 0.355, and this was similar to those frequencies observed in healthy Korean and Japanese (0.357 and 0.314, respectively) [27,30], but the frequencies were lower than those of American, African and European populations (0.761, 0.902 and 0.513, respectively) [31–33]. The frequencies of the − 353 A and +678 T alleles among the healthy controls were 0.388 and 0.312, respectively, and these were similar to those frequencies observed in healthy Korean (0.370 and 0.298, respectively) [27], but the frequencies were significantly different than those of European Caucasians (0.560 and 0.400, respectively) [34]. However, IL-8 −845 T/C and − 738 T/A did not show any polymorphism in our Chinese population as well as in the Korean population [27]. We also found that the −353 A/T, − 251 A/T and +678 T/C polymorphisms were in strong linkage disequilibrium. Two major haplotype frequencies of the TTC and AAT among the controls in the present study were 0.602 and 0.252, respectively, which were similar to those of study performed in Korea [27], suggesting that the distribution of IL-18 gene frequencies might vary among the different ethnic groups. The contribution of inflammation and inflammatory cells to the process of tumor development and progression is increasingly recognized [35,36]. It is now evident that a substantial proportion of cancer cases worldwide arise from

infection and chronic inflammation [37]. Both inflammatory and tumor cells produce an assorted array of cytokines and chemokines, which mediate all aspects of inflammation and profoundly affect the development and progression of cancer [38,39]. Inflammatory infiltrates may affect these processes via their ability to express a large variety of factors, including inflammatory chemokines. Members of the chemokine family have been observed to contribute to both the growth and the progression of different types of human cancer. IL-8 is a small basic protein that was purified as a neutrophil chemoattractant. It has been reported that, in addition to being a strong neutrophil chemoattractant, IL-8 possesses potent mitogenic, angiogenic, and motogenic properties in different cancer models. Most cancer cells, including NPC [15,40] . The cytokine IL-8 was originally identified as a leukocyte chemoattractant and a member of the CXC chemokine family. Although the precise mechanisms regulating IL-8 expression in tumor cells remain unknown, several studies have shown that IL-8 has some potential functions in different cancer types, including a role in angiogenesis, tumor growth, and metastasis [41–45]. Furthermore, it has been reported that serum IL-8 levels may be used as a serum marker for monitoring the clinical course of patients with some cancer types, including ovarian cancer [46], squamous cell cancer of the head and neck [47], and prostate cancer [48], indicating that IL-8 could play a part in the pathogenesis of cancer. The gene coding for IL-8 is within the CXC chemokine locus on chromosome 4q13–q21. The IL-8 gene consists of four exons, three introns, and a proximal promoter region. A common − 251 A/T polymorphism of the IL-8 promoter has been reported to influence production and expression of IL-8 [24,49]. Previous reports have demonstrated that IL-8 (−251) A allele was significantly associated with higher IL-8 level, and increased risk of gastric cancer [24,49]. These characteristics of IL-8 prompted us to evaluate whether genetic variation of the IL-8 gene affects the susceptibility to and prognosis significance of NPC. In the current study, we found − 251 A allele and its AAT haplotype were significantly higher in the NPC patients than in controls. Thus it is possible that a higher promoter activity of − 251 A allele and its AAT haplotype of the IL-8 might increase expression of IL-8, resulting in a high profuction of IL-8, which may induce a Th1predominant immune response, leading to be more susceptible to NPC than a low production of IL-8. Our results suggest that the −251 A allele and its AAT haplotype of IL-8 gene may play a facilitative role in the development of NPC. A number of molecular epidemiologic studies on the IL-8 genotypes and different cancer types susceptibility have been reported. In concordance with our study, Vairaktaris et al. [22] have reported that an increased risk of oral squamous cell carcinoma for − 251 A allele carriers versus non-carriers (P b 0.05). Taguchi et al. [24] have reported that the IL-8 −251 A/A genotype held a higher risk of atrophic gastritis (OR = 2.35; 95% CI, 1.12–4.94) and gastric cancer (OR = 2.22; 95% CI, 1.08–4.56) compared with the T/T genotype. Ohyauchi et al. [49] have reported that IL-8 − 251 A was associated with a higher risk of gastric cancer and gastric ulcer. Patients carrying IL-8 −251 A showed an increased risk of gastric cancer (OR = 2.01; 95% CI, 1.38–2.92) and gastric ulcer (OR = 2.07; 95% CI, 1.37–3.12). Lu et al. [50] have also reported that the risk of gastric cancer was significantly elevated in subjects with the IL-8 −251 AA (OR = 2.02; 95% CI,

SNP and haplotype association of the IL-8 gene with nasopharyngeal carcinoma 1.27–3.21). In contrast to these four previous studies and our current study, Shirai et al. [51] have reported that the IL-8 −251 T/T genotype was significantly associated with increased risk of gastric carcinoma compared to controls. Lee et al. [25] have reported that the IL-8 −251T allele was significantly associated with increased risk of gastric carcinoma. Kamangar et al. [52] and Savage et al. [33] have reported that there was no apparent relationship of the IL-8 −251 A/T polymorphism with the risk of gastric cancer. In addition, results with respect to prostate cancer are conflicting too [53,54]. Originally, IL-8 − 251 A/T polymorphism was reported to be associated with the progression of breast carcinoma, Snoussi et al. [55] and Smith et al. [56], demonstrated that a significant association between the IL-8 (− 251) AA homozygous genotype and the aggressive phenotype of breast carcinoma as defined by the high histological grade. However, there was no statistically significant association between this IL-8 gene polymorphism and the advanced stage of NPC in our data. Also, the reason for these discrepancies remains unclear, but several possibilities should be considered. First, it may be due to the genetic trait differences, IL-8 gene polymorphisms were distinct in specific population, various ethnicity and geographic region. Furthermore, cancer is a multi-factorial disease and individual exposure to various environmental factors, and genetic susceptibility might have caused different results. In addition, the inadequate study design such as non-random sampling and a limited sample size should also be considered. The possible selection bias that might have been present in the hospital-based, case-control study is a relevant issue. Finally, we cannot exclude that the observed association depends on a gene in linkage disequilibrium with the IL-8 gene or on the effect of IL-8 on another peptide. In conclusion, to our knowledge, this is the first study of the association of the IL-8 gene −845 T/C, −738 T/A, −353 A/T, −251 A/Tand +678 T/C polymorphisms, their haplotypes and NPC risk. Our results showed that the − 251 A/T polymorphism and AAT haplotype have a significant association with the presence of NPC. However, the numbers of cases and controls were relatively small in our study, so additional studies with larger sample sizes will be needed to validate the genetic effects of the IL-8 polymorphisms on NPC, further studies of the IL-8 sequence variants and their biologic function are also needed, which may help us to identify those individuals most susceptible to NPC in diverse ethnic populations.

Acknowledgments This study was supported by the National Natural Science Foundation (No. 30660159); by grant from the Nature Science Fund, Guangxi Province, China (No. 0499006); and the Foundation of the Education Department of Guangxi Province, China (No. 200420).

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