Human Immunology 75 (2014) 297–301

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Genetic polymorphism of interleukin-6 influences susceptibility to HBV-related hepatocellular carcinoma in a male Chinese Han population Shengli Tang a, Yufeng Yuan a, Yueming He a, Dingyu Pan a, Yongxi Zhang b, Yuanyuan Liu c, Quanyan Liu a, Zhonglin Zhang a, Zhisu Liu a,⇑ a

Department of General Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, China Department of Infectious Diseases, Zhongnan Hospital of Wuhan University, Wuhan 430071, China c State Key Laboratory of Virology, Institute of Virology, Medical School of Wuhan University, Wuhan 430071, China b

a r t i c l e

i n f o

Article history: Received 19 June 2013 Accepted 4 February 2014 Available online 12 February 2014

a b s t r a c t As a multifunctional cytokine, interleukin-6 (IL-6) plays a key role in chronic inflammation as well as tumor growth and progression of hepatitis B virus (HBV) infection. Recent studies have implicated that single nucleotide polymorphism (SNP) 572C>G (rs1800796) located within the promoter region of IL6 gene was associated with susceptibility to several diseases. Here, a case–control study was undertaken to investigate the association between this polymorphism and HBV-related hepatocellular carcinoma (HCC) susceptibility in a Chinese Han population. A total of 900 patients with chronic HBV infection, including 505 HBV-related HCC patients and 395 HBV infected patients without HCC were enrolled, and rs1800796 polymorphism was genotyped by the TaqMan method and DNA sequencing technology. The results indicated no significant association between rs1800796 polymorphism and the risk of HBV-related HCC in all subjects; however, a significant difference was identified in male subjects. Under the dominant model, male subjects with the G allele (CG/GG) have higher susceptibility to HBV-related HCC than those with CC genotype after adjusting confounding factors (P = 0.012, odds ratio [OR] 1.68, 95% confidence interval [95% CI] 1.15–2.42). Our results suggested that rs1800796 polymorphism of IL-6 gene was associated with susceptibility to HBV-related HCC in a male Chinese Han population. Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

1. Introduction Hepatocellular carcinoma (HCC) represents an international public health concern in that it is a common malignancy. The burden of HCC is expected to continue to increase, and it would be the tumor with the second highest increase in overall death rates [1]. The incidence rate of HCC varies considerably in the world, and Abbreviations: AFP, a-fetoprotein; ALB, albumin; ALT, alanine aminotransferase; CI, confidence interval; CT, computed tomography; DEN, diethylnitrosamine; HBcAb, hepatitis B virus core antibody; HBeAb, hepatitis B virus e antibody; HBeAg, hepatitis B virus e antigen; HBsAb, hepatitis B virus surface antibody; HBsAg, hepatitis B virus surface antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HIV, human immunodeficiency virus; IL-6, interleukin-6; MRI, magnetic resonance imaging; OR, odds ratio; PLT, platelet; SNP, single nucleotide polymorphism; T-Bil, total bilirubin. ⇑ Corresponding author. Address: Department of General Surgery, Zhongnan Hospital of Wuhan University, No. 169, Donghu Road, Wuhan 430071, China. Fax: +86 27 67812892. E-mail address: [email protected] (Z. Liu).

China alone accounts for approximately half of HCC malignancies [2,3]. HCC is etiologically associated with hepatitis B virus (HBV) in most cases, and is the dominant cause of death among HBV carriers with a lifetime risk of 27% for males and 4% for females [4]. Although chronic HBV infection is recognized as the most important causal factor for HCC in humans [5,6], it is known that only a minority of chronic carriers of HBV develop HCC [7], suggesting the presence of important cofactors in HBV-related HCC. The chronic HBV carriers with a family history of HCC have a twofold risk for HCC than those without the family history [8], strongly suggesting the importance of genetic susceptibility for HBV-related HCC. Therefore, a better understanding of the genes involved in tumor growth and progression is necessary for the development of novel diagnostic and therapeutic strategies that can improve the treatment of HCC. Interleukin-6 (IL-6) is a multifunctional cytokine that was originally characterized as a regulator of immune and inflammatory responses. Mounting evidences have confirmed that IL-6 play

http://dx.doi.org/10.1016/j.humimm.2014.02.006 0198-8859/Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

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S. Tang et al. / Human Immunology 75 (2014) 297–301

an important role in the pathophysiology and development of HCC. High concentration of IL-6 during liver injury can be detected in a murine model of HCC using diethylnitrosamine (DEN) as carcinogen [9]. In addition, high serum IL-6 level pedates the development of HCC in chronic hepatitis B patients, and has moderate accuracy in predicting future cancer [10], which demonstrates that increased IL-6 production is implicated in the pathogenesis of HCC. The gene encoding for IL-6 is located on chromosome 7p21 in humans, is composed of five exons, four introns, and a proximal promoter region [11]. A part of the single nucleotide polymorphisms (SNPs) identified in the IL-6 gene, especially within the non-coding promoter region, has been shown to have a powerful influence on the expression of the gene [12,13]. Recently, genetic polymorphisms of IL-6 have been extensively studied, and evidence demonstrates that several polymorphisms are associated with a range of diseases including cancer [14–19]. In light of the important role of IL-6 in HCC development and progression, we hypothesized that genetic polymorphisms of IL-6 was associated with the susceptibility of HBV-related HCC. To test this hypothesis, we performed a case–control study to investigate the association between rs1800796 (572C>G) polymorphism of IL-6 gene and HBV-related HCC susceptibility in a Chinese Han population.

knowing the subjects’ case or control status; more than 10% of samples were randomly selected for repeat analysis (which yielded 100% concordance). Finally, 10% of samples were analyzed using an ABI 3730 DNA sequencer (Applied Biosystems, Foster City, CA) to confirm the accuracy of this method. 2.3. Statistical analysis Continuous variables were expressed as mean (SD). Student’s t test or the v2 test was used to compare individual characteristics between subjects with and without HCC. Deviation from Hardy– Weinberg equilibrium was tested by using the v2 test for goodness of fit. The differences of genotypes and allelic frequencies between individuals with and without HCC were determined using a standard v2 test. Binary logistic regression was conducted to estimate the relative risk of this SNP adjusted for age (in years), gender and drinking history. Odds ratios (ORs) and their 95% confidence intervals (95% CI) were calculated. The v2 test was used to perform for the association of clinicopathologic characteristics

Table 1 General characteristics of the subjects.

2. Subjects and methods 2.1. Subjects A total of 900 subjects were periodically enrolled between December 2007 and February 2013 at the Zhongnan Hospital of Wuhan University, including 505 HBV-related HCC patients and 395 non-HCC patients with HBV infection. The subjects were exclusively unrelated Han Chinese and recruited without restrictions on gender and age. All of them were further confirmed to be HBsAg (hepatitis B virus surface antigen) positive, HBcAb (hepatitis B virus core antibody) positive, and HBeAg (hepatitis B virus e antigen) or HBeAb (hepatitis B virus e antibody) positive for at least 6 months. The diagnosis of HCC was histopathologically confirmed. The presence of HCC or liver cirrhosis was excluded in non-HCC patients by histology, computed tomography (CT), magnetic resonance imaging (MRI), ultrasonography, and laboratory tests. Moreover, patients with positive laboratory tests for human immunodeficiency virus (HIV), hepatitis C virus (HCV; anti-HCV and/or HCV-RNA), alcoholic liver disease, or autoimmune diseases were excluded. A one-time sample of approximately 3–5 ml of venous blood was collected from each participant. The following laboratory parameters were obtained for each participant at the time of whole-blood collection: serum albumin (ALB), total bilirubin (T-Bil), alanine aminotransferase (ALT), a-fetoprotein (AFP), and HBV DNA level, and HBsAg, HBsAb (hepatitis B virus surface antibody), HBeAg, HBeAb, HbcAb, and platelet count (PLT). All subjects provided informed consent to participate in the study, as approved by the ethical committee of Zhongnan Hospital of Wuhan University, Wuhan, China. 2.2. DNA extraction and genotyping Genomic DNA was extracted from peripheral whole blood using the QIAamp DNA Blood Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Samples were stored at 80° until genetic polymorphism analyses were performed. IL-6 polymorphism (rs1800796) genotyping was performed using the TaqMan 50 allelic discrimination assay technology in a 7500 Real-Time PCR System according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Genotyping was performed without

HBV patients with HCC (n = 505)

HBV patients without HCC (n = 395)

Age (y) Male, n (%) Drinkera, n (%) ALTb (U/L) T-Bilb (umol/l) ALBb (g/L) AFPb (ng/ml) PLTb (109/L) HBV DNAb (log10 copies/mL) HBeAg positiveb, n (%) HBeAb positvieb, n (%)

48.9 (10.5) 386 (76.4) 143 (28.3) 92.4 (42.8) 33.4 (8.9) 32.9 (4.6) 562.9 (201.6) 125.3 (34.8) 5.23 (2.71) 107 (21.2) 366 (72.5)

43.6 (9.3) 289 (73.3) 87 (22.1) 57.3 (12.7) 13.7 (6.2) 43.7 (3.5) 18.9 (9.7) 180.1 (42.6) 6.42 (3.53) 160 (40.5) 207 (52.4)

HBV genotype, n (%) B C No data

191 (37.8) 249 (49.3) 65 (12.9)

175 (44.3) 202 (51.1) 18 (4.6)

Tumor number (n = 505), n (%) Single Multiple

334 (66.1) 171 (33.9)

Tumor size (n = 505), n (%) 0.05, respectively). A higher proportion of drinkers was observed in the HBV-related HCC patients (28.3% vs. 22.1%, P = 0.033). In addition, the positive rate of HBeAb was significantly higher in patients with HCC than those of patients without HCC (72.5% vs. 52.4%, P < 0.001), and the two groups had statistically different laboratory results for HBV DNA, ALB, T-Bil, ALT, AFP and PLT (P < 0.001). 3.2. rs1800796 polymorphism of the subjects Genotype and allelic frequencies of IL-6 polymorphism (rs1800796) of the subjects are presented in Table 2. The genotype distributions were in Hardy–Weinberg equilibrium in each group. As the low frequency of GG genotype, the CG and GG genotype were combined for analysis. As shown in Table 2, the CC genotype of rs1800796 prevailed in two groups and the frequencies of the CC, CG and GG genotypes were 61.4%, 34.2% and 4.4% in the HBV-related HCC patients, and were 67.6%, 29.9% and 2.5% in HBV patients without HCC, respectively. Although the allele and genotype distribution (dominant model) of rs1800796 was not significantly different in patients with HCC and without HCC after adjusting confounding factors age, gender, and drinking history (P = 0.136, OR = 1.56, 95% CI = 0.79–1.85), a coincident trend can be observed that a high frequency of the CG or GG genotype was found in the HBV-related HCC patients, which revealed that subjects with the G allele seemed to have a higher susceptibility to HBV-related HCC than those with CC genotype. We further compared frequency distribution of genotypes/ alleles of rs1800796 polymorphism in subjects after segregation on the basis of gender. Interestingly, in male patients (Table 3), the frequencies of the CC, CG, and GG genotypes of rs1800796 were

HBV patients without HCC (n = 105)

OR (95% CI)a

Pa

Genotype, n (%) CC 78 (65.5) CG 34 (28.6) GG 7 (5.9)

64 (61.0) 35 (33.3) 6 (5.7)

1.00 0.76 (0.42–1.51) 0.83 (0.35–3.04)

0.536 0.986

Allele, n (%) C G

163 (77.6) 47 (22.4)

1.00 0.79 (0.62–1.58)

0.674

64 (61.0) 41 (39.0)

1.00 0.93 (0.52–1.64)

0.745

rs1800796

HBV-related HCC patients (n = 119)

190 (79.8) 48 (20.2)

Dominant model CC 78 (65.5) CG + GG 41 (34.5) a

P value or OR after adjusting for age and drinking history. OR, odds ratio; CI, confidence interval.

60.1%, 36.0% and 3.9% in HBV-related HCC patients, and 70.0%, 28.6% and 1.4% in patients without HCC, respectively. There were significant differences in the genotype and allele frequencies of rs1800796 polymorphism between the two groups. The rs1800796 CG and GG genotypes were associated with a significantly increased risk of HBV-related HCC compared with the CC genotype after adjusting for age and drinking history (P = 0.039, OR = 1.53, 95% CI = 1.09–2.12; P = 0.042, OR = 3.43, 95% CI = 1.23– 11.06, respectively). The data also revealed that male subjects with the G allele have higher susceptibility to HBV-related HCC than those with the C allele (P = 0.009, OR = 1.60, 95% CI = 1.09–2.24). Under the dominant model, genotype CG/GG was associated with an increased risk of HBV-related HCC (P = 0.012, OR = 1.68, 95% CI = 1.15–2.42); however, the association was not significant in female patients (P = 0.745) (see Table 4). In addition, we performed stratification analyses according to other clinical pathologic characteristics, including tumor number (single vs. multiple), size (P5 cm vs. C (rs1800795), -572C>G (rs1800796), and -596G>A (rs1800797) have been extensively studied and reported to be associated with susceptibility of a range of cancers, including cervical cancer [17], tobacco-related oral carcinoma [18], and colorectal cancer [19]. However, a different genetic background of ethnic diversity with respect to the allele frequencies of IL-6 promoter SNPs can be observed. Compared with Caucasian population, the allele frequencies of -174G>C and -596G>A are extremely rare in Asian populations [20,21], therefore, the two polymorphisms are unlikely to be contributing significantly to disease susceptibility in Chinese population. On the other hand, the G allele of rs1800796 polymorphism dominates in Caucasians [13], whereas the C allele is most common in Asian populations [21]. According to the results of this study, the minor allele frequency of rs1800796 polymorphism in all subjects (G allele: 19.7%) is consistent with that reported in previous study carried out in South China region (G allele: 19.3%) [22]. As high frequency population specific alleles are particularly useful for mapping genes the responsible for disease susceptibility [23], the SNP of IL6 gene rs1800796 can be selected as a more useful marker for an association study in Chinese population. In the case–control study described here, although the frequency of CG and GG genotype of rs1800796 among HCC patients was higher than that in the HBV patients without HCC group (34.2% vs. 29.9% and 4.4% vs. 2.5%, respectively), no significant association between rs1800796 polymorphism and the risk of HBV-related HCC was found in all subjects, which is consistent with the results of previous study performed in Korean population [21]. However, after stratification of all subjects based on gender, in male subjects, we observed that the risk of HCC was significantly higher among patients carrying the G allele for rs1800796 than among the patients with the CC genotype, suggesting that the risk of HBV-related HCC is conferred by the G allele only in male but not in female individuals. Previous report show that this important promoter variant can influence IL-6 transcription through a complex interaction, and the G allele of this locus has been found to be associated with increased transcription efficiency of IL-6 [24]. In conformity with this, the subjects with G allele had a greater IL-6 secretion capacity than those without it [25,26]. Although previous study demonstrated that high serum IL-6 level can predict the development of HCC in chronic hepatitis B patients; however, the significant association between rs1800796 and HBV-related HCC susceptibility was only detected in male subjects in this study, which indicated the effect of this polymorphism on developing HCC might be counteracted by the protective estrogenic hormone-related pathway in females. Recent reports demonstrate that, in a murine model, estrogen inhibited secretion of IL-6 from Kupffer cells exposed to necrotic hepatocytes and reduced circulating concentrations of IL-6 in DEN-treated male mice, which indicated that estrogenmediated inhibition of IL-6 production may reduce liver cancer risk in females [9]. Meanwhile, estrogen has the potential to inhibit lung metastasis from rat HCCs in vivo by suppression of IL-6 production [27].

In conclusion, our study found that rs1800796 polymorphism of IL-6 gene was associated with susceptibility to HBV-related HCC in a male Chinese Han population; however, in view of the relatively small sample size and only one polymorphism of IL-6 gene was genotyped in this study, the present results should be viewed cautiously. Further investigation with a larger sample size in which other SNPs were enrolled for multiple statistical tests may be requested to rule out the possible false positive of current study. Our finding suggested that polymorphism in the IL-6 gene might be a candidate risk factor for developing HBV-related HCC. Screening of these polymorphisms and functional studies would be useful in clinical practice to identify groups at high risk for HCC and might help to modify the design of HCC surveillance programs for patients with chronic HBV infection. Further studies of epidemiologic analysis and biologic function are needed to explore the role of IL-6 in HCC carcinogenesis. Author contributions SLT and ZSL conceived and designed the experiments. SLT and YYL performed the experiments. SLT performed the statistical analysis and wrote the first draft. YFY, YMH, DYP, YXZ, QYL, and ZLZ provided the specimens and clinical data of the patients. All authors contributed to further drafts, and have read and approved the final manuscript. Acknowledgments The work was supported by the National Natural Science Foundation of China (Nos. 81172349 and 81272692) and Science and Technology Project of Wuhan (No. 2013060501010153). References [1] Parkin DM. The global health burden of infection-associated cancers in the year 2002. Int J Cancer 2006;118:3030–44. [2] Sener SF. Disease without borders. CA Cancer J Clin 2005;55:7–9. [3] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69–90. [4] Dickinson JA, Wun YT, Wong SL. Modelling death rates for carriers of hepatitis B. Epidemiol Infect 2002;128:83–92. [5] Yang JD, Roberts LR. Hepatocellular carcinoma: a global view. Nat Rev Gastroenterol Hepatol 2010;7:448–58. [6] El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007;132:2557–76. [7] Yu MW, Hsu FC, Sheen IS, Chu CM, Lin DY, Chen CJ, et al. Prospective study of hepatocellular carcinoma and liver cirrhosis in asymptomatic chronic hepatitis B virus carriers. Am J Epidemiol 1997;145:1039–47. [8] Yu MW, Chang HC, Liaw YF, Lin SM, Lee SD, Liu CJ, et al. Familial risk of hepatocellular carcinoma among chronic hepatitis B carriers and their relatives. J Natl Cancer Inst 2000;92:1159–64. [9] Naugler WE, Sakurai T, Kim S, Maeda S, Kim K, Elsharkawy AM, et al. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science 2007;317:121–4. [10] Wong VW, Yu J, Cheng AS, Wong GL, Chan HY, Chu ES, et al. High serum interleukin-6 level predicts future hepatocellular carcinoma development in patients with chronic hepatitis B. Int J Cancer 2009;124:2766–70. [11] Georges JL, Loukaci V, Poirier O, Evans A, Luc G, Arveiler D, et al. Interleukin-6 gene polymorphisms and susceptibility to myocardial infarction: the ECTIM study. Etude Cas-Temoin de l’Infarctus du Myocarde. J Mol Med (Berl) 2001;79:300–5. [12] Jordanides N, Eskdale J, Stuart R, Gallagher G. Allele associations reveal four prominent haplotypes at the human interleukin-6 (IL-6) locus. Genes Immun 2000;1:451–5. [13] Ota N, Nakajima T, Nakazawa I, Suzuki T, Hosoi T, Orimo H, et al. A nucleotide variant in the promoter region of the interleukin-6 gene associated with decreased bone mineral density. J Hum Genet 2001;46:267–72. [14] Tong Y, Wang Z, Geng Y, Liu J, Zhang R, Lin Q, et al. The association of functional polymorphisms of IL-6 gene promoter with ischemic stroke: analysis in two Chinese populations. Biochem Biophys Res Commun 2010;391:481–5. [15] Fishman D, Faulds G, Jeffery R, Mohamed-Ali V, Yudkin JS, Humphries S, et al. The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 1998;102:1369–76.

S. Tang et al. / Human Immunology 75 (2014) 297–301 [16] Bao S, Yang W, Zhou S, Ye Z. Relationship between single nucleotide polymorphisms in 174G/C and 634C/G promoter region of interleukin-6 and prostate cancer. J Huazhong Univ Sci Technol Med Sci 2008;28:693–6. [17] Gangwar R, Mittal B, Mittal RD. Association of interleukin-6 174G>C promoter polymorphism with risk of cervical cancer. Int J Biol Markers 2009;24:11–6. [18] Gaur P, Mittal M, Mohanti B, Das S. Functional variants of IL4 and IL6 genes and risk of tobacco-related oral carcinoma in high-risk Asian Indians. Oral Dis 2011;17:720–6. [19] Yu Y, Wang W, Zhai S, Dang S, Sun M. IL6 gene polymorphisms and susceptibility to colorectal cancer: a meta-analysis and review. Mol Biol Rep 2012;39:8457–63. [20] Zhai R, Liu G, Yang C, Huang C, Wu C, Christiani DC. The G to C polymorphism at 174 of the interleukin-6 gene is rare in a Southern Chinese population. Pharmacogenetics 2001;11:699–701. [21] Park BL, Lee HS, Kim YJ, Kim JY, Jung JH, Kim LH, et al. Association between interleukin 6 promoter variants and chronic hepatitis B progression. Exp Mol Med 2003;35:76–82.

301

[22] Qiu XQ, Bei CH, Yu HP, Zeng XY, Zhong QA. Study on the relationship between single-nucleotide polymorphisms in IL-6, IL-10 genes and HBV-related hepatocellular carcinoma. Chin J Epidemiol 2011;32:510–3. [23] Stephens JC, Schneider JA, Tanguay DA, Choi J, Acharya T, Stanley SE, et al. Haplotype variation and linkage disequilibrium in 313 human genes. Science 2001;293:489–93. [24] Terry CF, Loukaci V, Green FR. Cooperative influence of genetic polymorphisms on interleukin 6 transcriptional regulation. J Biol Chem 2000;275:18138–44. [25] Malarstig A, Wallentin L, Siegbahn A. Genetic variation in the interleukin-6 gene in relation to risk and outcomes in acute coronary syndrome. Thromb Res 2007;119:467–73. [26] Jang Y, Kim OY, Hyun YJ, Chae JS, Koh SJ, Heo YM, et al. Interleukin-6-572C>G polymorphism-association with inflammatory variables in Korean men with coronary artery disease. Transl Res 2008;151:154–61. [27] Wang YC, Xu GL, Jia WD, Han SJ, Ren WH, Wang W, et al. Estrogen suppresses metastasis in rat hepatocellular carcinoma through decreasing interleukin-6 and hepatocyte growth factor expression. Inflammation 2012;35:143–9.