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Keywords:

  • cytokine;
  • genetic susceptibility;
  • IL-16 polymorphism;
  • inflammation;
  • renal cell carcinoma

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Objectives:  Interleukin-16 (IL-16) plays a fundamental role in inflammatory diseases, as well as in the development and progression of tumors. A T-to-C polymorphism at the -295 position in the promoter region of the IL-16 gene has been described. This variation might lead to altered IL-16 expression, and might modulate an individual's susceptibility to cancer. The objective of the present study was to determine if IL-16 polymorphism is associated with risk of renal cell carcinoma (RCC).

Methods:  A case–control study including 335 RCC cases and 340 cancer-free controls was carried out. All subjects were genetically unrelated ethnic Han Chinese recruited from a single institution between July 2006 and July 2009. The IL-16 -295 T>C polymorphism was determined by using the polymerase chain reaction-restriction fragment length polymorphism method. Serum samples were available for 70 RCC cases and 96 controls to detect IL-16 concentration.

Results:  Compared with the IL-16 -295 TT genotype, the CC genotype had a significantly decreased RCC risk (adjusted odds ratio [OR] = 0.34, 95% confidence interval [CI] = 0.18–0.66). Furthermore, a significant decreased risk of RCC was found in the combined variant genotypes CT + CC compared with the TT genotype (adjusted OR = 0.68, 95% CI = 0.50–0.93). In addition, the serum IL-16 levels in RCC patients were significantly lower than those in controls (P < 0.001). Furthermore, patients carrying CC genotype or CT genotype had higher serum IL-16 levels than TT carriers.

Conclusion: IL-16 -295 T>C polymorphism is significantly associated with a higher risk of developing RCC in Chinese population.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Cytokines are a group of modulatory proteins or glycoproteins that bind to their respective receptor in response to a variety of stimuli, resulting in the activation of a second messenger and signal transduction pathways within a cell.1 Interleukin-16 (IL-16) is a multifunctional cytokine that was initially identified as lymphocyte chemoattractant factor (LCF) in 1982.2 The IL-16 gene is located on chromosome 15q26.3,3 and is initially translated into a precursor protein consisting of 631 amino acids, which is cleaved by caspase-3 to form the active C-terminal domain containing 121 amino acids.4–6 In the non-diseased state, IL-16 is almost exclusively expressed in lymphatic tissue, and in high levels in T cells.1 As a cytokine with chemotactic properties, IL-16 is considered to be a T helper (Th) cell chemoattractant factor. After binding to the CD4 molecule, IL-16 can activate CD4+ T cells, macrophages, monocytes, eosinophils, and dendritic cells.7,8 Furthermore, IL-16 can promote the secretion of tumor-associated inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and IL-15, all these cytokines have been shown to play a important role in tumorigenesis.9–11

Renal cell carcinoma (RCC) represents 2–3% of all cancers and is the third leading cause of death among genitourinary malignancies, with the highest incidence occurring in the developed countries.12 It is estimated that approximately 37.7 men and 16.6 women per 100 000 Chinese individuals are diagnosed with RCC every year.13 Accumulative epidemiological studies have suggested that cigarette smoking, gender, obesity and a history of hypertension, along with some other less certain factors, such as alcohol consumption, occupational exposures, physical activity and family history of cancer, are associated with RCC.14,15

In 2000, Nakayama et al.16 reported a new polymorphism in the promoter region of the human IL-16 gene, which involves a T-to-C substitution at position -295. This single nucleotide polymorphism (SNP) was reported to be associated with autoimmune diseases.17–20 Recently, the role of IL-16 in tumorigenesis has been explored, with upregulation of IL-16 expression shown in both human and rat gliomas in vivo,21 and higher serum levels of IL-16 in many kinds of tumors including RCC.22–24 These data highlight the important role that IL-16 might play in tumorigenesis. To the best of our knowledge, no reports have been published regarding the role of the IL-16 -295 T>C polymorphism in RCC. Therefore, our present study was designed to investigate the association of IL-16 -295 T>C polymorphism (rs4778889) with risk of RCC and the influence of this SNP on IL-16 serum levels in patients with RCC versus healthy controls in our ongoing, hospital-based, case–control study in a Chinese population.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Study subjects

The present case–control study consisted of 335 RCC cases and 340 cancer-free controls. Cases were patients with histopathologically confirmed incident RCC. All subjects were genetically unrelated ethnic Han Chinese recruited from the First Affiliated Hospital of Nanjing Medical University between July 2006 and July 2009. Those cases that had previous cancer, metastasized cancer from other or unknown origin and previous radiotherapy or chemotherapy were excluded. The cancer-free control subjects were recruited from those who were seeking health care in the outpatient departments at the hospital. We used a short questionnaire to obtain demographic and risk factor information, and frequency matched the controls to the cases by age and sex. Before recruitment, we described the purpose of our research to each subject. If the subject agreed and signed their names, then a standard questionnaire was given by trained interviewers to obtain information on demographic data and related risk factors through face-to-face interviews. After interview, an approximately 5-mL venous blood sample was collected from each subject. The response rate of those control subjects we approached for participation in this study was over 85%. All selected controls had no individual history of cancers or other diseases, such as inflammatory diseases and so on. The study was approved by the institutional review board of Nanjing Medical University.

Genotyping

The IL-16 -295 T>C polymorphism was determined by using the polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) method, as described previously.24 The PCR primers for the IL-16 -295 T>C polymorphism were 5′-CTCCACACTCAAAGCCTTTTGTTCCTATGA-3′ (forward) and 5′-CCATGTCAAAACGGTAGCCTCAAGC-3′ (reverse). The PCR reactions were carried out in a total volume of 20 uL containing 50 ng genomic DNA, 10 × Taq Buffer, 0.02 mmol/L of MgCl2, 0.05 mmol/L of dNTP mix, 10 pmol/uL of each primer and 1 U Taq DNA polymerase. After initial denaturation at 95°C for 5 min, the reaction was carried out at 95°C denaturation for 30 s, 60°C annealing for 40 s, and 72°C extension for 45 s for a total of 34 cycles, and a final elongation at 72°C for 10 min. The 280 bp amplified products were incubated with 5 U of AhdI (New England Biolabs, Beijing, China) restriction enzyme at 37°C overnight. The restriction fragments were then analyzed by electrophoresis in 3% agarose gel stained with 0.5% ethidium bromide and photographed under UV illumination. Digestion with AhdI yielded 246 bp, and 34 bp fragments only when C allele was present. The polymorphism analysis was carried out by two persons independently, in a blind fashion. More than 15% of the samples were randomly selected for confirmation, and the results were 100% concordant.

Serum IL-16 levels

Serum samples were available for 70 RCC cases and 96 controls. When blood samples were obtained, the serum was allowed to clot for 30 min at 4°C before centrifugation at 2000 rpm for 10 min at 4°C. Serum was isolated and stored at −80°C before use. We detected IL-16 concentration by using a sandwich enzyme immunoassay (Quantikine EGF immunoassay kit, R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The minimum level of detection for IL-16 was 5 pg/mL. No cross-detection of other cytokines was observed. The intra-assay variation was <10%.

Statistical analyses

Student's t-test (for continuous variables) or χ2-test (for categorical variables) were used to evaluate differences in the distributions of selected demographic variables, and frequencies of genotypes of IL-16 -295 T>C polymorphism between the cases and controls. Hardy–Weinberg equilibrium (HWE) was tested using a goodness-of-fit χ2-test. The associations between IL-16 -295 T>C genotypes and risk of RCC were estimated by computing odds ratios (OR) and their 95% confidence intervals (CI) from unconditional logistic regression analysis with the adjustment for age, sex, body mass index (BMI), pack-years of smoking, and drinking status. P < 0.05 was considered statistically significant and all statistical tests were two sided. All of the statistical analyses were performed with Statistical Analysis System software (9.1.3; SAS Institute, Cary, NC, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Characteristics of the study population

The frequency distributions of selected characteristics of the cases and controls are presented in Table 1. There were no differences between the cases and controls on age, sex, BMI, smoking and drinking status (all P > 0.05). The majority of patients (81.8%) had the conventional clear cell carcinoma. Papillary carcinoma presented in 12 (3.6%) patients, and 20 (6.0%) had chromophobe carcinoma. In addition, there were 29 (8.6%) unclassified RCC patients. Approximately 74.3% of patients were in a localized stage (stage I + II), and 25.7% of patients were in an advanced stage (stage III + IV). The nuclear grade of well differentiated (grade I + II), moderately differentiated (grade III), and poorly differentiated (grade IV) were 60.9%, 27.5%, and 11.6%, respectively.

Table 1.  Distribution of selected variables between the renal cell carcinoma cases and control subjects
VariablesCases (n = 335)Controls (n = 340)P*
n%n%
  • *

    Student's t-test for age and BMI distributions between cases and controls; two-sided χ2-test for others selected variables between cases and controls. BMI, body mass index.

Age (years) (mean ± SD)55.1 ± 11.454.6 ± 12.70.592
BMI (kg/m2) (mean ± SD)23.9 ± 2.923.7 ± 3.30.382
Sex     
 Male22266.321563.20.410
 Female11333.712536.8 
Smoking status     
 Never20862.121964.40.423
 Ever12737.912135.6 
 Former267.8185.3 
 Current10130.110330.3 
Pack-years of smoking     
 020862.121964.40.194
 0–209026.99728.5 
 >203711.0247.1 
Drinking status     
 No22968.423167.90.907
 Yes10631.610932.1 
Stage     
 Localized (I + II)24974.3   
 Advanced (III + IV)8625.7   
Grade     
 Well-differentiated (I + II)20460.9   
 Moderately-differentiated (III)9227.5   
 Poorly-differentiated (IV)3911.6   
Histology     
 Clear cell27481.8   
 Papillary123.6   
 Chromophobe206.0   
 Unclassified298.6   

Association between the IL-16 -295 T>C polymorphism and risk of RCC

The genotype and allele distribution of the IL-16 -295 T>C polymorphism in the cases and controls are shown in Table 2. The observed genotype frequencies for this polymorphism were in Hardy–Weinberg equilibrium in the controls (χ2 = 0.92, P = 0.34). For the IL-16 -295 T>C polymorphism, the frequencies of the TT, CT, and CC genotypes were 59.4%, 36.4%, and 4.2%, respectively, among the cases, and 50.3%, 39.7%, 10.0%, respectively, among the controls (P = 0.004). When we used the IL-16 TT genotype as the reference, we found that CC genotype was associated with a statistically significantly decreased risk of RCC (adjusted OR = 0.34, 95% CI = 0.18–0.66). Furthermore, a significant decreased risk of RCC was found in the combined variant genotypes CT + CC compared with the TT genotype (adjusted OR = 0.68, 95% CI = 0.50–0.93). The IL-16 C allele frequency was 0.224 among the cases and 0.299 among the controls, and the difference was statistically significant (P = 0.002).

Table 2.  Genotype and allele frequencies of the IL-16 polymorphism among the cases and controls and the associations with risk of renal cell carcinoma
GenotypeCases (n = 335)Controls (n = 340)P*Adjusted OR (95% CI)
n%n%
  • *

    Two-sided χ2-test for either genotype distributions or allele frequencies between the cases and controls.

  • †Adjusted for age, sex, body mass index, pack-years of smoking and drinking status in logistic regression model. CI, confidence interval; OR, odds ratio.

TT19959.417150.30.0041.00 (reference)
CT12236.413539.7 0.76 (0.55–1.05)
CC144.23410.0 0.34 (0.18–0.66)
CT + CC13640.616949.7 0.68 (0.50–0.93)
T allele52077.647770.10.0021.00 (reference)
C allele15022.420329.9 0.68 (0.53–0.87)
Ptrend    0.002 

Stratified analysis of IL-16 -295 T>C polymorphism and risk of RCC

In the present study, we further evaluated the effect of the IL-16 -295 T>C polymorphism on RCC risk stratified by age, BMI, sex, smoking status, pack-years of smoking, and drinking status. As shown in Table 3, the association between IL-16 -295 T>C polymorphism and RCC risk did not vary by drinking status; however, the association appeared stronger in subgroups of younger patients (adjusted OR = 0.62, 95% CI = 0.40–0.96), BMI >23 Kg/m2 (adjusted OR = 0.60, 95% CI = 0.41–0.90), female patients (OR = 0.49, 95%CI = 0.29–0.83), and non-smokers (adjusted OR = 0.60, 95% CI = 0.41–0.89). Furthermore, we evaluated the effect of IL-16 -295 T>C polymorphism on clinicopathological characteristics of RCC. As shown in Table 4, a statistically significant deceased risk was found in RCC patients with a localized stage (adjusted OR = 0.65, 95% CI = 0.47–0.91). And in the stratification of grade, we also found that a significantly decreased risk was only in patients with well differentiated RCC (adjusted OR = 0.62, 95% CI = 0.44–0.89). These results clearly showed that the IL-16 -295 C allele might reduce RCC risk.

Table 3.  Stratification analyses between IL-16 genotypes and risk of renal cell carcinoma in cases and controls
VariablesCases/controlsGenotypes (cases/controls)P*Adjusted OR (95% CI) CT + CC vs TT
CT + CCTT
n%n%
  • *

    Two-sided χ2-test for genotype distributions between the cases and controls.

  • †Adjusted for age, sex, BMI, pack-years of smoking and drinking status in logistic regression model. BMI, body mass index; CI, confidence interval; OR, odds ratio.

Total335/340136/16940.6/49.7199/17159.4/50.30.0170.68 (0.50–0.93)
Age       
 ≤55160/17561/8638.1/49.199/8961.9/50.90.0420.62 (0.40–0.96)
 >55175/16575/8342.9/50.3100/8257.1/49.70.1690.74 (0.48–1.14)
Pinteraction      0.081
BMI       
 ≤23124/14450/6640.3/45.874/7859.7/54.20.3640.80 (0.49–1.30)
 >23211/19686/10340.8/52.5125/9359.2/47.50.0170.60 (0.41–0.90)
Pinteraction      0.041
Sex       
 Male222/21592/10041.4/46.5130/11558.6/53.50.2860.80 (0.55–1.17)
 Female113/12544/6938.9/55.269/5661.1/44.80.0120.49 (0.29–0.83)
Pinteraction      0.005
Smoking status       
 Never208/21981/11238.9/51.1127/10761.1/48.90.0110.60 (0.41–0.89)
 Ever127/12155/5743.3/47.172/6456.7/52.90.5480.85 (0.51–1.40)
Pinteraction      0.904
Pack-years       
 0208/21981/11238.9/51.1127/10761.1/48.90.0110.60 (0.41–0.89)
 0–2090/9741/4345.6/44.349/5454.4/55.70.8661.07 (0.59–1.91)
 >2037/2414/1437.8/58.323/1062.2/41.70.1170.43 (0.15–1.24)
Pinteraction      0.941
Drinking status       
 Never229/23196/11541.9/49.8133/11658.1/50.20.0910.72 (0.50–1.04)
 Ever106/10940/5437.7/49.566/5562.3/50.50.0810.60 (0.35–1.04)
Pinteraction      0.140
Table 4.  Association between IL-16 polymorphism and clinicopathological characteristics of renal cell carcinoma
GenotypeControlsCases
LocalizedAdvancedGrade (I + II)Grade (III)Grade (IV)
nAdjusted OR (95% CI)nAdjusted OR (95% CI)nAdjusted OR (95% CI)nAdjusted OR (95% CI)nAdjusted OR(95% CI)
  • Adjusted for age, sex, BMI, pack-years of smoking and drinking status in logistic regression model. BMI, body mass index; CI, confidence interval; OR, odds ratio.

TT1711501.00 (reference)491.00 (reference)1251.00 (reference)541.00 (reference)201.00 (reference)
CT + CC169990.65 (0.47–0.91)370.78 (0.48–1.27)790.62 (0.44–0.89)380.70 (0.44–1.12)191.02 (0.52–2.00)
P0.0170.2680.0130.1520.907

IL-16 serum levels and RCC risk

The median serum level of IL-16 detected was 283.80 pg/mL (±80.66) in cases and 409.80 pg/mL (±71.11) in controls (P < 0.001) (Fig. 1), suggesting that serum IL-16 level might serve as an independent protective marker against RCC. Furthermore, serum IL-16 levels in patients carrying CC (374.68 pg/mL) genotype or CT (290.20 pg/mL) genotype were higher than those with TT (273.49 pg/mL) genotype (CC vs TT, P = 0.048; CT vs TT, P = 0.394; CC + CT vs TT, P = 0.193) (Fig. 2).

image

Figure 1. Distribution of serum IL-16 levels in 70 cases and 96 controls. The mean level of serum IL-16 in cases was significantly lower than controls (P < 0.001).

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image

Figure 2. Distribution of serum IL-16 levels in different genotypes in patients.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

In the present study, we investigated whether or not IL-16 -295 T>C polymorphism in the promoter region of IL-16 gene is associated with the risk of developing RCC in a case–control study. We found that the functional polymorphism of IL-16 -295 T>C has a significant effect on the risk of RCC; individuals carrying the IL-16 -295 C allele were associated with a significantly decreased risk of developing RCC compared with the IL-16 -295 T allele. To the best of our knowledge, no reported study has investigated the association between IL-16 -295 T>C polymorphism and the risk of RCC in a Chinese population.

Increasing evidence from preclinical and clinical studies has confirmed the contribution of inflammation and inflammatory cells to the process of tumor development and progression,25,26 and approximately 25% of all cancer cases worldwide are related to chronic infection and inflammation.27 Studies of cytokines and their role in cancer development and progression are complicated by pleiotropy and the apparent redundancy of cytokine action. Two distinct Th cytokine cell subsets, Th1 and Th2, are characterized by distinct and mutually exclusive patterns of cytokine production and different functions.28 The cytokine profiles produced by Th1 and Th2 lymphocytes show distinct patterns in which the Th1 lymphocytes produce inflammatory cytokines while the Th2 lymphocytes produce anti-inflammatory cytokines.29 A key feature of these cytokine patterns is that they reciprocally regulate one another because Th1 cytokines can inhibit the proliferation and functions of the Th2 lymphocytes, whereas Th2 cytokines can suppress cytokine production by the Th1 lymphocytes.30,31

IL-16 is considered to be a proinflammatory cytokine and a Th cell chemoattractant factor. After binding to the CD4 molecule, IL-16 can promote the secretion of tumor-associated inflammatory cytokines, such as TNF-α, IL-1β, IL-6 and IL-15; all these cytokines have been shown to play a important role in tumorigenesis.9–11 In 2000, Nakayama et al.16 first identified a new polymorphism in the promoter region of the human IL-16 gene, -295 T>C, and Burkart et al.18 examined the effect of this SNP on gene expression using a luciferase reporter assay, and found a sixfold increased expression from the C allele compared with the T allele. Recently, the role of IL-16 in tumorigenesis has been explored and the high levels of IL-16 have been shown in several malignant cancers, both in vitro and in vivo.21–24 These findings seemed to be contradictory to our present results. The discrepancy could be interpreted to suggest that different mechanisms underlie the development of different malignant cancers, and IL-16 gene might play a different role in different tumorigenesis. Additionally, studies on chronic inflammatory diseases have found that individuals carrying the IL-16 -295 TT genotype had a significantly increased risk of these diseases compared with those who carrying the CC genotype.17–19 Furthermore, in a murine model of allergic asthma, intraperitoneal administration of IL-16 inhibited antigen-induced airway hyperresponsiveness and decreased the number of eosinophils in bronchoalveolar lavage fluid.32 These data support that IL-16 might not only can augment inflammation, but also can inhibit inflammation. However, the exact mechanism is unclear.

We also evaluated the influence of IL-16 -295 T>C polymorphism on IL-16 serum levels in patients with RCC versus healthy controls, and found that the median serum level of IL-16 in cases was significantly lower than controls (P < 0.001). Additionally, serum IL-16 levels in patients carrying CC genotype or CT genotype were also significantly higher than those with TT genotype. Our data suggest that higher serum IL-16 level might serve as an independent protective marker against RCC.

In our present study, we found that the association between IL-16 -295 T>C polymorphism and RCC risk appeared stronger in young patients and female patients. This might be attributed to the exposure of young patients to lower levels of risk factors, such as some anticancer drugs and chemical carcinogens in the workplace, during their lifetime. For female patients, they might not only be exposed to fewer risk factors than male patients, such as tobacco smoking and heavy drinking, but this association might also be a result of female patients' estrogenic hormone. Estrogen might interact with IL-16 and reduce the possibility for developing RCC. However, it should be noted that our sample size was relatively small, especially for female cases, so our findings might be a false positive.

Additionally, we found that the IL-16 -295 C allele has a statistically significant protective role in RCC patients only with localized stage, and this protective role also existed only in patients with well differentiated RCC. These findings suggested that there might be different mechanisms underlying the early development of RCC and the subsequent progression of RCC, and IL-16 might affect these two mechanisms differentially. Thus, increased IL-16 production in patients with the IL-16 -295 C allele might inhibit onset of RCC in the early period of the disease, for example, through anti-inflammatory activity, but might be detrimental once the disease is established, for example, by hampering tumor immune surveillance. However, our sample size was relatively small, when our sample size was larger, a protective effect could be seen with the TT genotype in the advanced cases as well.

Our present study has some limitations. First, our sample size was small, and because we did not have detailed information on environment factors, such as occupational exposures, diet and physical activity, the statistical power of our study might be limited, especially for gene-environment interaction analyses. Second, our study was a hospital-based study design, because we could not rule out the possibility of selection bias of subjects that might have been associated with a particular genotype. However, the agreement with the Hardy–Weinberg equilibrium suggested that the selection bias in terms of genotype distribution would not be substantial.

In conclusion, our present study showed that functional polymorphism of the IL-16 -295 T>C is associated with risk of RCC. We found that the variant CC genotype was associated with decreased risk of RCC compared with their wild-type TT homozygote. These findings suggested that the IL-16 -295 T>C polymorphism might be a marker for genetic susceptibility to RCC. These findings, after validation by larger studies, might help identify at-risk populations for primary cancer prevention.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

This study was supported by the Foundation of Medical Key Department of Jiangsu Province-Department of Urology of the First Affiliated Hospital of Nanjing Medical University (BK2008473).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References