Association of IL2-330 Gene Polymorphism with Lung Cancer

Sitokinler, bagisiklik sisteminin homeostazini duzenlemek icin hucreler arasi sinyallesmenin aracilari olarak gorev yapan, salgilanan veya zara bagli proteinlerdir. Mikroplara ve tumor antijenlerine yanit olarak dogustan gelen bagisikliga sahip hucreler tarafindan uretilirler. IL2-330 gen polimorfizminin bircok kanser turu ile iliskili oldugunu gosteren birkac calisma olmasina ragmen, bildigimiz kadariyla akciger kanseri ile IL2-330 gen polimorfizmi arasindaki iliskiyi arastiran az sayida calisma vardir. Bu calismada IL2-330 gen polimorfizminin akciger kanseri patogenezindeki rolu arastirilmistir. Calismaya akciger kanseri tanisi almis 96 hasta ile yas ve cinsiyet bakimindan uyumlu 96 saglikli, gonullu birey katildi. Genomik DNA izolasyon kiti kullanilarak izole edildi ve IL2 -330 gen polimorfizmi polimeraz zincir reaksiyonu ile iki cift primer yontemiyle karsilastirilarak belirlendi. Akciger kanserinde IL2-330 gen polimorfizmine gore hasta grubu ve saglikli grup incelendiginde genotip ve allel frekanslarinin her iki grupta benzer oldugu goruldu (p>0,05). Sonuc olarak gruplar arasinda istatistiksel olarak anlamli bir fark yoktu. Akciger kanserinin etnik cesitliligi goz onune alindiginda, calismanin diger populasyonlarda da dogrulanmasi gerekmektedir.


INTRODUCTION
According to the World Health Organization (WHO) report, lung cancer is the most common cancer type in males worldwide, and the second most common cancer type in females also causes about 1.3 million deaths worldwide each year. The most common cause of lung cancer is long-term exposure to tobacco smoke, and nearly 15% of all lung cancer patients are non-smokers. Therefore, it is known that genetic factors play important role in the pathogenesis of lung cancer (1). The immune system cells secrete a large number of reporter proteins, which regulate the division of the host cell and are involved in innate and acquired immune responses. These reporters are called cytokines (2). IL2, a potent immune regulatory cytokine involved in cellmediated immune response, is produced by T cells when it is activated by mitogens, or by the interaction of antigen with major histocompatibility complexes and also like as a T-cell growth factor (3). The immune system is organized such that it does not respond to our antigens (4). However, as cancer cells acquire many mutations and changes (5), they express tumor-specific antigens with sporadic mutations, thereby activating the immune system, eventually leading to the killing of cancer cells. Thus, the immune system can prevent the formation of primary tumors (6). However, mutational changes occurred as cancer cells continued to divide, accumulating either by chance or in response to immune-induced inflammation. Also, due to genetic instability, fixed tumor cell division can produce low immunogenicity that can escape immune elimination (7). The human IL2 gene is localized on chromosome 4q26. In the human IL2 gene, two major polymorphisms have been identified: one is located in the promoter region at nucleotide -330 (8) and the other in the first exon at position +114 (9). G -330T (rs2069762), identified upstream of the IL2 promoter-enhancer domain, reportedly affects protein production. Individuals that were homozygous for the G allele (G/G) in the promoter region of the IL-2 gene produced more than three times more IL-2 than individuals that G/T and T/T genotypes (10). We hypothesized that this SNP may be associated with lung cancer risk, as IL2 plays important role in the regulation of immune response and the process of inflammation-mediated carcinogenesis, and also because of its functional association with IL2-330 T/G. To test this hypothesis, we performed genotyping analyzes for this SNP in a case-control study of lung cancer patients and healthy controls in a Turkish population.

METHOD Study Population
The case-control group consisted of 96 patients diagnosed with lung cancer in Zonguldak Bülent Ecevit University Medical Oncology Department and 96 healthy individuals. The patient group was diagnosed with chest X-ray, sputum cytology, and computed tomography imaging tests. This study was approved by the Zonguldak Bulent Ecevit University Clinical Research Ethics Committee (2017-05-11 / 01) in 2017, and written informed consent was obtained from each participant.

DNA Isolation and Genotyping
Genomic DNA was extracted from 200 microliter of peripheral blood using the E.Z.N.A Blood DNA isolation Kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer's instructions. The polymorphism in the -330 region of the IL2 gene was analyzed by the Confronting two pairs primer method (8). The PCR mix contains 20 pmol from each of the primers (Table 1), 125 mM dNTP, 125 mM Taq DNA buffer, 125 mM MgCl2, 15 mM Taq DNA Polymerase in a final volume of 25 µl. PCR reaction conditions; 1 cycle 95 ° C for 5 minutes, then 35 cycles of 95 ° C for 1 minute and 57 °C for 1 minute, 72 °C for 1 minute followed 1 cycle 72 °C for 7 minutes. PCR products were visualized on 3% agarose gel electrophoresis. For the G allele, a band of 152 bp was obtained and for the T allele, a band of 215 bp was obtained. A 312 bp band is common in all samples ( Figure  1).

Primers
Sequence

Statistical Analysis
A case-control study was performed and the allelic frequency of the polymorphism was calculated in both cases and controls. Deviations from Hardy-Weinberg equilibrium were evaluated by comparing observed and expected genotype frequencies. The x2 test was used to compare the genotype frequency of IL2 gene polymorphism in lung cancer patients and controls. The odds ratio (OR) and 95% confidence interval (CI) were calculated to compare the lung cancer risk for the alleles. p values <0.05 were considered to indicate statistical significance. The SPSS software was used (ver. 18.0; SPSS Inc., Chicago, IL).

RESULTS
The mean age of the lung cancer patient group was 43.26±10.54, the mean age of the control group was 45.8±10.62. The patient and control groups were compared in terms of gender. There was no statistically significant difference was found between the groups (p>0.05) ( Table  2). In this study, the association between lung cancer and IL2 -330 (rs2069762) polymorphism was investigated and it was found that the frequencies of TT, GT, and GG genotypes were 21.9%, 67.7%, and 10.4% in the patient group and were 17.7%, 79.2% and 3.1% in the control group, respectively. There was no statistically significant difference between these two groups (Table 3). For IL2 -330 T / G polymorphism, T allele frequencies were 55.7% in lung cancer patients and 56.5% in the control group. G allele frequency was determined as 44.3% in lung cancer patients and 43.5% in the controls (Table 4). There was no statistically significant difference between the groups.

DISCUSSION
Cytokines are small glycoproteins that bind to cell surface receptors and regulate the development, survival, and function of immune cells (11). IL2 is one of the key cytokines with pleiotropic effects on the immune system. This immunoregulatory cytokine IL2 plays an important role in the antitumor immunoreactivity of T cells, and the polymorphism in the -330 promoter region is the most investigated (12). However, in the present case-control study, our results indicated that there was no association between IL2 -330 rs2069762 gene polymorphism and lung cancer. Few reports regarding the association between the IL2 gene polymorphism and lung cancer have been published. A case-control study by Eaton KD et al., it was investigated the role of genetic polymorphisms in inflammation-related genes in the risk for development of lung cancer in 625 cases and 625 matched controls.  Similarly, with our results, the study did not find any correlation between genotype and lung cancer risk for the IL2 rs2069762 and rs2069763 polymorphisms (13).
In several studies, genetic polymorphisms of the IL2 -330 T/G have been implicated in the susceptibility to a range of inflammatory diseases and cancer, including gastric atrophy from Helicobacter pylori infection, myelogenous leukemia (14,15,16,17), gastric cancer (18), lung cancer (19), and breast cancer (20). However, other studies have shown that IL2 -330T / G polymorphism is not associated with gastric cancer or cutaneous malignant melanoma. The first meta-analysis which provided comprehensive information of the association on IL2 -330 T/G polymorphism and cancer risk was performed by  (23). There are some limitations to this study. First, we did not adjust other compounding variables for risk factors of lung cancer, such as tobacco smoke, environmental factors, and family history. Second, the number of samples was relatively small for the investigation of genetic polymorphisms. Further studies with large samples and including environmental, other host genetic factors will be needed.

CONCLUSION
Our study is the first study investigating the association between lung cancer and IL2 rs2069762 polymorphism. Because lung cancer shows ethnic variation, repeating the study with more samples in different populations will contribute to the understanding of the relationship.