ABSTRACT

Objective:

Gastric cancer is a complex malignant tumor associated with chronic inflammation. In the present study, we aimed to investigate the frequency of interleukin 1B (IL-1B) gene polymorphisms affecting gene expression in patients with gastroesophageal cancer (GC) diagnosed in the Hakkari region.

Methods:

Blood samples of 17 patients with GC (group 1) and 59 healthy controls (group 2) were enrolled in the study. The single-nucleotide polymorphisms (SNPs) rs1143627 c.-118C>T, rs16944 c.-598C>T, and rs1143634 c.315C>T polymorphisms in the IL-1B gene were studied among groups via polymerase chain reaction and restriction fragment length polymorphism. Results were analyzed by descriptive statistics and the x² test. The association between SNPs and GC risk was evaluated by odd ratios (ORs) and 95% confidence intervals.

Results:

The frequencies of the three genotypes in the SNP rs1143627, rs16944, and rs1143634 were similar between the groups, and C>T transition was not found to be significant [(p=0.69, OR: 1.16 95%, confidence interval (CI): 0.54-2.51; p= 0.16, OR: 0.58 95%, CI: 0.26- 1.25; p=0.7, OR: 0.83 95%, CI: 0.32-2.11, respectively].

Conclusion:

Our results did not reveal any significant association between IL-1B gene SNPs and gastroesophageal cancer in the Hakkari region.

Keywords:

Gastro-esophageal cancer, IL-1B gene, restriction fragment length polymorphism, rs1143627, rs16944, rs1143634

INTRODUCTION

Gastroesophageal cancer (GC) is one of the most harmful cancers in the world, predominantly in specific geographies such as East Asia, South America, and Eastern Europe (1-3). The risk factors for GC include genetic susceptibility, insufficient high-fiber food consumption, a stationary lifestyle, and chronic exposure to inflammation and oxidative stress (4-7). In particular, the chronic inflammatory microenvironment is a crucial contributing factor to GCs (8,9). The prevalence of GC is high in the Hakkari region. Because cancer is a multifactorial chronic disease, we aimed to investigate polymorphisms affecting the expression of one of these genes, interleukin (IL)-1B, in patients and control samples obtained from this region.

The IL-1B gene, located on chromosome 2q14, is a powerful inflammatory biomarker and participates in a variety of cellular activities, including immune response to pathogens, cell proliferation, differentiation, and apoptosis (6). In addition, IL-1B is a potent inhibitor of gastric acid secretion (10,11-13). Recently, single-nucleotide polymorphisms (SNPs) or mutations in the IL-1 gene have been proposed as a key factor in determining gastric tumor morphogenesis (10-14). Several studies have supported the association of increased IL-1B secretion with SNP polymorphisms in the promoter of the IL-1B gene (15,16). Interestingly, the transition between C and T alleles in the three SNPs of the IL-1B gene promoter, including rs1143627, rs16944, and rs1143634, have been associated with IL-1B levels (4,5,16-20).

This study was designed to investigate the frequency of genotype distribution in the SNPs rs1143627, rs16944, and rs1143634 within the IL-1B gene in patients diagnosed with gastric cancer and controls living in the Hakkari region.

MATERIALS and METHODS

Patients and control subjects

A total of 17 patients diagnosed with GC aged between 31 and 88 years (5 females and 12 males) (group 1) and 59 healthy controls aged between 21 and 78 years (17 females and 42 males) living in the same region (group 2) were included in the current study. This study was approved by the Başkent University Ethics Committee (approval number: KA 18/354) and supported by the Başkent University Research Fund. The present study was conducted by following the principles of the Declaration of Helsinki. Participants were enrolled after verbal and written informed consent was obtained. Patients in group 1 who were diagnosed with GC via histopathological examination were eligible for this study. Healthy controls in group 2 who were cancer-free subjects and living in the same region were randomly selected, and subjects in this group with any signs and symptoms of digestive system diseases and other systemic disorders were excluded.

Genotyping

Genomic DNA was extracted from peripheral blood using a commercial genomic DNA extraction kit (Invitrogen®, USA). The SNPs rs1143627 c.-118C>T, rs16944 c.-598C>T, and rs1143634 c.315C>T (reference genome NM_000576.2) polymorphisms in the IL-1B gene were analyzed using polymerase chain reaction (PCR) - restriction fragment-length polymorphism. The primer sequences are listed in Table 1. Thermal cycling consisted of several steps: starting with an initial denaturation step at 95 °C for 3 min, followed by 35 cycles at 95 °C for 30 seconds; 53 °C for 30 seconds (the SNP rs1143627 and rs1143634), 50 °C for 30 seconds (the SNP rs16944), 72 °C for 45 s, and a final step of 72 °C for 5 min. PCR products were digested with specific restriction enzymes (Table 2). Finally, the end products were electrophoresed on 3% agarose gel for determining genotypes.

Statistical Analysis

The SPSS software package version 22.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Descriptive statistics were applied to demographic characteristics. Quantitative results are shown as means ± standard deviation. The frequencies of the genotypes and alleles in the two groups were analyzed according to the Hardy-Weinberg equilibrium and compared using the x² test. P<0.05 was considered significant.

RESULTS

In the present study, 17 patients with GC (group 1) and 59 healthy subjects (group 2) were enrolled to analyze the functional polymorphisms of rs1143627 C>T, rs16944 C>T, and rs1143634 C>T in the promoter region of the IL-1B gene.

The mean age was 65.58±13.26 years in group 1 and 37.28±13.37 years in group 2.

The ratio of female (n)/male (n) was 5/12 in group 1 and 17/42 in group 2. There was no significant difference between groups (p=0.96).

In terms of SNP rs1143627 C>T, the frequencies of wild-type (CC), variant heterozygote (CT), and variant homozygote (TT) genotype was 23.5%, 58.8%, 17.6% in group 1, 35.6%, 42.4%, 22% in group 2, respectively (p=0.48) (Table 3). The total C allele frequency was 56.8% in group 1 and 52.9% in group 2, whereas the total T allele frequency was 43.2% in group 1 and 47.1% in group 2. However, there was not a statistically significant difference between the two groups (p=0.69, OR: 1.16, 95% CI: 0.54-2.51) (Table 3).

In addition, the frequencies of wild-type (CC), variant heterozygote (CT), and variant homozygote (TT) genotype in SNP rs16944 C>T were 29.4%, 52.9%, 17.6% in group 1, 22%, 40.7%, 37.3% in group 2, respectively (p=0.31) (Table 3). The total C allele frequency was 55.9% in group 1 and 42.4% in group 2, whereas the total T allele frequency was 44.1% in group 1 and 57.6% in group 2 (p=0.16, OR: 0.58 95% CI: 0.26-1.25) (Table 3).

Finally, the frequencies of wild-type (CC), variant heterozygote (CT), and variant homozygote (TT) genotype in the SNP rs1143634 C>T were 58.8%, 41.2%, 0.0% in group 1, 54.2%, 44.1%, 1.7% in group 2, respectively (Table 3). There was a predominance of homozygote genotype (CC) in all groups, without significant difference between groups (p=0.83) (Table 3). Total C allele frequency was 79.4% in group 1, 76.3% in group 2, and the total T allele frequency was 20.6% in group 1, and 23.7% in group 2 (p=0.7, OR: 0.83 95% CI: 0.32-2.11) (Table 3).

DISCUSSION

GC is a multi-factorial and complex malignant tumor with a low survival rate (6). In the pathophysiology of the disease, IL-1B-induced chronic inflammatory microenvironment is reported as a key element in tumorigenesis, vascular, and extracellular changes for tumor invasion (21-26). In addition, IL-1B exacerbates the inflammatory microenvironment via induction of COX-2 and iNOS production (27).

An increase in GC incidence is positively associated with increased life prospects. Dysregulation of the immune system in aging populations is a critical risk factor for cancer morphogenesis. Indeed, the accumulation of inflammatory cytokines and mediators in advanced age aggravates the dysplastic transformation of gastroesophageal cells (23,24).

In recent years, association studies of the SNPs of the IL-1B gene have been the focus in GC susceptibility (28-32). To the best of our knowledge, for the first time in the literature, we investigated the association between GC and the SNPs rs1143627 C>T, rs16944 C>T, and rs1143634 C>T in a sample from the Hakkari population. First, we found a higher frequency of the heterozygote variant (CT) genotype in the SNP rs1143627 in group 1 although the difference was insignificant. In addition, total T allele frequency was induced in group 1 without any significant difference. The results of this study were comparable with those of other polymorphism studies (4,33-37). Yang et al. (38) found that the T allele in the SNP rs1143627 was correlated with induced GC in the Chinese population. Additionally, He et al. (39) determined that the homozygote genotype (TT) in the SNP rs1143627 contributes to GC morphogenesis in China. Qiu et al. reviewed the clinical notes of 52 patients with GC and 52 healthy controls. They found that the CT and TT genotype was significantly higher in patients with GC, and they pointed to a significant positive association between the T allele and GC susceptibility in the Hakka population (6). The results of this study demonstrated a discordant distribution of the genotype frequencies in other studies. Several studies have noted that the CC genotype is a crucial risk factor for GC in the Chinese, Hispanic, and Caucasian populations (5,32,40-42). For instance, Takagi et al. (43) suggested that the CC genotype in the SNP rs1143627 was significantly associated with GC development in the Japan population via increased inflammatory cytokines such as IL-1B and IL-8. Collectively, these different outcomes may have arisen from the allelic heterogeneity in different geographic regions.

Second, we analyzed the SNP rs16944 C>T between the groups and found that the frequency of the wild-type (CC) and heterozygote variant (CT) genotype was higher in group 1. The CT genotype was predominant in both the groups. Additionally, the total C allele frequency was higher in group 1 than in group 2. However, there were no significant differences between the groups in terms of genotype and allele distribution. In support of this, Yang et al. (44) determined a significant interaction between the C allele and GC risk in Asians. In addition, this association was found dominantly with Helicobacter pylori infection in China (20). Similar to this study, Kato et al. (36) reported the clinical notes of 699 patients with gastric cancer. The bottom line of their study was that the C allele did not provide an additional impact on GC in the Japan population (36). In contrast, TT homozygosity has been proven to be a significant determinant in gastroesophageal tumorigenesis via induced IL-1B secretion in the Caucasian, Chinese, and Japanese populations (5,15,38,43,45,46). Moreover, it has been emphasized that the impact of the T allele in the SNP rs16944 may be nebulous in the high-risk areas of China, unlike in the low-risk areas (5,40). Eventually, the global differences in allele distribution display why it is so difficult to determine the impacts of the C>T transition in GC.

CONCLUSION

Finally, we compared the rs1143634 C>T polymorphism within the IL-1B gene between groups and found that wild type (CC) was higher in group 1 without statistical significance between the two groups. The majority of the three genotypes were also the wild type (CC) in both groups. This means that the C>T transition is uncommon in the Hakkari population. In the study by Al-Moundhri et al. (33), there was no association between the T allele and GC. On the other hand, other previous studies pointed out that the heterozygote (CT) and homozygote (TT) variant genotypes were significantly linked to GC development (47,48). Furthermore, it has been postulated that the aforementioned variants were correlated with induced IL-1B production in the gastric mucosa (49).

The importance of this study is that the patient and control populations were selected from a very limited geographic area of Türkiye, Hakkari. The city is rather a low-populated region of a similar ethnic background, and consanguineous marriages are frequent. In summary, we observed a similar distribution of the three genotypes among the groups. As the study population comprised a limited number of patients, future large-scale controlled studies will be more promising to shed light on how IL-1B gene polymorphisms are distributed in gastric cancer patients.

Acknowledgments: We thank Gözde Özer for the statistical analysis of this study.

Ethics

Ethics Committee Approval: This study was approved by the Başkent University Ethics Committee (approval numner: KA 18/354).

Informed Consent: Participants were enrolled after verbal and written informed consent was obtained.

Peer-review: Externally peer-reviewed.

Authorship Contributions

Concept: D.Y., S.A-D., Y.K., G.Ü., Y.K.T., F.İ.Ş., Design: D.Y., S.A-D., Y.K., G.Ü., Y.K.T., F.İ.Ş., Data Collection or Processing: D.Y., S.A-D., Y.K., G.Ü., Y.K.T., F.İ.Ş., Analysis or Interpretation: D.Y., S.A-D., Y.K., G.Ü., Y.K.T., F.İ.Ş., Literature Search: D.Y., S.A-D., Y.K., G.Ü., Y.K.T., F.İ.Ş., Writing: D.Y., S.A-D., Y.K., G.Ü., Y.K.T., F.İ.Ş.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: This study was supported by the Başkent University Research Fund.

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Frequency of IL-1B Gene Polymorphisms in Patients with Gastroesophageal Cancer in the Hakkari Region