2Deparment of Genetics, Ondokuz Mayis University Faculty of Veterinary, Samsun-Türkiye
3Deparment of Medical Genetics, Hitit University Faculty of Medicine, Çorum-Türkiye
4Deparment of Medical Biology, Amasya University Faculty of Medicine, Amasya-Türkiye
5Deparment of General Surgery, Bezmialem University Faculty of Medicine, İstanbul-Türkiye
6Deparment of Pathology, Ondokuz Mayis University Faculty of Veterinary, Samsun-Türkiye DOI : 10.5505/tjo.2022.3624
Summary
OBJECTIVEOral squamous cell carcinoma (OSCC), with its low survival rates and increasing incidence, is due to various etiological factors including environmental, genetic, and epigenetic changes. Interleukin 6 (IL- 6) is a cytokine with both pro- and anti-inflammatory effects. Therefore, we investigated the possible association of the IL-6 gene variants with risk for OSCC in a Turkish cohort.
METHODS
This study included 42 patients with OSCC and 110 age-and gender-matched healthy controls. Three
variants (-174G/C [rs1800795], -572G/C (rs1800796), and -597G/A [rs1800797]) in the IL-6 promoter
region were analyzed by polymerase chain reaction-restriction fragment length polymorphism
(PCR-RFLP) method.
RESULTS
There was a significant difference in the genotype and allele frequencies of the IL-6 -174G/C variant
between OSCC patients and controls. While the IL-6 -174G/C G/C genotype was higher in the
patient group than in the control group, the G/G and C/C genotypes were lower in the patients
compared to the control group (p=0.016, OR:0.653, 95% CI: 0.38-1.11). The genotype and allele distributions
of -572G/C and -597G/A variants of the IL-6 gene were not statistically different between
OSCC patients and the control group.
CONCLUSION
Our current investigation is focused on the role of variants of IL-6 gene on OSCC. To the best of our
knowledge, this study is the first study to evaluate the genotype and allele frequencies of IL-6 -174G/C,
-572G/C, and 597G/A variants in patients with OSCC in a Turkish population. The results support that
the IL-6 -174G/C variant may play an important role in susceptibility to OSCC in our population.
Introduction
Oral squamous cell carcinoma (OSCC) is classified among the most common solid tumors, accounting for 4% of all malignancies.[1] OSCC is more common in men (male:female =2:1). Many risk factors have been associated with the development of OSCC, including low intake of fresh fruits-vegetables, alcoholism, and cigarette smoking.[<2>] Many studies have shown that the inflammatory process leads to an increase in the risk of developing cancer. The chronic inflammatory process causes permanent tissue damage and changes the microenvironment by affecting inflammatory cells and cytokines. These changes lead to a cascade of events such as tumorigenesis, then malignant invasion, and finally metastasis.Interleukin 6 (IL-6), which is among the most studied cytokine types, is both an important pro-inflammatory and anti-inflammatory mediator. It is also considered to induce immune reactions and host defense.[3] IL-6 plays a role in various biological reactions through stimulation of B and T lymphocytes, production of acute-phase protein synthesis in the liver, regulation of hematopoiesis. IL-6 also acts as a pyrogen, activates the coagulation system, and suppresses the synthesis of tumor necrosis factor-alpha (TNF-α) and IL-1β in vitro. [4] The gene coding for IL-6 is located on chromosome 7p21-24 and consists of four introns and five exons. The data showed that IL-6 expression can be partially genetically modulated by variants located at positions in the promoter region of IL-6 (rs1800795, rs1800796, and rs1800797 [also known as IL-6 -174G/C, -572G/C, and -597G/A]).[5] The IL-6 -174G/C variant is one of the most studied polymorphisms. The IL-6 gene functional -174G/C variant can increase the transcriptional activity of the IL-6 promoter, leading to IL-6 upregulation in stress or infection. Some studies have shown that the IL-6 -174G/C gene variant is significantly associated with certain cancers.[6]
There was no previous study showing the association between IL-6 -174G/C, -572G/C, and -597G/A variants and OSCC in the Turkish population. Therefore, we aimed to investigate the role of IL-6 gene variants in the pathogenesis of OSCC in the Turkish population.
Methods
Study PopulationForty-two patients with OSCC (27 male, 15 women, mean age: 63.05±13.25 years) and 110 healthy controls (72 male, 38 women, mean age: 56.02±11.03 years) were included in this study. The patients were recruited from the Department of Medical Oncology, Faculty of Medicine, Gaziosmanpaşa University, Tokat, Turkey. All patients with OSCC were pathologically confirmed. Patients with oral precancerous diseases such as oral submucous fibrosis, leukoplakia, erythroplakia, or verrucous hyperplasia were not included in the study. The age-and gender-matched control group (n=110) consisted of healthy individuals with no personal or family history of cancer. Informed consent was obtained from each participant interviewed to gather detailed information on demographic information. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Hitit University, Faculty of Medicine (2017/200).
Genotyping
Genomic DNA was extracted from whole venous
blood samples using a commercial DNA isolation kit
(Sigma-Aldrich, Taufkirchen, Germany). The IL-6
gene -174G/C, -572G/C, and -597G/A variants were
analyzed by polymerase chain reaction (PCR) based
restriction fragment length polymorphism analysis.
The IL-6 -174G/C variant was analyzed as previously
described by Tseng et al.[7] using forward (F) 5´-
TTG TCA AGA CAT GCC AAA GTG CGG AG-3´
and reverse (R) 5´-GTG CAA TGT GAC GTC CCT
TAG CAT-3´ primers. The amplification conditions
consisted of an initial melting step of 5 min at 94°C;
followed by 40 cycles of 30 s at 94°C, 30 s at 56°C, and
1 min at 72°C. After amplification, the 156 bp PCR
product was digested with Fast Digest BseL-I restriction
endonuclease (Fermentas) at 37°C for 30 min
and analyzed on a 3% agarose gel stained with ethidium
bromide. Two fragments (139 and 17 bp) for G
allele and three fragments (117, 22, and 17 bp) for C
allele were observed.
The primers used for IL-6-572G/C and -597G/A variants were (F): 5´-CAG CAG CCA ACC TCC TCT AA-3´ and (R) 5´-AAA CCA GAC CCT TGC ACA AC-3´. The cycling conditions for IL-6 -572G/C and -597G/A variants were 40 cycles of 30 s at 95°C, 30 s at 62°C, and 1 min at 72°C. For the IL-6-572G/C, the G to C transversion was identified by digestion with BsrBI, producing for the G allele fragments of 150 and 74 bp. For the IL-6 -597GA, the G to A transition was identified with FokI, the A allele yields fragments of 136 and 88 bps. A second PCR was performed to confirm samples whose results were not clear.
STRING Analysis
STRING is a proteomics database focused on networks
and interactions of proteins of a wide variety
of species. In this study, IL-6 protein was examined
by the STRING analysis, and the proteins it interacts
with were investigated.
Statistical Analysis
All statistical analyses were performed with the Statistical
Package for the Social Science for Windows
(version 22.0; SPSS Inc., Chicago, IL, USA) Statistical
Program. The χ2 test was used to measure significance
of differences in the allele frequency and the genotype
distribution between the groups. Odds ratio (OR) and
95% confidence intervals (CI) were calculated. A value
of p<0.05 was considered statistically significant.
Results
A total of 152 subjects (42 OSCC patients and 110 healthy controls) were included in this study. The sex, age, smoking, age of onset of smoking, duration, daily cigarette consumption, alcohol consumption, frequency of alcohol consumption, family history, response of treatment, patient status, disease status, and disease area of OSCC patients were analyzed. The demographic and clinical characteristics of the study participants are shown in Table 1. Allelic and genotypic distributions of IL-6 gene -174 G/C, -572G/C, and -597G/A variants are presented in Table 2.;){kind=link}
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Table 1: Baseline demographical and clinical features of the patients with OSCC
Table 2: Genotype and allele distribution of IL-6 gene variants in groups
IL-6 gene -174G/C Variant (rs1800795)
There was a significant difference in the genotype distribution
of the IL-6 -174G/C variant between OSCC
patients and controls. IL-6 -174G/C G/C genotype was
higher in the patients compared to the control group
and G/G and C/C genotypes were lower in the patients
compared to the control group (p=0.016, OR:0.653,
95%CI: 0.38-1.11). No significant difference was observed
in patients and healthy controls in terms of allele
frequencies of the IL-6 -174G/C variant.
IL-6 gene-572G/C Variant (rs1800796)
The frequencies of the G/G, G/C, and C/C genotypes
of IL-6 -572G/C were 76.2%, 19.0%, and 33.3% in
OSCC patients and 74.5%, 21.8%, and 3.6% in controls,
respectively. The G and C allele frequencies were
85.71% and 14.28% in the OSCC patients, and 84.54%
and 14.54% in controls, respectively. The genotype and
allele distributions of IL-6 gene -572G/C variant were not statistically different between OSCC patients and
the control group.
IL-6 gene -597G/A Variant (rs1800797)
The frequencies of the G/G, G/A, and A/A genotypes
of IL-6 -597G/A were 64.3%, 33.3%, and 2.4% in the
OSCC patients and 74.5%, 24.5%, and 0.9% in controls,
respectively. G and A allele frequencies were 80.95%
and 19.04% in the OSCC patients, and 86.81% and
13.18% in controls, respectively. There was no significant
difference between patients and controls in terms
of genotype and allele distribution of the IL-6 gene
-597G/A variant.
STRING Analysis
Analyzing the IL-6 protein with the STRING database,
predicted the functional partners of the protein with
high confidence (score: 0.7) was found as follows: Signal
transducer and activator of transcription 3 (STAT3),
Proto-Oncogene C-Jun (JUN), Interleukin-4 (IL-4), Interleukin-10 (IL-10), Interleukin-6 Receptor (IL6R),
IL-6 Cytokine Family Signal Transduce (IL6ST), Interleukin
13 (IL13), Suppressor of Cytokine Signaling
3 (SOCS3), Interleukin 1B (IL1B), and C-X-C Motif
Chemokine Ligand 8 (CXCL8). The interaction network
of these proteins is shown in Figure 1.
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Discussion
Cancer development is a multistep process attributed to the accumulation of the risk factors in addition to the genetic susceptibility.[8] The molecular changes responsible for the initiation and progression of malignancy in normal epithelial cells are not yet well known. Other factors associated with the inflammatory response, angiogenic process, immune reactions, and thrombotic activity have also been associated with an increased risk of developing oral cavity cancer. The key element in this cancer development process is cytokines produced by activated innate immune cells that stimulate growth and progression in tumor cells. Recent studies have shown that genetic markers such as single-nucleotide polymorphisms (SNPs) may be associated with increased risk for specific diseases. The pathogenesis of OSCC is attributed to many factors, such as environmental, infections, diet, primarily tobacco, alcohol consumption, and viral agents. In addition, genetic and epigenetic changes are also effective in the development of malignant lesions of the oral cavity.IL-6 is produced by various endothelial cells, malignant cells and immune cell types primarily macrophages, dendritic cells, and B-cells.[9] IL-6 mostly stimulates the JAK1/STAT3 signaling pathway and acts as a pro-inflammatory factor.[10] IL-6 promotes carcinogenesis in a variety of ways, including apoptosis, survival, proliferation, angiogenesis, invasion, metastasis, and metabolism.[11] IL-6 level is elevated in plasma and cancer tissue where it plays a role in the proliferation and differentiation of malignant cells.[11] Circulating high IL-6 level is generally associated with poor prognosis and indicates a high degree of tumor aggressiveness, while low serum IL-6 is thought to respond better to treatment.[12,13]
There have been many studies investigating possible genetic mutations that could be used as a predictive and diagnostic tool for OSCC.[14] Many of these mutations have been identified in different tissues such as exfoliated oral epithelial cells,[15] saliva,[16] or serum.[17] It is well know that OSCC tumor cells produce different types of cytokines primarily IL-1, IL-6, IL-8, and TNF-α.[18] Lee et al.[19] found increased IL-1β, IL-6, and IL-8 in saliva in OSCC carcinogenesis. In addition, a significant overexpression of IL-6 and IL-8 was demonstrated in OSCC using microarray.[20] Wang et al.[21] showed a remarkable expression of IL-6R and IL-6 mRNA in tissue samples from OSCC patients compared to normal mucosa. Furthermore, Chen et al.[22] showed that overexpression of IL-6 in affected cancer cells correlated with aggressive clinical behavior, particularly in the male population with OSCC, so IL-6 can be used as a predictive independent the factor for survival.
The regulation and expression of the IL-6 gene are modulated by the binding of several transcriptional factors to the specific promoter site. The promoter region of IL-6 contains localized SNPs that can induce some modification at the level of specific DNA sequences thereby altering the binding of various transcription factors to these cis-acting elements, resulting in gene expression and synthesis of IL-6. This may also explain differences in susceptibility to various diseases such as cancer among different individuals.[23] IL-6 -174G/C is close to the binding sites of several transcriptional factors, including NF-κB and NF-IL-6.[24] IL-6 -174G/C promoter variant, a critical moderator of Th2 reactions, have been associated with high IL-6 production.[25] Oligonucleotides containing guanine at -174 position have a greater DNA-protein interaction than those containing cytosine. Therefore, they have a greater affinity for transcription factors and subsequently cause increased expression of the IL-6 gene. [24] During the inflammation, the C allele of the IL-6 -174G/C gene variant elevates the level of IL-6 protein. The IL-6 -174G/C variant was observed to be significantly associated with many malignities including breast, ovarian, prostatic, gastric, cervical, and colorectal cancer.[26] Vairaktaris et al.[27] reported that IL-6 -174G/C variant G/C- C/C genotypes and C allele were associated with an increased risk of OSCC. Furthermore, Singh et al.[26] found significant differences in G/C genotype and C allele frequencies of the IL-6 -174 G/C variant between OSCC patients and controls in the Indian population. Zafar et al.[28] demonstrated that there was a significant association between OSCC stages (III and IV) and IL-6 -174G/C variant. Hsu et al.[29] showed an association between an increased risk of oral precancerous lesions and IL-6 -174G/C C allele. However, in another study performed by Gaur et al.,[30] it was reported that IL-6 -174G/C CC genotype and C allele appeared to be protective in patients with tobocco-related OSCC. These inconsistencies can be attributed to various factors considered in these studies including race, population size, sex, and disease status. In this study, we found that the IL-6 -174G/C G/C genotype was significantly associated with an increased risk of OSCC (Table 2). Our results were consistent with many studies.
The relationship between IL-6 -572G/C and circulating IL-6 levels has been inconclusive in previous genetic studies. Shin et al.[31] observed that the IL-6 -572 G/G genotype was associated with significantly higher levels of circulating IL-6 than the C/C or C/G genotype. Furthermore, Seow et al.[32] reported that the IL-6 -572G/C G allele was associated with increased IL-6 protein level in serum compared to the C allele. Zhang et al.[33] found that IL-6 -572C/G genotype distribution differed between patients with breast cancer and controls. However, there was no significant difference between IL-6 -572C/G genotypes and serum IL-6 levels in these patients and healthy controls. In studies in patients with hepatocellular carcinoma and gastric adenocarcinoma, the IL-6 -572 G/C genotype and allele frequency were not different between patients and controls.[34,35] Singh et al.[26] reported that the IL-6 -572G/C variant was not significantly associated with OSCC patients. In this study, there was no significant difference between OSCC patients and controls in terms of genotype and allele distribution of the IL-6 -572G/C variant.
IL-6 -597G/A is another variant in the promoter region of the IL-6 gene that can modulate the effect of the IL-6-174G/C on transcription activity.[24] De Michele et al.[36] observed that IL-6 -597G/A G/G genotype was significantly associated with worse disease and free survival in patients with breast cancer. In addition, they found that IL-6 -597G/A genotype distribution was significantly associated with the number of positive lymph nodes. Snoussi et al.[37] showed that IL-6 -597G/A heterozygous genotype was related to breast carcinoma. Wilkening et al.[38] found that patients with skin basal cell carcinoma had a higher IL-6 -597G/A G/G genotype. Gupta et al.[39] reported that IL-6 -597G/A variant G/G and G/A genotype have been reported to be more common in patients with OSCC than in controls. In this study, genotype and allele distributions of IL-6 -597G/A were not significantly different between OSCC patients and controls.
There are several limitations to this analysis. First, only three variants of IL-6 were evaluated. Second, the gene-gene and gene-environment interactions were not investigated. Finally, IL-6 expression level was not examined in this study.
Conclusion
Our current investigation is focused on the role of variants of IL-6 gene on OSCC. To the best of our knowledge, this study is the first study to evaluate the genotype and allele frequencies of IL-6 -174G/C, -572G/C, and -597G/A variants in patients with OSCC in a Turkish population. Our results support that the IL-6 -174G/C variant may play an important role in susceptibility to OSCC in our population. Peer-review: Externally peer-reviewed.Conflict of Interest: All authors declared no conflict of interest.
Ethics Committee Approval: The study was approved by the Hitit University, Faculty of Medicine Ethics Committee (no: 2017/200, date: 19/12/2017).
Financial Support: None declared.
Authorship contributions: Concept - Ö.G., S.Y.; Design - Ö.G., H.D., S.Y.; Supervision - S.Y., A.F.N.; Funding - None; Materials - Ö.G., H.D.; Data collection and/or processing - Ö.G., H.D.; Data analysis and/or interpretation - S.Y., N.K.; Literature search - A.F.N., A.T.; Writing - A.F.N., A.T.; Critical review - A.F.N., H.D.
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