Introduction
Oral squamous cell carcinoma (OSCC) is classified
among the most common solid tumors, accounting
for 4% of all malignancies.[] 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.[]
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.
[] 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]).[] 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.[]
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 Population
Forty-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.[] 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.
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.
Fig 1: Interactions of IL-6 protein, according to STRING database predictions. (a) Confidence network: stronger associations
are represented by thicker lines, (b) this network represents the types of evidences for the association, (c)
presentation of the different modes of action, involving in the protein?protein interactions.
SOCS3: Suppressor of Cytokine Signaling 3; STAT3: Signal transducer and activator of transcription 3; IL6R: Interleukin-6 Receptor;
IL6ST: IL-6 Cytokine Family Signal Transduce; IL-4-6-10-13: Interleukin-4-6-10-13; IL1B: Interleukin 1B; CXCL8: C-X-C Motif Chemokine
Ligand 8.
Discussion
Cancer development is a multistep process attributed
to the accumulation of the risk factors in addition
to the genetic susceptibility.[] 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.[] IL-6 mostly
stimulates the JAK1/STAT3 signaling pathway and
acts as a pro-inflammatory factor.[] IL-6 promotes
carcinogenesis in a variety of ways, including apoptosis,
survival, proliferation, angiogenesis, invasion, metastasis,
and metabolism.[] IL-6 level is elevated in
plasma and cancer tissue where it plays a role in the
proliferation and differentiation of malignant cells.[]
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.[,]
There have been many studies investigating possible
genetic mutations that could be used as a predictive
and diagnostic tool for OSCC.[] Many of these
mutations have been identified in different tissues
such as exfoliated oral epithelial cells,[] saliva,[] or serum.[] It is well know that OSCC tumor cells
produce different types of cytokines primarily IL-1,
IL-6, IL-8, and TNF-α.[] Lee et al.[] 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.[] Wang et al.[] 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.[] 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.[] IL-6
-174G/C is close to the binding sites of several transcriptional
factors, including NF-κB and NF-IL-6.[]
IL-6 -174G/C promoter variant, a critical moderator
of Th2 reactions, have been associated with high IL-6
production.[] 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.
[] 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.[] Vairaktaris et al.[] 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.[] 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.[] demonstrated
that there was a significant association between OSCC
stages (III and IV) and IL-6 -174G/C variant. Hsu et
al.[] 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.,[] 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.[] 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.[] 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.[] 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.[,] Singh et al.[] 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.[] De
Michele et al.[] 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.[]
showed that IL-6 -597G/A heterozygous genotype
was related to breast carcinoma. Wilkening et al.[]
found that patients with skin basal cell carcinoma
had a higher IL-6 -597G/A G/G genotype. Gupta et
al.[] 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.
