BioMed Research International
Volume 2018 (2018), Article ID 9531315, 8 pages
https://doi.org/10.1155/2018/9531315

Research Article

Association of Resistin Gene Polymorphisms with Oral Squamous Cell Carcinoma Progression and Development

Wei-Hung Yang,1,2,3 Shoou-Jyi Wang,⁴ Yung-Sen Chang,³ Chen-Ming Su,⁵ Shun-Fa Yang,6,7 and Chih-Hsin Tang8,9,10,11

¹School of Chinese Medicine, China Medical University, Taichung, Taiwan
²Department of Nursing, National Taichung University of Science and Technology, Taichung, Taiwan
³Department of Orthopedic Surgery, Taichung Hospital, Ministry of Health and Welfare, Taichung, Taiwan
⁴Department of Orthopedic Surgery, Chang-Hua Hospital, Ministry of Health and Welfare, Puhsin Township, Changhua County, Taiwan
⁵Department of Biomedical Sciences Laboratory, Affiliated Dongyang Hospital of Wenzhou Medical University, Dongyang, Zhejiang, China
⁶Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan
⁷Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan
⁸Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan
⁹Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan
¹⁰Chinese Medicine Research Center, China Medical University, Taichung, Taiwan
¹¹Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan

Correspondence should be addressed to Shun-Fa Yang; ysf@csmu.edu.tw and Chih-Hsin Tang; chtang@mail.cmu.edu.tw

Received 28 May 2018; Accepted 27 September 2018; Published 14 October 2018

Academic Editor: Konstantinos Michalakis

Copyright © 2018 Wei-Hung Yang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Oral squamous cell carcinoma (OSCC) accounts for over 90% of malignant neoplasms of the mouth. In Taiwan, OSCC is the fourth most common male cancer and the fourth leading cause of male cancer death. Resistin (RETN) is an adipokine that is associated with obesity, inflammation, and various cancers. Here, we examine the association between four single nucleotide polymorphisms (SNPs) of the RETN gene (rs3745367, rs7408174, rs1862513, and rs3219175) and OSCC susceptibility as well as clinical outcomes in 935 patients with OSCC and in 1200 cancer-free healthy controls. We found that, in 1465 smokers, RETN polymorphisms carriers with the betel-nut chewing habit had a 6.708–10.882-fold greater risk of having OSCC compared to RETN wild-type carriers without the betel-nut chewing habit. Patients with OSCC who had A/A homozygous of RETN rs3219175 polymorphism showed a high risk for an advanced tumor size (> T2), compared to those patients with G/G homozygotes. In addition, A/T/G/G haplotype significantly increased the risks for OSCC by 1.376-fold. This study is the first to examine the risk factors associated with RETN SNPs in OSCC progression and development in Taiwan.

1. Introduction

Oral squamous cell carcinoma (OSCC) accounts for over 90% of malignant neoplasms of the mouth [1]. In Taiwan, OSCC is the fourth most common male cancer and the fourth leading cause of male cancer death [2]. Despite combination treatment with radiation, surgery, and chemotherapy, the prognosis of OSCC remains poor [3, 4]. OSCC occurs through multiple genetic alterations due to chronic exposure to environmental carcinogens. Chronic inflammation, alcohol and tobacco consumption, betel quid chewing, and viral infection are all implicated as risk factors for OSCC [5–7]. Single nucleotide polymorphisms (SNPs) constitute the most common type of DNA sequence variation influenced gene expression and protein production and function as well as disease susceptibility in particular individuals [8, 9]. Previous research has suggested that gene polymorphisms may increase the risk of developing oral cancer. For instance, individuals carrying specific C-C chemokine ligand 4 (CCL4), interleukin-18 (IL-18), or intercellular adhesion molecule-1 (ICAM-1) SNPs have a higher susceptibility to OSCC compared with wild-type controls [2, 10, 11]. Thus, identifying genes that impact upon OSCC susceptibility is important for early detection of disease.

Resistin is a 12.5-kDa cysteine-rich adipokine that is constitutively secreted by adipose tissue [12]; resistin levels in plasma correlate with inflammatory markers and coronary artery calcification, a measure of coronary atherosclerosis [13]. Several SNPs have been discovered in the RETN promoter and 3′-untranslated regions [14]. Genetic variation in RETN is associated with a greater risk of various diseases, including metabolic syndrome and colon cancer [15, 16], while a functional RETN gene polymorphism at -420 (rs186513) appears to promote susceptibility to type 2 diabetes [17] and RETN SNPs have been found to correlate with lung cancer progression in a Chinese Han population [18]. Moreover, upregulation of resistin gene expression in human breast cancer tissues has been reported [19]. Up until now, no correlation has been established between RETN gene polymorphisms and OSCC prognosis. This case-control study sought to determine the role of four RETN SNPs and clinicopathological features in OSCC susceptibility in a cohort of Taiwan individuals.

2. Materials and Methods

2.1. Participants

We enrolled 935 patients (cases) presenting with OSCC to Chung Shan Medical University Hospital in Taichung or Changhua Christian Hospital in Changhua, Taiwan, between 2007 and 2015. A total of 1,200 anonymized healthy controls were randomly selected from the Taiwan Biobank Project; none had a previous history of cancer at any sites. The study exclusion criteria specified that subjects with oral precancerous disease, such as oral submucous fibrosis, leukoplakia, erythroplakia, or verrucous hyperplasia, were ineligible for enrollment. Data pertaining to primary tumor size, lymph node involvement, and histologic grade were extracted from medical records. Tumors were staged according to the American Joint Committee on Cancer (AJCC) Tumor, Node, Metastasis (TNM) staging criteria [20]. Prior to enrollment, all participants provided informed written consent and completed a structured questionnaire surveying sociodemographic data and cigarette and alcohol use.

2.2. Determination of Genotypes

Total genomic DNA was isolated from whole blood specimens using QIAamp DNA Blood Mini Kits (Qiagen, Valencia, CA), as per the manufacturer’s instructions. DNA was dissolved in TE buffer (10 mM Tris pH 7.8, 1 mM EDTA) and stored at −20°C until quantitative polymerase chain reaction (PCR) analysis. Four RETN SNPs were selected (rs3745367, rs7408174, rs1862513, and rs3219175) with minor allele frequencies >5% in the HapMap population. Primers and probes consisted of rs3745367 “CTCCGACTGTCCCCACCTTATCCAC[A/G]GCTCCAAACCCAA”, rs7408174 “TTTTACCACAAAAAGGCCCGTTGTA[C/T]TGGAAACAAAGAA”, rs1862513 “CCTGACCAGTCTCTGGACATGAAGA[C/G]GGAGGCCCTGTTG”, rs3219175 “CTCCAGCCCTTACTGTCTGCTCAGG[A/G]GCTTCCTCTTGGC”. These SNPs have previously been found to correlate with progression of lung and breast cancer as well as development of rheumatoid arthritis [18, 19, 21]. We genotyped the SNPs with the commercially available TaqMan SNP genotyping assay (Applied Biosystems, Warrington, UK), according to the manufacturer’s protocols [22, 23].

2.3. Statistical Analysis

The genotype distribution of each SNP was analyzed for Hardy-Weinberg equilibrium and confirmed by Chi-square analysis. Demographic characteristics were compared between patients and controls using the Mann–Whitney U-test and Fisher’s exact test. Associations between genotypes, OSCC risk, and clinicopathological characteristics were estimated using adjusted odds ratios (AORs) and 95% confidence intervals (CIs) obtained from age- and gender-adjusted multiple logistic regression models. The statistical analysis about haplotype was according to previous studies [24, 25]. A value of <0.05 was considered statistically significant. Data were analyzed using SAS statistical software (Version 9.1, 2005; SAS Institute Inc., Cary, NC).

3. Results

Mean age did not differ significantly between men with (n=935) and without OSCC (n=1,200) (Table 1). Significant between-group differences were found for betel quid chewing (p<0.001), cigarette smoking (p<0.001), and alcohol drinking (p<0.001); all behaviors were significantly more prevalent among the OSCC cohort compared with controls (Table 1). Around half of the patients (49.8%) had stage I/II disease and half (50.2%) had stage III/IV disease (Table 1). One-third (32.8%) had N1–N3 lymph node metastasis. Nearly all tumors (99.0%) were classified as M0 status; the majority of tumors (85.1%) were moderately and poorly differentiated (Table 1).

Table 1: Demographic and clinical characteristics of 1,200 cancer-free, healthy males (controls) and 935 Taiwanese male patients with oral squamous cell carcinoma.

Variable
Controls (N=1,200)
Patients (N=935)
p-value

Age (years)

    ≤54
566 (47.2%)
453 (48.5%)
p=0.556

    >54
634 (52.8%)
482 (51.5%)

Betel quid chewing

    No
1,001 (83.4%)
190 (20.3%)

    Yes
199 (16.6%)
745 (79.7%)
p <0.001

Cigarette smoking

    No
564 (47.0%)
106 (11.3%)

    Yes
636 (53.0%)
829 (88.7%)
p <0.001

Alcohol drinking

    No
963 (80.3%)
440 (47.1%)

    Yes
237 (19.7%)
495 (52.9%)
p <0.001

Histological grade

    I+II

466 (49.8%)

    III+IV

469 (50.2%)

Tumor status (T)

    T1+T2

540 (57.8%)

    T3+T4

395 (42.2%)

Lymph node metastasis (N)

    N0

628 (67.2%)

    N1+N2+N3

307 (32.8%)

Distal metastasis (M)

    M0

926 (99.0%)

    M1

9 (1.0%)

Cell differentiation

    Well differentiated

139 (14.9%)

    Moderately or poorly differentiated

796 (85.1%)

The Mann-Whitney U test or Fisher’s exact test was used to compare values between healthy controls and patients with OSCC.   p value < 0.05 was considered statistically significant.

Table 2 shows the RETN genotypes in OSCC patients and controls. In the controls, all genotypic frequencies were in Hardy-Weinberg equilibrium (p>0.05). In both patients and controls, most of those with the rs3745367 SNP were homozygous for the G/G genotype, most of those with the rs7408174 SNP were homozygous for the T/T genotype, most of those with the rs1862513 SNP were homozygous for G/G, and most of those with the rs3219175 SNP were homozygous for G/G (Table 2). After adjusting for potential confounders, we found no significant differences in the incidences of OSCC patients with the rs3745367, rs7408174, rs1862513, and rs3219175 polymorphisms compared to controls.

Table 2: Genotyping and allele frequency of RETN single nucleotide polymorphisms in oral squamous cell carcinoma and cancer-free, healthy males (controls).

Variable
Controls (N=1,200)
Patients (N=935)
OR (95% CI)
AOR (95% CI)

rs3745367

GG
461 (38.4%)
349 (37.3%)
1.000 (reference)
1.000 (reference)

GA
561 (46.8%)
449 (48.0%)
1.057 (0.877-1.274)
1.132 (0.888-1.444)

AA
178 (14.8%)
137 (14.7%)
1.017 (0.782-1.322)
1.066 (0.755-1.506)

GA+AA
739 (61.6%)
586 (62.7%)
1.047 (0.878-1.249)
1.116 (0.887-1.406)

rs7408174

TT
603 (50.3%)
499 (53.4%)
1.000 (reference)
1.000 (reference)

TC
497 (41.4%)
369 (39.5%)
0.897 (0.750-1.074)
0.891 (0.705-1.126)

CC
100 (8.3%)
67 (7.1%)
0.810 (0.581-1.128)
0.901 (0.588-1.380)

TC+CC
597 (49.7%)
436 (46.6%)
0.883 (0.744-1.047)
0.893 (0.714-1.116)

rs1862513

GG
443 (36.9%)
352 (37.7%)
1.000 (reference)
1.000 (reference)

GC
575 (47.9%)
452 (48.3%)
0.989 (0.821-1.192)
0.991 (0.777-1.265)

CC
182 (15.2%)
131 (14.0%)
0.906 (0.696-1.181)
0.908 (0.644-1.280)

GC+CC
757 (63.1%)
583 (62.3%)
0.969 (0.812-1.157)
0.971 (0.771-1.223)

rs3219175

GG
761 (63.4%)
590 (63.1%)
1.000 (reference)
1.000 (reference)

GA
397 (33.1%)
304 (32.5%)
0.988 (0.822-1.187)
0.918 (0.722-1.168)

AA
42 (3.5%)
41 (4.4%)
1.259 (0.808-1.962)
0.994 (0.559-1.767)

GA+AA
439 (36.6%)
345 (36.9%)
1.014 (0.849-1.210)
0.926 (0.735-1.168)

The adjusted odds ratios (AORs) with their 95% confidence intervals (CIs) were estimated by multiple logistic regression models that controlled for betel quid chewing, tobacco smoking, and alcohol consumption.

Tobacco consumption and betel quid chewing are important risk factors for the development of OSCC [5–7]. The effect of the interaction between tobacco consumption and betel quid chewing with RETN genotypes on OSCC progression is shown in Table 3. Among all 1,465 smokers (healthy controls and patients combined), those who either had at least one A allele of rs3745367, one C allele of rs74081741, one C allele of rs1862513, one A allele of rs3219175 or chewed betel nuts were 5.838-fold (95% CI: 3.985-8.552), 4.105-fold (95% CI: 2.960-5.692), 5.090-fold (95% CI: 3.493-7.418), and 4.134-fold (95% CI: 3.028-5.644) more likely, respectively, to develop OSCC compared to smokers with wide-type homozygotes who did not chew betel nuts. Moreover, smokers with at least one A allele of rs3745367, one C allele of rs74081741, one C allele of rs1862513, or one A allele of rs3219175 and who chewed betel nuts had respective risks that were 8.544-fold (95% CI: 5.668-12.880), 8.583-fold (95% CI: 6.026-12.225), 6.708-fold (95% CI: 4.467-10.074), and 10.882-fold (95% CI: 7.861-15.064) higher, respectively, for developing OSCC compared with the wild-type homozygous smokers. These results suggest that RETN gene polymorphisms have a strong impact upon oral cancer susceptibility in men who smoke tobacco and/or chew betel nuts.

Table 3: Associations of the combined effect of RETN gene polymorphisms and betel nut chewing with the susceptibility to oral cancer among 1,465 smokers.

Variable
Controls (n=636) ()
Patients (n=829) ()
OR (95 CI)
AOR (95 CI)

rs3745367

GG genotype & non-betel nut chewing
178 (28.0%)
39 (4.7%)
1.00
(reference)
1.000
(reference)

GA or AA genotype or betel nut chewing
135 (21.2%)
291 (35.1%)
9.838 (6.580-14.708)
8.544 (5.668-12.880)

GA or AA genotype with betel nut chewing
323 (50.8%)
499 (60.2%)
7.051 (4.852-10.246)
5.838 (3.985-8.552)

rs7408174

TT genotype & non-betel nut chewing
218 (34.3%)
62 (7.5%)
1.00
(reference)
1.000
(reference)

TC or CC genotype or betel nut chewing
127 (20.0%)
385 (46.4%)
10.659 (7.540-15.068)
8.583 (6.026-12.225)

TC or CC genotype with betel nut chewing
291 (45.7%)
382 (46.1%)
4.616 (3.349-6.361)
4.105 (2.960-5.692)

rs1862513

GG genotype & non-betel nut chewing
166 (26.1%)
41 (5.0%)
1.00
(reference)
1.000
(reference)

GC or CC genotype or betel nut chewing
138 (21.7%)
289 (34.9%)
8.479 (5.698-12.617)
6.708 (4.467-10.074)

GC or CC genotype with betel nut chewing
332 (52.2%)
499 (60.1%)
6.085 (4.208-8.800)
5.090 (3.493-7.418)

rs3219175

GG genotype & non-betel nut chewing
287 (45.1%)
78 (9.4%)
1.00
(reference)
1.000
(reference)

GA or AA genotype or betel nut chewing
125 (19.7%)
448 (54.0%)
13.187 (9.583-18.147)
10.882 (7.861-15.064)

GA or AA genotype with betel nut chewing
224 (35.2%)
303 (36.6%)
4.977 (3.672-6.746)
4.134 (3.028-5.644)

The adjusted odds ratios (AORs) with their 95% confidence intervals (CIs) were estimated by multiple logistic regression models that controlled for alcohol consumption.

Next, we compared associations between the RETN rs3219175 polymorphism and clinical status in OSCC patients aged >54 years. Compared with patients with the G/G genotype, those with the A/A genotype at the rs3219175 SNP were 2.480-fold (95% CI: 1.058-5.814) more likely to develop large tumors (>T2) (Table 4). No significant between-group differences were observed for clinical stage, lymph node metastasis, distant metastasis, or cell differentiation at the rs3219175 SNP (Table 4).

Table 4: Associations between the RETN rs3219175 polymorphism and clinical status in men aged >54 years with OSCC.

Clinical Stage

OR (95% CI)
AOR (95% CI)

RETN rs3219175
Stage I/II (n=243)
Stage III/IV (n=239)

GG
160 (65.8%)
150 (62.8%)
1.000 (reference)
1.000 (reference)

GA
74 (30.5%)
73 (30.5%)
1.052 (0.711-1.558)
1.030 (0.693-1.529)

AA
9 (3.7%)
16 (6.7%)
1.896 (0.813-4.421)
1.896 (0.808-4.452)

Tumor size

RETN rs3219175
T2 (n=272)
> T2 (n=210)

GG
181 (66.5%)
129 (61.4%)
1.000 (reference)
1.000 (reference)

GA
82 (30.2%)
65 (31.0%)
1.112 (0.748-1.653)
1.113 (0.747-1.658)

AA
9 (3.3%)
16 (7.6%)
2.494 (1.069-5.820)
2.480 (1.058-5.814)

Lymph node metastasis

RETN rs3219175
No (n=333)
Yes (n=149)

GG
215 (22.8%)
95 (63.8%)
1.000 (reference)
1.000 (reference)

GA
102 (54.5%)
45 (30.2%)
0.998 (0.652-1.528)
0.969 (0.631-1.488)

AA
16 (22.7%)
9 (6.0%)
1.273 (0.543-2.983)
1.240 (0.525-2.930)

Metastasis

RETN rs3219175
M0 (n=477)
M1 (n=5)

GG
308 (64.6%)
2 (40.0%)
1.000 (reference)
1.000 (reference)

GA
144 (30.2%)
3 (60.0%)
3.208 (0.530-19.411)
3.009 (0.491-18.451)

AA
25 (5.2%)
0 (27.2%)
-
0.783 (0.172-3.559)

Cell differentiation

RETN rs3219175
Well (n=76)
Moderate/poor (n=406)

GG
51 (67.1%)
259 (63.8%)
1.000 (reference)
1.000 (reference)

GA
21 (27.6%)
126 (31.0%)
1.181 (0.681-2.050)
1.176 (0.675-2.047)

AA
4 (5.3%)
21 (5.2%)
1.034 (0.340-3.139)
0.963 (0.315-2.945)

Multivariate analysis adjusted for the effects of betel quid chewing, tobacco smoking, and alcohol consumption.

p = 0.0345; p = 0.0367.

An evaluation of the haplotypes sought to determine the combined effect of the four polymorphisms on OSCC susceptibility. In an analysis of distribution frequencies of RETN rs3745367, rs7408174, rs1862513, and rs3219175 haplotypes, the most common haplotype in healthy controls was G/T/G/G, which was therefore selected as the reference. The A/T/G/G RETN haplotype significantly increased the risk for developing OSCC by 1.376-fold (95% CI: 1.039-1.823, p<0.05) (Table 5). Furthermore, A/T/G/G RETN haplotype also enhanced the risk for developing OSCC in those who chewed betel nuts by 18.39-fold (95% CI: 12.333-27.421, p<0.05) (Table 6).

Table 5: RETN haplotype frequencies in healthy controls and OSCC patients.

Haplotype block

Controls
Patients

rs3745367 G/A
rs7408174 T/C
rs1862513 G/C
rs3219175 G/A
n = 2400
n = 1870
OR (95% CI)
AOR (95% CI)

G
T
G
G
679 (28.3%)
492 (26.3%)
1.000 (reference)
1.000 (reference)

G
C
G
G
475 (19.8%)
422 (22.6%)
1.226 (1.029-1.461)
1.212 (0.965-1.522)

A
T
C
A
318 (13.2%)
291 (15.6%)
1.263 (1.037-1.538)
1.150 (0.890-1.486)

A
T
G
G
257 (10.7%)
226 (12.1%)
1.214 (0.981-1.502)
1.376 (1.039-1.823)

A
T
C
G
189 (7.9%)
153 (8.2%)
1.117 (0.876-1.424)
1.219 (0.890-1.672)

G
T
C
G
191 (8.0%)
146 (7.8%)
1.055 (0.826-1.347)
1.062 (0.773-1.460)

G
T
C
A
67 (2.8%)
57 (3.0%)
1.174 (0.810-1.703)
1.051 (0.644-1.716)

A
C
C
A
86 (3.6%)
36 (1.9%)
0.578 (0.385-0.867)
0.712 (0.427-1.188)

G
C
C
G
63 (2.6%)
30 (1.6%)
0.657 (0.419-1.031)
0.889 (0.509-1.552)

A
C
G
G
46 (1.9%)
14 (0.7%)
0.420 (0.228-0.773)
0.343 (0.161-0.728)

A
C
C
G
19 (0.8%)
1 (0.1%)
0.073 (0.010-0.544)
0.176 (0.019-1.643)

G
C
C
A
6 (0.2%)
0 (0.0%)
-
-

A
T
G
A
2 (0.1%)
2 (0.1%)
1.380 (0.194-9.831)
1.620 (0.105-24.958)

G
C
G
A
2 (0.1%)
0 (0.0%)
-
-

Multivariate analysis adjusted for the effects of betel quid chewing, tobacco smoking, and alcohol consumption.

p=0.0225; p=0.0201; p=0.0081; p=0.0053; p=0.0107; p=0.0258; p=0.0054.

OSCC: oral squamous cell carcinoma; OR: odds ratio; AOR: adjusted odds ratio.

Table 6: Combined effect of betel quid chewing and RETN haplotypes on OSCC development.

Betel quid chewing
RETN haplotype
Controls
Patients
AOR (95% CI)

n = 2400
n = 1870

Yes
A-T-G-G
33 (1.4%)
179 (9.6%)
18.390 (12.333-27.421)

Yes
Others
365 (15.2%)
1311 (70.1%)
12.774 (10.666-15.300)

No
A-T-G-G
224 (9.3%)
47 (2.5%)
1.203 (0.856-1.690)

No
Others
1778 (74.1%)
333 (17.8%)
1.000 (reference)

Other haplotypes included G-T-G-G, G-C-G-G, A-T-C-A, A-T-C-G, G-T-C-G, G-T-C-A, A-C-C-A, G-C-C-G, A-C-G-G, A-C-C-G, G-C-C-A, A-T-G-A, and G-C-G-A.

Multivariate analysis adjusted for the effects of tobacco smoking and alcohol consumption.

p<0.001.

OSCC: oral squamous cell carcinoma.

4. Discussion

Resistin is an adipokine that is associated with obesity, inflammation, and various cancers [26–29]. Furthermore, evidence suggests that in patients with lung cancer, high serum resistin levels may play a role in the pathogenesis of cancer cachexia [30]. Upregulation of resistin in serum has been detected in OSCC patients [31]. RETN polymorphisms have been identified in various cancers, including colon, breast, and lung [16, 18, 19], although scant data exist on the involvement of RETN polymorphisms in OSCC. To the best of our knowledge, this current study is the first to examine the distribution of the rs3745367, rs7408174, rs1862513, and rs3219175 SNPs and their possible association with susceptibility to the development of OSCC. We also investigated susceptibility to OSCC when these RETN SNPs were combined with environmental carcinogens. Interestingly, we did not find any significant differences between the frequencies of OSCC patients and controls with the rs3745367, rs7408174, rs1862513, and rs3219175 polymorphisms. We did discover that OSCC patients aged >54 years carrying the rs3219175 G/G homozygous polymorphism had a significantly high risk of developing large-size tumors compared to those carrying the rs3219175 A/A homozygous polymorphism. However, we did not recruit the survival results of OSCC patients. Future research could evaluate the association of RETN polymorphisms with survival of OSCC patients. In addition, it would be advisable to collect data on a larger number of patients for analysis of the functions of RETN polymorphisms in OSCC.

The linkage disequilibrium is expressed across the human genome and therefore could be used as a genetic marker to locate adjacent variants that participate in the detection and treatment of disease. Haplotype analyses can provide data on disease susceptibility [32]. Our evaluation of the impact of different haplotype combinations of four RETN SNPs (rs3745367, rs7408174, rs1862513, and rs3219175) upon the risk of OSCC revealed that the A/T/G/G haplotype is associated with a high risk for OSCC when compared with the reference haplotype, G/T/G/G. Furthermore, compared with other SNPs, the A/T/G/G RETN haplotype also enhanced the risk for developing OSCC in those who chewed betel nuts. We speculate that this haplotype is in linkage disequilibrium with other functional polymorphisms that are responsible for the susceptibility to OSCC.

Exposure to environmental carcinogens might lead to an earlier onset of oral cancer development. It is known that genomic changes during hepatocarcinogenesis progressively alter the hepatocellular phenotype to produce cellular intermediates that evolve into malignancy [33]. Polymorphisms of several genes are known to be associated with the risk of OSCC [34]. Thus, genetic components may play a pivotal role in carcinogenesis. Our study demonstrates a synergistic effect between betel quid chewing and tobacco smoking with four RETN SNPs (rs3745367, rs7408174, rs1862513, and rs3219175) on the risk of developing OSCC. Smokers with at least one A allele of rs3745367, one C allele of rs74081741, one C allele of rs1862513, or one A allele of rs3219175 and who chewed betel nuts were more likely to develop OSCC, with respective odds of 8.544 (95% CI: 5.668-12.880), 8.583 (95% CI: 6.026-12.225), 6.708 (95% CI: 4.467-10.074), and 10.882 (95% CI: 7.861-15.064). Long-term exposure to tobacco smoke and betel-nut chewing has been found to promote chronic inflammation reactions in oral tissue, subsequently leading to random genetic alteration and the development of oral cancer [35]. We suggest that tobacco smoking and betel-nut chewing in combination with RETN polymorphisms can increase the risk of rapid progression to oral cancer; thus, patients with a higher RETN polymorphism need to be aware of their higher risk of oral cancer.

Our results demonstrate an association between RETN gene variants and risk of OSCC. Compared with OSCC patients carrying the G/G genotype, those with the A/A genotype at the rs3219175 SNP were prone to developing large-size tumors. In an analysis of haplotype combinations of four RETN SNPs (rs3745367, rs7408174, rs1862513, and rs3219175), the A/T/G/G haplotype was found to be associated with a high risk of OSCC. Thus, RETN could serve as a genetic prognostic marker for OSCC treatment.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

All authors confirm that they have no financial or personal relationships with other people or organizations that could inappropriately influence this work.

Acknowledgments

This work was supported by grants from the Ministry of Science and Technology of Taiwan (MOST 106-2320-B-039-005; 107-2320-B-039-019-MY3); China Medical University under the Higher Education Sprout Project, Ministry of Education, Taiwan (CMRC-CHM-3-1); and Taichung Hospital, Ministry of Health and Welfare (107023).

References

S. V. K. Rao, G. Mejia, K. Roberts-Thomson, and R. Logan, “Epidemiology of oral cancer in Asia in the past decade - An update (2000-2012),” Asian Pacific Journal of Cancer Prevention, vol. 14, no. 10, pp. 5567–5577, 2013.

M.-Y. Lien, C.-W. Lin, H.-C. Tsai et al., “Impact of CCL4 gene polymorphisms and environmental factors on oral cancer development and clinical characteristics,” Oncotarget , vol. 8, no. 19, pp. 31424–31434, 2017.

S. Xie, H. Xu, X. Shan et al., “Clinicopathological and Prognostic Significance of Survivin Expression in Patients with Oral Squamous Cell Carcinoma: Evidence from a Meta-Analysis,” PLoS ONE, vol. 10, no. 2, p. e0116517, 2015.

C. Lee, N. Chiang, Y. Lu et al., “Benzyl isothiocyanate (BITC) triggers mitochondria-mediated apoptotic machinery in human cisplatin-resistant oral cancer CAR cells,” Biomedicine, vol. 8, no. 3, p. 15, 2018.

M. C. Chang, C. P. Chiang, C. L. Lin, J. J. Lee, L. J. Hahn, and J. H. Jeng, “Cell-mediated immunity and head and neck cancer: With special emphasis on betel quid chewing habit,” Oral Oncology, vol. 41, no. 8, pp. 757–775, 2005.

Y.-J. Chen, J. T.-C. Chang, C.-T. Liao et al., “Head and neck cancer in the betel quid chewing area: Recent advances in molecular carcinogenesis,” Cancer Science, vol. 99, no. 8, pp. 1507–1514, 2008.

V. Ajila, H. Shetty, S. Babu, V. Shetty, and S. Hegde, “Human Papilloma Virus Associated Squamous Cell Carcinoma of the Head and Neck,” Journal of Sexually Transmitted Diseases, vol. 2015, Article ID 791024, 5 pages, 2015.

B. S. Shastry, “SNP alleles in human disease and evolution,” Journal of Human Genetics, vol. 47, no. 11, pp. 561–566, 2002.

M. Yang, K. Lin, M. Lu et al., “Contribution of matrix metalloproteinases-1 genotypes to gastric cancer susceptibility in Taiwan,” Biomedicine, vol. 7, no. 2, p. 10, 2017.

H. Tsai, C. Hsin, Y. Hsieh et al., “Impact of Interleukin-18 Polymorphisms -607A/C and -137G/C on Oral Cancer Occurrence and Clinical Progression,” PLoS ONE, vol. 8, no. 12, p. e83572, 2013.

C. Lin, C. Chuang, C. Tang et al., “Combined Effects of ICAM-1 Single-Nucleotide Polymorphisms and Environmental Carcinogens on Oral Cancer Susceptibility and Clinicopathologic Development,” PLoS ONE, vol. 8, no. 9, p. e72940, 2013.

C. M. Steppan, S. T. Bailey, S. Bhat et al., “The hormone resistin links obesity to diabetes,” Nature, vol. 409, no. 6818, pp. 307–312, 2001.

M. P. Reilly, M. Lehrke, M. L. Wolfe, A. Rohatgi, M. A. Lazar, and D. J. Rader, “Resistin is an inflammatory marker of atherosclerosis in humans,” Circulation, vol. 111, no. 7, pp. 932–939, 2005.

C. M. Steppan, E. J. Brown, C. M. Wright et al., “A family of tissue-specific resistin-like molecules,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 98, no. 2, pp. 502–506, 2001.

S. Kumar, V. Gupta, N. Srivastava et al., “Resistin 420C/G gene polymorphism on circulating resistin, metabolic risk factors and insulin resistance in adult women,” Immunology Letters, vol. 162, no. 2, pp. 287–291, 2014.

R. N. Alharithy, “Polymorphisms in RETN gene and susceptibility to colon cancer in Saudi patients,” Annals of Saudi Medicine, vol. 34, no. 4, pp. 334–339, 2014.

C.-M. Chung, T.-H. Lin, J.-W. Chen et al., “Common quantitative trait locus downstream of RETN gene identified by genome-wide association study is associated with risk of type 2 diabetes mellitus in Han Chinese: A Mendelian randomization effect,” Diabetes/Metabolism Research and Reviews, vol. 30, no. 3, pp. 232–240, 2014.

W. Hu, C. Tang, Y. Sun et al., “Correlation between resistin gene polymorphism and clinical aspects of lung cancer,” Medicine, vol. 96, no. 52, p. e9485, 2017.

K. A. Vallega, N. Liu, J. S. Myers, K. Yu, Q. A. Sang, and M. Tan, “Elevated Resistin Gene Expression in African American Estrogen and Progesterone Receptor Negative Breast Cancer,” PLoS ONE, vol. 11, no. 6, p. e0157741, 2016.

J.-N. Vauthey, G. Y. Lauwers, N. F. Esnaola et al., “Simplified staging for hepatocellular carcinoma,” Journal of Clinical Oncology, vol. 20, no. 6, pp. 1527–1536, 2002.

L. Wang, C. Tang, T. Lu et al., “Resistin polymorphisms are associated with rheumatoid arthritis susceptibility in Chinese Han subjects,” Medicine, vol. 97, no. 12, p. e0177, 2018.

Y.-J. Lin, T.-J. Ho, T.-H. Lin et al., “P-coumaric acid regulates exon 12 splicing of the ATP7B gene by modulating hnRNP A1 protein expressions,” BioMedicine (Netherlands), vol. 5, no. 2, pp. 22–30, 2015.

T.-C. Li, C.-I. Li, L.-N. Liao et al., “Associations of EDNRA and EDN1 polymorphisms with carotid intima media thickness through interactions with gender, regular exercise, and obesity in subjects in Taiwan: Taichung Community Health Study (TCHS),” BioMedicine (Netherlands), vol. 5, no. 2, pp. 8–14, 2015.

B. Wang, Y.-E. Chou, M.-Y. Lien, C.-M. Su, S.-F. Yang, and C.-H. Tang, “Impacts of CCL4 gene polymorphisms on hepatocellular carcinoma susceptibility and development,” International Journal of Medical Sciences, vol. 14, no. 9, pp. 880–884, 2017.

C.-Q. Wang, C.-H. Tang, Y. Wang et al., “FSCN1 gene polymorphisms: Biomarkers for the development and progression of breast cancer,” Scientific Reports, vol. 7, no. 1, p. 15887, 2017.

C.-H. Tsai, H.-C. Tsai, H.-N. Huang et al., “Resistin promotes tumor metastasis by down-regulation of miR-519d through the AMPK/p38 signaling pathway in human chondrosarcoma cells,” Oncotarget , vol. 6, no. 1, pp. 258–270, 2015.

C.-M. Su, C.-J. Hsu, C.-H. Tsai, C.-Y. Huang, S.-W. Wang, and C.-H. Tang, “Resistin promotes angiogenesis in endothelial progenitor cells through inhibition of microRNA206: Potential implications for rheumatoid arthritis,” Stem Cells, vol. 33, no. 7, pp. 2243–2255, 2015.

C. M. Su, C. Y. Huang, and C. H. Tang, “Characteristics of resistin in rheumatoid arthritis angiogenesis,” Biomarkers in Medicine, vol. 10, no. 6, pp. 651–660, 2016.

C. Su, C. Tang, M. Chi et al., “Resistin facilitates VEGF-C-associated lymphangiogenesis by inhibiting miR-186 in human chondrosarcoma cells,” Biochemical Pharmacology, vol. 154, pp. 234–242, 2018.

G. Demiray, S. Değirmencioğlu, E. Uğurlu, and A. Yaren, “Effects of Serum Leptin and Resistin Levels on Cancer Cachexia in Patients With Advanced-Stage Non–Small Cell Lung Cancer,” Clinical Medicine Insights: Oncology, vol. 11, p. 117955491769014, 2017.

C.-C. Wu, H.-W. Chu, C.-W. Hsu, K.-P. Chang, and H.-P. Liu, “Saliva proteome profiling reveals potential salivary biomarkers for detection of oral cavity squamous cell carcinoma,” Proteomics, vol. 15, no. 19, pp. 3394–3404, 2015.

W. Hu, P.-Y. Liu, Y.-C. Yang et al., “Association of HMGB1 gene polymorphisms with lung cancer susceptibility and clinical aspects,” International Journal of Medical Sciences, vol. 14, no. 12, pp. 1197–1202, 2017.

S. S. Thorgeirsson and J. W. Grisham, “Molecular pathogenesis of human hepatocellular carcinoma,” Nature Genetics, vol. 31, no. 4, pp. 339–346, 2002.

K.-W. Chang, T.-C. Lee, W.-I. Yeh et al., “Polymorphism in heme oxygenase-1 (HO-1) promoter is related to the risk of oral squamous cell carcinoma occurring on male areca chewers,” British Journal of Cancer, vol. 91, no. 8, pp. 1551–1555, 2004.

C.-C. Wang, H.-L. Lin, S.-P. Wey, and T.-R. Jan, “Areca-nut extract modulates antigen-specific immunity and augments inflammation in ovalbumin-sensitized mice,” Immunopharmacology and Immunotoxicology, vol. 33, no. 2, pp. 315–322, 2011.