Association Between Genetic Polymorphisms of WNT1 Inducible Signaling Pathway Protein 1 and Uterine Cervical Cancer

Abstract

To date, no study has investigated the involvement of the single-nucleotide polymorphisms (SNPs) of WNT1 inducible signaling pathway protein 1 (WISP1) in uterine cervical cancer. Therefore, we conducted this study to explore the clinical implications of WISP1 SNPs in cervical cancer. One hundred and fifteen patients with invasive cervical cancer, 95 patients with preinvasive lesions, and 316 normal controls were enrolled. The WISP1 SNPs rs62514004, rs2929973, rs2977530, and rs2977537 were selected, and their genotypic distributions were determined through real-time polymerase chain reaction. Our findings showed that genotypes AG/GG in WISP1 SNP rs2977530 reduced the risk of invasive cervical cancer with AA as a reference; however, these genotypes did not reduce the risk of preinvasive lesions. By contrast, genotype AA in WISP1 SNP rs2977537 elevated the risk of invasive cervical cancer with GG/GA as a reference, but it did not elevate the risk of preinvasive lesions. Moreover, an additional integrated in silico analysis indicated that WISP1 rs2977537 altered the WISP1 expression recorded in the Genotype-Tissue Expression database. In conclusion, genotypes AG/GG in WISP1 SNP rs2977530 reduce the susceptibility of Taiwanese women to invasive cervical cancer, whereas genotype AA in rs2977537 increases the said risk.

Keywords

WNT1 inducible signaling pathway protein 1 single-nucleotide polymorphism uterine cervical cancer clinicopathological variables patient survival

Introduction

Uterine cervical cancer is the most common type of gynecological cancer in Taiwan. A report by the Health Promotion Administration of the Ministry of Health and Welfare, Taiwan, revealed that the age-standardized incidence rate of cervical cancer in 2013 was 30.68 per 100 000 women. The age-standardized mortality rate in 2013 was 3.89 per 100 000 women. The incidence rate ranked third and the mortality rate ranked seventh among Taiwanese women in 2013. Cytologic interpretations of cervical dysplasia are classified into low-grade squamous intraepithelial lesions (LSILs) and high-grade SILs.¹ Histologic diagnoses of LSILs are regarded as cervical intraepithelial neoplasia 1 (CIN1; low-grade CIN) or high-grade CIN consisting of CIN2 and CIN3.² Cervical carcinogenesis is currently thought to display a continuum of neoplastic transition from CIN to invasive squamous cell carcinoma.³

The WNT1 inducible signaling pathway protein 1 (WISP1), also referred to as CCN4, is a member of the CCN family.⁴ The acronym “CCN” is derived from the names of its first 3 family members, cysteine rich 61 (CCN1), connective tissue growth factor (CCN2), and nephroblastoma overexpressed gene (CCN3); another 3 family members are WISP1, WISP2, and WISP3 (CIN4, CIN5, and CIN6, respectively).^4
–6 Five exons and 4 introns constitute the WISP1 gene, which is located on chromosome 8q24.1 to 8q24.3.⁷ This gene was first defined as a downstream target of WNT1 and β-catenin, and it contributes to β-catenin-mediated carcinogenesis.^6,8

When a single-nucleotide difference is present in the shared sequence of a gene between the individuals of a species or the paired chromosomes in an individual, a single-nucleotide polymorphism (SNP) develops.^9
–11 Genetic polymorphisms can affect promoter activity or change the encoded amino acids in a protein when arising in the related coding sequence, thus influencing gene expression.¹² The SNPs may play critical roles in the occurrence and development of diseases. A study revealed that WISP1 variants could induce cellular transformation and increase the proliferation and invasiveness of cancer cells.¹³ To our knowledge, no study has established a connection between WISP1 genetic polymorphisms and cervical cancer. Therefore, we conducted this study to explore the association between WISP1 SNPs and the development of cervical cancer, and we further explored clinicopathological variables and patient survival among Taiwanese women.

Materials and Methods

Population

One hundred and fifteen patients with invasive cancer of the uterine cervix were consecutively recruited, and they received routine treatment protocols at the Department of Obstetrics and Gynecology in Chung Shan Medical University Hospital (CSMUH), Taiwan, from February 1994 to October 2014. Ninety-five patients with high-grade CIN were recorded as having preinvasive cervical lesions and received an abdominal total hysterectomy, a vaginal total hysterectomy, or a large loop excision of the transformation zone. The diagnosis of all patients with invasive cervical cancer or preinvasive lesions was confirmed by a pathological report before the treatment was started. Moreover, 316 healthy women without cervical lesions were included as normal controls. They received Papanicolaou smears at the outpatient department of CSMUH, and the cytologic diagnosis was further verified using colposcopy. All study participants were Taiwanese women living in central Taiwan. The institutional review board of CSMUH approved this study (CSMUH No: CS14014), and we obtained informed written consents from all participants.

Selection of WISP1 Genetic Polymorphisms

In this study, 4 WISP1 SNPs were selected based on data from the International HapMap Project and the findings of Chen et al.¹⁴ These genetic polymorphisms included WISP1 SNPs rs62514004 (exon 1, -899 A>G), rs2929973 (exon 5, 3′ untranslated region), rs2977530 (intron 1, -9995 [for exon 2] A>G), and rs2977537 (intron 1, -5044 [for exon 2] G>A).

Blood Sample Collection, Genomic DNA Extraction, and WISP1 Genotyping

Blood specimens were obtained from all participants using a standard venipuncture technique, placed into Vacutainer tubes containing EDTA, and stored at 4°C immediately after collection. DNA was extracted from peripheral vein blood leukocytes based on the manufacturer’s protocol, as detailed previously.¹⁵

Genotypes of WISP1 SNPs rs62514004, rs2929973, rs2977530, and rs2977537 were determined using the ABI StepOne Real-Time PCR System (Applied Biosystems, Foster City, California) and analyzed using SDS software version 3.0, as described previously.¹⁶

Bioinformatics Analysis

Data from the Genotype-Tissue Expression (GTEx) database were used to identify the correlations between rs2977537 and WISP1 expression in vaginal tissue. The expression of WISP1 in cervical dysplasia and cervical cancer was evaluated on the basis of publicly available microarray data sets from the GSE63514 and GSE26511 projects.

Statistical Analyses

Analysis of variance was used to analyze the age distribution of patients with invasive cervical cancer, those with preinvasive lesions, and the control group, and a post hoc analysis was further applied using Scheffe Test. The Hardy-Weinberg equilibrium was used to check the genotypic distributions of WISP1 SNPs rs62514004, rs2929973, rs2977530, and rs2977537 in the normal controls (degree of freedom = 2). The χ² test or Fisher exact test was applied to analyze the association between the genotypic frequencies of WISP1 SNPs and the incidence of cervical neoplasias (including preinvasive lesions and invasive cancer). The adjusted odds ratios (AORs) and 95% confidence intervals (CIs) were calculated to analyze the relationships between the genotypic distributions of WISP1 SNPs and the incidence of cervical neoplasias (including preinvasive lesions and invasive cancer) using logistic and multiple logistic regression models after controlling for age. A χ² test was used to assess the relationship between WISP1 SNPs and various clinicopathological parameters. The Kaplan-Meier model and Cox proportional hazard model were used to determine the association between WISP1 SNPs and cervical cancer survival relative to survival time or until the closing date of the study (October 31, 2015).

Results

A significant difference was observed in age distributions of patients with cervical neoplasia and normal controls (50.3 ± 13.5 vs 44.0 ± 10.1; P < .001). The age differences between patients with invasive cervical cancer and those with preinvasive lesions (55.1 ± 12.4 vs 44.4 ± 12.5; P < .001) and between those with cervical cancer and normal controls (55.1 ± 12.4 vs 44.0 ± 10.1; P < .001) were also significant. However, no significant difference was observed between those with preinvasive lesions and normal controls (44.4 ± 12.5 vs 44.0 ± 10.1; P = .936). Genotypic distributions of WISP1 SNPs rs62514004, rs2929973, rs2977530, and rs2977537 in the normal control women all satisfied the Hardy-Weinberg equilibrium. The χ² values for WISP1 SNPs rs62514004, rs2929973, rs2977530, and rs2977537 were 2.144, 0.020, 0.472, and 2.004, respectively, with all P > .05 (degree of freedom = 2).

Association Between the Genotypic Distribution of WISP1 SNPs and Cervical Neoplasia

A significant difference was found in the genotypic frequency of WISP1 SNP rs2977530 (P = .039) between women with cervical neoplasia and normal controls (Table 1). The AG/GG genotypes of WISP1 SNP rs2977530 were differently distributed between patients with cervical neoplasia and normal controls with AA as a reference (P = .018). After controlling for age, women with AG/GG genotypes tended to display a lower risk of developing cervical neoplasia (AOR: 0.69, 95% CI: 0.46-1.03). Similarly, a difference was found in the genotypic frequency of rs2977537 among women with cervical neoplasia and normal controls (P = .008; Table 1). However, genotype AA of WISP1 SNP rs2977537 was distributed differently between patients with cervical neoplasia and normal controls with GG/GA as a reference (P = .003). Moreover, after controlling for age, women with genotype AA exhibited a higher risk of developing cervical neoplasia (AOR: 1.68, 95% CI: 1.11-2.55) with GG/GA as a reference. No significant differences were observed in the genotypic distributions of WISP1 SNPs rs62514004 and rs2929973 (P = .343 and P = .631, respectively) among women with cervical neoplasia and normal controls.

Table 1.

Genotypic Distribution of Single-Nucleotide Polymorphisms of WNT1 Inducible Signaling Pathway Protein 1 in Patients With Uterine Cervical Neoplasia and Normal Women.^a

Variables	Normal Controls (n = 316)	Cervical Neoplasia^b (n = 210)	P Value	OR (95% CI)	AOR (95% CI)^c
rs62514004
Co-dominant AA^d	254	168	.343	1.00	1.00
AG	56	41		1.11 (0.71-1.73)	1.10 (0.70-1.75)
GG	6	1		0.25 (0.03-2.11)	0.21 (0.02-1.78)
Dominant AA^d	254	168	.915	1.00	1.00
AG/GG	62	42		1.02 (0.66-1.59)	1.01 (0.64-1.58)
Recessive AA/AG^d	310	209	.252	1.00	1.00
GG	6	1		0.25 (0.03-2.07)	0.20 (0.02-1.74)
rs2929973
Codominant TT^d	129	85	.631	1.00
TG	145	91		0.95 (0.65-1.39)	0.95 (0.64-1.41)
GG	42	34		1.23 (0.72-2.08)	1.33 (0.77-2.30)
Dominant TT^d	129	85	.937	1.00	1.00
TG/GG	187	125		1.01 (0.71-1.45)	1.03 (0.71-1.50)
Recessive TT/TG^d	274	176	.354	1.00	1.00
GG	42	34		1.26 (0.77-2.06)	1.37 (0.82-2.27)
rs2977530
Codominant AA^d	77	71	.039^e	1.00	1.00
AG	163	88		0.59 (0.39-0.89)	0.65 (0.43-1.00)
GG	76	51		0.73 (0.45-1.18)	0.76 (0.46-1.25)
Dominant AA^d	77	71	.018^e	1.00	1.00
AG/GG	239	139		0.63 (0.43-0.93)	0.69 (0.46-1.03)
Recessive AA/AG^d	240	159	.951	1.00	1.00
GG	76	51		1.01 (0.67-1.52)	0.99 (0.65-1.51)
rs2977537
Codominant GG^d	83	55	.008^e	1.00	1.00
GA	168	88		0.79 (0.52-1.21)	0.85 (0.55-1.32)
AA	65	67		1.56 (0.96-2.52)	1.52 (0.92-2.50)
Dominant GG^d	83	55	.985	1.00	1.00
GA/AA	233	155		1.00 (0.68-1.49)	1.04 (0.69-1.58)
Recessive GG/GA^d	251	143	.003^e	1.00	1.00
AA	65	67		1.81 (1.22-2.69)	1.68 (1.11-2.55)

Abbreviations: AOR, adjusted odds ratio; OR, odds ratio; 95% CI, 95% confidence interval; WISP1, WNT1 inducible signaling pathway protein 1.

^aStatistical analysis: logistic regression model, χ² or Fisher exact tests.

^bCervical neoplasia included preinvasive lesions and invasive cancer of uterine cervix.

^cThe AORs with their 95% confident intervals were estimated by logistic regression after controlling for age.

^dUsed as references for comparison to evaluate the odds ratios of other genotypes.

^e P < .05.

Association Between the Genotypic Distribution of WISP1 SNPs and Invasive Cervical Cancer

When the cervical neoplasia group was further divided into subgroups according to the presence of invasive cervical cancer or preinvasive lesions, significant differences were found in the genotypic distributions of WISP1 SNP rs2977530 in codominant and dominant conditions (P = .011 and P = .028, respectively) among patients with cervical invasive cancer, patients with preinvasive lesions, and normal control women (Table 2). However, after controlling for age, genotype AG in WISP1 SNP rs2977530 reduced the risk of invasive cervical cancer (OR: 0.52, 95% CI: 0.30-0.90) with AA as a reference, but it did not reduce the risk of preinvasive lesions (OR: 0.83, 95% CI: 0.49-1.42; Table 2). The AG/GG genotypes in SNP rs2977530 also reduced the risk of cervical invasive cancer (OR: 0.54, 95% CI: 0.34-0.85) with AA as a reference, but they did not reduce the risk of preinvasive lesions (OR: 0.77, 95% CI: 0.46-1.28). After controlling for age, AG/GG still tended to reduce the risk of invasive cervical cancer. Conversely, genotype AA in WISP1 SNP rs2977537 elevated the risk of invasive cervical cancer (OR: 1.86, 95% CI: 1.11-3.11; Table 2) using GG/GA as a reference after controlling for age, but it did not elevate the risk of preinvasive lesions (OR: 1.45, 95% CI: 0.86-2.46). No significant differences were observed in the genotypic distributions of WISP1 SNPs rs62514004 and rs2929973 among patients with invasive cancer, patients with preinvasive lesions, and normal controls.

Table 2.

Genotypic Distribution of Single-Nucleotide Polymorphisms of WNT1 Inducible Signaling Pathway Protein 1 in Patients With Invasive Cancer or Preinvasive Lesions of Uterine Cervix and Normal Women.^a

Variables	Controls (n = 316)	Preinvasive (n = 95)	Invasive (n = 115)	P Value	OR (95% CI)^b	OR (95% CI)^c	AOR (95% CI)^d	AOR (95% CI)^e
rs62514004
Co-dominant AA^f	254	77	91	.646	1.00	1.00	1.00	1.00
AG	56	18	23		1.06 (0.59-1.91)	1.15 (0.67-1.97)	1.06 (0.59-1.91)	1.20 (0.67-2.15)
GG	6	0	1		UA	0.47 (0.06-3.92)	UA	0.35 (0.04-3.30)
Dominant AA^f	254	77	91	.936	1.00	1.00	1.00	1.00
AG/GG	62	18	24		0.96 (0.53-1.72)	1.08 (0.64-1.83)	0.95 (0.53-1.71)	1.10 (0.62-1.95)
Recessive AA/AG^f	310	95	114	.326	1.00	1.00	1.00	1.00
GG	6	0	1		UA	0.45 (0.05-3.81)	UA	0.34 (0.04-3.17)
rs2929973
Codominant TT^f	129	37	48	.564	1.00	1.00	1.00	1.00
TG	145	39	52		0.94 (0.56-1.56)	0.96 (0.61-1.52)	0.94 (0.56-1.56)	0.97 (0.59-1.60)
GG	42	19	15		1.58 (0.82-3.03)	0.96 (0.49-1.89)	1.59 (0.83-3.05)	1.05 (0.50-2.20)
Dominant TT^f	129	37	48	.917	1.00	1.00	1.00	1.00
TG/GG	187	58	67		1.08 (0.68-1.73)	0.96 (0.62-1.49)	1.08 (0.68-1.73)	0.99 (0.62-1.58)
Recessive TT/TG^f	274	76	100	.235	1.00	1.00	1.00	1.00
GG	42	19	15		1.63 (0.90-2.97)	0.98 (0.52-1.84)	1.64 (0.90-2.99)	1.06 (0.53-2.13)
rs2977530
Codominant AA^f	77	28	43	.011^f	1.00	1.00	1.00	1.00
AG	163	49	39		0.83 (0.48-1.42)	0.43 (0.26-0.71)	0.83 (0.49-1.42)	0.52 (0.30-0.90)
GG	76	18	33		0.65 (0.33-1.28)	0.78 (0.45-1.35)	0.65 (0.33-1.28)	0.84 (0.46-1.55)
Dominant AA^f	77	28	43	.028^g	1.00	1.00	1.00	1.00
AG/GG	239	67	72		0.77 (0.46-1.28)	0.54 (0.34-0.85)	0.77 (0.46-1.29)	0.62 (0.38-1.03)
Recessive AA/AG^f	240	77	82	.259	1.00	1.00	1.00	1.00
GG	76	18	33		0.74 (0.42-1.31)	1.27 (0.79-2.05)	0.74 (0.42-1.31)	1.24 (0.74-2.10)
rs2977537
Codominant GG^e	83	23	32	.012^g	1.00	1.00	1.00	1.00
GA	168	46	42		0.99 (0.56-1.74)	0.65 (0.38-1.10)	0.99 (0.56-1.74)	0.76 (0.43-1.34)
AA	65	26	41		1.44 (0.76-2.76)	1.64 (0.93-2.88)	1.44 (0.76-2.76)	1.57 (0.84-2.91)
Dominant GG^f	83	23	32	.839	1.00	1.00	1.00	1.00
GA/AA	233	72	83		1.12 (0.66-1.90)	0.92 (0.57-1.49)	1.12 (0.66-1.90)	1.00 (0.59-1.68)
Recessive GG/GA^f	251	69	74	.005^g	1.00	1.00	1.00	1.00
AA	65	26	41		1.46 (0.86-2.47)	2.14 (1.34-3.42)	1.45 (0.86-2.46)	1.86 (1.11-3.11)

Abbreviations: AOR, adjusted odds ratio; OR, odds ratio; UA, unavailable; 95% CI, 95% confidence interval.

^aStatistical analysis: multiple logistic regression or χ² or Fisher exact tests.

^bComparison between patients with cervical preinvasive lesions and control women.

^cComparison between patients with cervical invasive cancer and control women.

^dThe adjusted ORs with their 95% CIs were estimated by multiple logistic regression models after controlling for age between patients with cervical preinvasive lesions and control women.

^eThe adjusted ORs with their 95% CIs were estimated by multiple logistic regression models after controlling for age between patients with cancer and control women.

^fUsed as references for comparison to evaluate the odds ratios of other genotypes.

^g P < .05.

Because AG/GG in WISP1 SNP rs2977530 exhibited a dominant protective effect and AA in rs2977537 exhibited a recessive promoting effect for the development of invasive cervical cancer, we further investigated the relationship of the WISP1 SNPs rs2977530 and rs2977537 with clinicopathological variables and patient survival. We found that neither rs2977530 nor rs2977537 was associated with clinicopathological parameters, such as stromal invasive depth and lymph node metastasis (Table 3). For genotypes AG/GG and AA in WISP1 SNP rs2977530, a nonsignificant difference of 83.3% versus 86.1% was observed for 5-year cervical cancer survival rates (P = .740); the hazard ratio of AG/GG was 1.21 with AA as a reference (95% CI: 0.39-3.82; Table 4). Moreover, comparing genotypes AA and GG/GA in rs2977537 revealed 5-year survival rates of 81.6% versus 85.4%, which was a nonsignificant difference (P = .634); the hazard ratio of AA was 1.30 with GG/GA as a reference (95% CI: 0.44-3.83).

Table 3.

The Association of Genotypic Distribution of Single-Nucleotide Polymorphisms of WNT1 Inducible Signaling Pathway Protein 1 With Clinicopathological Variables in the Patients With Cervical Cancer.^a

Clinicopathological Variables^b	rs2977530		P Value	OR (95% CI)	rs2977537		P Value	OR (95% CI)
Clinicopathological Variables^b	AA^c	AG/GG	P Value	OR (95% CI)	GG/GA^c	AA	P Value	OR (95% CI)
Stage			.160				.051
I^c	29	42		1.00	42	29		1.00
Others	11	29		1.82 (0.73-4.69)	31	9		0.42 (0.15-1.08)
Pathologic type			.945				.920
Squamous cell carcinoma^c	34	60		1.00	62	32		1.00
Adenocarcinoma	6	11		1.04 (0.32-3.74)	11	6		1.06 (0.29-3.47)
Depth of stromal invasion			.779				.563
≤1/2 stromal depth^c	22	41		1.00	40	23		1.00
>1/2 stromal depth	18	30		0.89 (0.38-2.11)	33	15		0.79 (0.33-1.88)
Tumor diameter			.395				.160
≤4 cm^c	28	44		1.00	44	28		1.00
>4 cm	12	27		1.43 (0.58-3.62)	29	10		0.54 (0.20-1.37)
Tumor grade			.346				.242
Well^c	11	14		1.00	14	11		1.00
Moderate or poor	29	57		1.54 (0.56-4.19)	59	27		0.58 (0.21-1.63)
Parametrial invasion			.425				.131
No invasion^c	32	52		1.00	52	32		1.00
Invasion	8	19		1.46 (0.53-4.32)	21	6		0.46 (0.14-1.36)
Vaginal invasion			.191				.311
No invasion^c	34	52		1.00	55	31		1.00
Invasion	6	18		1.96 (0.66-6.63)	18	6		0.59 (0.17-1.78)
Pelvic lymph node metastasis			.679				.849
Negative^c	29	54		1.00	55	28		1.00
Positive	11	17		0.83 (0.32-2.24)	18	10		1.09 (0.39-2.89)

Abbreviations: OR, odds ratio; 95% CI, 95% confidence interval.

^aStatistical analysis: χ² test.

^bSome clinicopathological data could not be collected from the patients with cervical cancer due to incomplete medical charts or records.

^cUsed as references for comparison to evaluate the odds ratio of another subtype.

Table 4.

The Association of Genotypic Distribution of Single-Nucleotide Polymorphisms of WNT1 Inducible Signaling Pathway Protein 1 With Survival of Patient With Cervical Cancer.^a

WISP1 SNPs	Median Survival Months	5-Year Survival Rate (%)	Hazard Ratio	95% CI	P Value
rs2977530 AA^b	192	86.1	1.00		.740
AG/GG	195	83.3	1.21	0.39-3.82
rs2977537 GG/GA^b	195	85.4	1.00		.634
AA	192	81.6	1.30	0.44-3.83

Abbreviation: CI, confidence interval; SNP, single-nucleotide polymorphism.

^aStatistical analysis: Kaplan-Meier curve and Cox proportional hazards models.

^bUsed as references for comparison to another SNP subtype.

Association Between WISP1 Expression and Clinicopathological Characteristics in Patients With Cervical Neoplasia

To further support our findings, we evaluated WISP1 expression using publicly available microarray data sets from the GSE63514 and GSE26511 projects. We found that WISP1 mRNA expression was higher in cervical tumor tissue than in normal tissue (Figure 1A). Moreover, WISP1 expression was associated with pelvic lymph node metastasis (Figure 1B). Furthermore, to conduct a preliminary assessment of the putative functional relevance of WISP1 SNP rs2977537, we assessed WISP1 expression in vaginal tissue using the GTEx database (Figure 1C). The data suggested that changes in WISP1 expression due to genetic polymorphisms may affect the development of cervical cancer.

Discussion

Our study revealed that genotype AG reduces the susceptibility of Taiwanese women to cervical neoplasia and that genotypes AG/GG tend to reduce the susceptibility with AA as a reference in WISP1 SNP rs2977530. However, AG and AG/GG reduced and tended to reduce the risk, respectively, with AA as a reference between patients with cervical invasive cancer and normal control women but not between patients with preinvasive lesions and normal control women after the subdivision of neoplasia into invasive cancer and preinvasive lesions. The protective effect of AG or AG/GG against cervical neoplasia may be attributable to the number of cases with invasive cervical cancer, because no significant difference was found in the genotypic distribution of patients with preinvasive lesions and normal control women in WISP1 SNP rs2977530. Only 1 mutant G was needed to contribute to the dominant protective effect against cervical neoplasia of WISP1 SNP rs2977530. Further studies may support this finding.

By contrast, AA elevated the risk of cervical neoplasia in Taiwanese women with GG/GA as a reference in WISP1 SNP rs2977530. This may also be related to the number of cases with invasive cervical cancer, because patients with invasive cervical cancer had a significantly higher risk than normal control women, but this difference was not observed between patients with preinvasive lesions and normal control women. Both mutant AA alleles were needed for the recessive effect of WISP1 SNP rs2977537 to be exhibited.

Haplotype association mapping has provided a method for identifying susceptibility genes, and it demonstrates associations between susceptibility and diseases. Moreover, this method yields higher statistical power than individual SNP analysis.^17

–20 However, in this study, haplotype association mapping may have become complex because of the inverse effect of WISP1 SNP rs2977530 (protective) and rs2977537 (promoting) for the susceptibility for invasive cervical cancer. Therefore, we did not apply the haplotype method in this study.

Genetic polymorphisms occur in promoter regions or exons, and they may affect gene expression.¹² If an SNP arises in the 3′-untranslated region of a gene, it may affect biological processes. However, SNPs in introns can also play important roles in genes and may be associated with diseases.²¹ WISP1 rs2977530 and rs2977537 were identified in introns in this study. Our findings demonstrate the inverse effect of these SNPs on the development of invasive cervical cancer. The WISP1 rs2977530 and rs2977537 were reported to be involved in regulating the structure or function of the WISP1 gene, and they may be related to susceptibility for lung cancer.¹⁴ In this study, we could not identify a significant association between invasive cervical cancer and WISP1 SNP rs62514004, which was in exon 1. However, Chen et al revealed that the rs62514004 genetic polymorphism is associated with susceptibility for lung cancer.¹⁴ Lau et al revealed that WISP1 SNPs rs62514004, rs2977530, and rs2977530 are unrelated to oral squamous cell carcinoma.¹⁶ After stratifying individuals into smokers and nonsmokers, participants with AG/GG in WISP1 rs2977530 displayed a 0.598-fold lower risk (95% CI: 0.364-0.980) of oral squamous cell carcinoma (a protective effect in agreement with our findings) among the 664 nonsmokers. Thereafter, we further assessed the relationships between WISP1 SNPs rs2977530 and rs2977537 and the relevant clinicopathological variables for cervical cancer as well as patient survival.

To our knowledge, no study has investigated the relationships of WISP1 expression and WISP1 SNPs with the relevant clinicopathological parameters for cervical cancer. Pennica et al revealed that WISP1 overexpression in colon cancer was involved in colon tumorigenesis.⁶ They demonstrated that WISP1 expression was increased in lung and prostate cancer tissues compared to normal tissue counterparts.^22,23 By contrast, another study identified a correlation between reduced WISP1 expression and breast cancer and melanoma.²⁴ In this study, we did not identify associations between WISP1 SNPs rs2977530 and rs2977537 and any clinicopathological parameters of cervical cancer. Another study reported that the WISP1 SNPs rs62514004, rs2977530, and rs2977537 had no significant association with the clinicopathological variables of oral squamous cell carcinoma, which was consistent with our findings.¹⁶ In other studies, WISP1 was considered an important factor for stimulating aggressiveness in colon cancer and oral squamous cell carcinoma cells.^25,26 Chen et al revealed that WISP1 might be implicated in the development and progression of primary lung cancers, and its expression might serve as a valuable prognostic marker.²² However, we did not identify significant associations between WISP1 SNPs rs2977530 and rs2977537 and cervical cancer survival. This could be because the majority of patients with cervical invasive cancer had early-stage cervical cancer (stage I) and had a good prognosis in general. This reduced the influence of WISP1 genetic polymorphisms rs2977530 and rs2977537 on patient survival.

In conclusion, genotypes AG/GG in WISP1 SNP rs2977530 protect Taiwanese women from uterine cervical cancer. Conversely, genotype AA in WISP1 SNP rs2977537 promotes the risk of cervical cancer in Taiwanese women. However, genotypes AG/GG in WISP1 SNP rs2977530 and genotype AA in WISP1 SNP rs2977537 are not associated with the clinicopathological variables of cervical cancer or with patient survival.

Footnotes

Authors’ Note

Y.-H.L. and Y.-H.H. contributed equally to this work.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research and/or authorship of this article: This study was supported by research grants from Ministry of Science and Technology (MOST 105-2314-B-040-016-MY2) and Chung Shan Medical University Hospital (CSH-2017-D-002).

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