Significant association between interleukin-10 gene polymorphisms and cervical cancer risk: a meta-analysis

Previous studies have suggested that interleukin-10 (IL-10) polymorphisms may be associated with an increased risk of developing cervical cancer. However, the published results on this subject matter are controversial. The aim of this study was to conduct a meta-analysis of published reports to more precisely investigate the relationship between IL-10 polymorphisms and cervical cancer risk. Five online databases (PubMed, Embase, Web of SCI, CNKI and Wanfang) were searched, and seventeen articles with sufficient quantitative information were included in our meta-analysis. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the association between IL-10 polymorphisms and cervical cancer risk. Publication bias, sensitivity and cumulative analyses were also performed to support our findings. Overall, there was a significant association between the IL-10 -1082A > G polymorphism and cervical cancer risk observed in the total population (G vs. A: OR = 1.60, 95% CI = 1.12–2.29, P = 0.01, I2 = 92.3%; AG vs. AA: OR = 1.34, 95% CI = 1.04-1.74, P = 0.03, I2 = 65.9%; AG + GG vs. AA: OR = 1.58, 95% CI = 1.11–2.25, P = 0.01, I2 = 84.4%), and the same results were obtained in the subgroup analysis. Moreover, the IL-10 -819 T > C polymorphism exhibited a significant, protective effect against cervical cancer. In summary, our meta-analysis suggests that IL-10 polymorphisms may play a variety of roles in regard to cervical cancer risk, especially in Asians.


INTRODUCTION
Cervical cancer is the second most common form of cancer diagnosed in women and the third leading cause of death from cancer. This accounts for approximately 8% of total cancer cases and cancer deaths in women [1].
In 2012, there were an estimated 527,600 new cervical cancer cases and 265,700 deaths from cervical cancer worldwide, mostly affecting developing countries [1]. Cervical cancer affects the cervix and encompasses squamous cell carcinomas (90%), adenocarcinoma (10%), and other subtypes [2,3]. Currently, a combination of Meta-Analysis surgery, radiotherapy and chemotherapy is still the most effective form of treatment for cervical cancer [4][5][6]. However, any form of treatment inevitably implies severe trauma to the patient, as well as an economic burden and mental stress [7][8][9]. A variety of risk factors, such as chronic inflammation, unhealthy living conditions, and human papillomavirus (HPV) infections have been proven to increase the risk of cervical tumorigenesis [10][11][12][13]. However, although numerous epidemiological and molecular biology-related studies have been conducted, the precise effects of these factors on the process of tumorigenesis process are still poorly understood.
In the last decade, numerous studies have suggested that certain cytokines may play critical roles in the processes of inflammatory cell infiltration and malignant cell transformation [14,15]. Interleukin-10 (IL-10) is a multifunctional cytokine that is mainly secreted by T helper type2 cells (Th2 cells), monocytes/macrophages, keratinocytes and tumor cells as well, as well as human helpertype2 cells (Th1 cells) [16,17]. IL-10 exhibits complex biological effects, including the capacity to stimulate mast cells maturation and accelerate the proliferation and differentiation of B cells, restrain type 1 immune responses by inhibiting the production of cytokines such as IL-2, IFN-gamma, and other cytokines, decrease the IFN-gamma production by natural killer cells and interfere with macrophage activation [18]. IL-10 exhibits a dual role during cancer development, inducing both cancer-promoting (immunosuppressive) and cancerinhibiting (anti-angiogenic) effects [19].
Single nucleotide polymorphisms are one of the most common, heritable variations in the human genome, accounting for more than 90% of all variation [20]. The IL-10 gene is located on chromosome 1q31-32, which spans 4.8 kb, and contains 5 exons and 4 introns that encode 178 amino acids [21]. To date, at least 50 polymorphic loci have been reported, such as -2849, -2776, -2769 and -2763 [22]. The three most common SNPs in the IL-10 promoter region that have been reported to significantly influence gene transcription and expression are -1082A > G (rs1800870), -819T > C (rs1800871) and -592C > A (rs1800872) [23]. Some molecular research has shown that these polymorphisms can influence and/or change the susceptibility of individuals to different forms of cancer, such as head and neck cancer, gastric cancer, leukemia, and others [24][25][26]. In 2001, Stanczuk et al. published the first case-control study examining the effects of the IL-10 -1082A > G polymorphism on cervical cancer risk, and the results suggested that African women with an AG genotype were at an increased risk for cervical cancer [27]. To date, two meta-analyses have been conducted, examining previously published studies to elucidate the association between IL-10 polymorphisms and cervical cancer risk [28,29]. However, these meta-analyses have not been comprehensive and have not yielded consistent results. Therefore, we conducted the present meta-analysis to provide a more precise and comprehensive assessment of the relationship between IL-10 polymorphisms and cervical cancer susceptibility. This meta-analysis was performed according to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [30]. No ethical issues were implicated in this study because our data were based on previously published reports.

Association between the IL-10 -1082A > G polymorphism and cervical cancer risk
Publication bias was assessed with Funnel plots. Plot asymmetry was observed only in the AG+GG vs. AA model, which was redressed in the subgroup analysis by ethnicity ( Figure 2D

Association between the IL-10 -819T > C polymorphism and cervical cancer risk
Six studies involving 953 cases and 1,082 controls were included in this meta-analysis to assess the association between the IL-10 -819T > C polymorphism and cervical cancer risk. Interestingly, all genetic models indicated that they confer a protective effect against cervical cancer occurrence (C vs. T, OR = 0.74, 95% CI  Figure 3A for TC+CC vs. TT model). Furthermore, subgroup analyses based on HWE status, the ethnicity, the control design and the genotyping method all indicated the same protective effect (Supplementary Table 1).
The cumulative analysis also indicated some change only in the recessive model ( Figure 3B for TC+CC vs. TT model). A sensitivity analysis was conducted, and no conspicuous change in the pooled ORs was detected except in the recessive model ( Figure 3C for TC+CC vs. TT model). Moreover, no publication bias was observed, indicating that the results were statistically robust (C vs. T: P = 0.23; TC vs. TT: P = 0.06; CC vs. TT: P = 0.96; TC+CC vs. TT: P = 0.11; CC vs. TT+TC: P = 0.26) ( Figure 3D for TC+CC vs. TT model).

Association between the IL-10 -592C > A polymorphism and cervical cancer risk
For the IL-10 -592C > A polymorphism, ten studies consisting of 3,149 cases and 2,237 controls were pooled in the meta-analysis to assess whether this IL-10 -592C > A polymorphism was associated with cervical cancer risk. Overall, no significant association was observed in any of the five models (Supplementary Table 1; Figure 4A for CA+AA vs. CC model). Only two genetic models (for AA vs. CC, OR = 1.86, 95% CI = 1.02-3.39, P = 0.04, I 2 = 69.6%; for AA vs. CC+CA, OR = 1.62, 95%CI = 1.06-2.49, P = 0.03, I 2 = 46.6%) revealed an increased risk for cervical cancer in the Caucasian population group. Additional subgroup analyses were conducted based on HWE status, the control design and the genotyping method, but no significant associations were observed. The pooled ORs did not exhibit any changes following sensitivity or cumulative analyses, and no publication bias was observed ( Figure 4B

DISCUSSION
Genetic factors are now known to be important variables affecting the susceptibility of patients to various diseases and have been paid an increasing amount of attention [47,48]. Inflammatory and immune cytokines play important roles during the process of tumorigenesis transition from normal epithelium to malignant tumors, and these cytokines can promote this process by inducing angiogenesis, compensatory cell proliferation, DNA damage, or the accumulation of gene mutations [49]. Gene mutations, especially polymorphisms in the promoter region, can affect the process of gene transcription, resulting in abnormal expression of the corresponding mRNAs and dysfunction of the expressed proteins. These mutations can also influence the susceptibility of individuals to cancer [50,51].
Since 2001, a large number of molecular epidemiological case-control studies have been conducted to explore the association between IL-10 polymorphisms and cervical cancer risk, but the results have been inconsistent. In terms of the IL-10 -1082A > G polymorphism, Singhal [38]. A similarly increased risk was also observed in other studies. In contrast, other studies reported a negative association between the IL-10-1082A > G polymorphism and cervical cancer risk. For -819T > C and -592C > A polymorphisms, there is still controversy regarding the relationship between these two polymorphisms and cervical cancer susceptibility.
In 2013, Ni et al. published the first metaanalysis examining the association between IL-10 gene polymorphisms and cervical cancer risk. Their metaanalysis included eight studies comprising 1,498 cases and 1,608 controls for the IL-10 -1082A > G polymorphism, as well as five studies involving 2,396 cases and 1,388 controls for the IL-10 -592C > A polymorphism [28]. The researchers found that the IL-10 -592C > A polymorphism was associated with an increased risk of cervical cancer development. No significant association was found for the IL-10 -1082A > G polymorphism [28]. In 2014, Zhang et al. conducted another meta-analysis to evaluate the association between the IL-10 -1082A > G polymorphism and cervical cancer risk. This study included eight case-control studies comprising 1,983 cases and 1,618 controls; they also reported no association between this polymorphism and cervical cancer susceptibility [29]. Moreover, the association between the IL-10-819T > C polymorphism and cervical cancer risk was also explored in the subgroup analysis by Yu et al. in 2013. This analysis only included three case-control studies, with 1,895 subjects, and indicated that there was a positive association with cervical cancer in some genetic models [52].
Compared with the previous meta-analyses, our meta-analysis relied on a more scientifically sound retrieval strategy and included more research studies (seventeen publications involving 7,286 individuals) to accurately assess the associations between the IL-10 -1082A > G, -819T > C and -592C > A polymorphisms and cervical cancer risk. Our results suggest that the IL-10-1082A > G polymorphism may be associated with an increased risk of cervical cancer development. In contrast, the IL-10-819T > C polymorphism may have a protective effect against cervical cancer development, especially in Asian populations. In the stratified analysis, the heterogeneity between the included IL-10-1082A > G polymorphism studies was moderately, and successfully reduced by the subgroup analysis based on ethnicity. This suggests that ethnic diversity maybe a potential heterogeneity factor. Moreover, after conducting the sensitivity and publication bias analyses, no significant alterations or bias were observed for any of the three polymorphisms, presenting the stability of the results of our meta-analysis.

Representativeness of cases
Consecutive/randomly selected cases with clearly defined sampling frame Not consecutive/randomly selected case or without clearly defined sampling frame Not described 2 1 0

Source of controls
Population-base control Hospital-base control and/or healthy base control Not described

Hardy-Weinberg equilibrium in controls
Hardy-Weinberg equilibrium Hardy-Weinberg disequilibrium Not available

Genotyping examination
Genotyping done under "blinded" condition and repeated again Genotyping done under "blinded" condition or repeated again Unblinded done or not mentioned and unrepeated 2 1 0 Association assessment Assess association between genotypes and cancer with appropriate statistics and adjustment for confounders Assess association between genotypes and cancer with appropriate statistics and without adjustment for confounders Inappropriate statistics used 2 1 0 www.impactjournals.com/oncotarget To our knowledge, this is the first meta-analysis to examine the association between IL-10 polymorphisms and cervical cancer risk, including the three most common polymorphic loci (IL-10 -1082A > G, -819T > C and -592C > A). However, there are some limitations of this study that should be addressed. First, only seventeen publications were included in our meta-analysis. The limited number of studies and sample size for each polymorphic locus may reduce the reliability of the results and affect the assessment of associations between these IL-10 polymorphisms and cervical cancer susceptibility. Second, additional risk factors, such as smoking, drinking, and HPV infection, were not considered. The interactions between genetic and environmental factors and cancer development could not be evaluated in our analysis. Third, the association between the IL-10 -1082A > G, -819T > C and -592C > A polymorphisms and cervical cancer risk were analyzed separately, and the influence of the haplotype and gene-gene interactions was not analyzed due to an insufficient amount of data. Fourth, the heterogeneity that exists between the IL-10-1082A > G polymorphism studies could influence the current results and distort the conclusions. In this meta-regression analysis, we could not find the source of the heterogeneity, although it could be reduced in the subgroup analysis.
In summary, our meta-analysis suggests that the IL-10-1082A > G and -819T > C polymorphisms are associated with cervical cancer susceptibility, but with contradictory effects. In contrast, no significant association was found between the IL-10-592C > A polymorphism and cervical cancer susceptibility.

Eligibility criteria
To be included in this study, publications had to meet the following inclusion criteria: 1) the study design had to be a case-control study (including retrospective or prospective studies); 2) the focus of the study had to be on IL-10 promoter polymorphisms (-1082A > G, -819T > C and -592C > A); 3) the case group had to include women with cervical cancer and the control group had to consist of women without cervical cancer; 4) the reports had to include sufficient information on the frequency distribution of different genotypes in order to calculate the odds ratios (ORs) and 95% confidence intervals (CIs); and 5) in the case that there were duplicate studies, we included the most recent or those with the largest samples sizes.

Data extraction and quality evaluation
Two investigators (Guo and Wen), independently extracted the following information from all of the included studies: first author, year of publication, study country or region where the study was performed, ethnicity (Asian, Caucasian or African), the source of the controls, the sample sizes of patients and controls, data on the frequency distribution of different genotypes, the Hardy-Weinberg equilibrium (HWE) for the controls, and the genotyping method. In addition, the modified Newcastle-Ottawa scale (NOS) was employed by the first two authors in order to evaluate the quality of the included studies [53]. The scores ranged from 0 points (worst) to 10 points (best). Studies with a score of 7 or higher were classified as high quality (Table 2).

Statistical analysis
ORs with 95% CIs were calculated to assess the strength of the association between the IL-10 -1082A > G, -819T > C and -592C > A polymorphisms and cervical cancer susceptibility. For the IL-10 -1082A > G polymorphism, the five following genetic models were used: allele contrast model (G vs. A), co-dominant models (AG vs. AA and GG vs. AA), dominant model (AG+GG vs. AA), and recessive model (GG vs. AA+AG). The same genetic models were also used to assess the IL-10 -819T > C and -592C > A polymorphisms. Subgroup analyses were performed according to HWE status, ethnicity difference, control designs, and genotyping methods. Heterogeneity between studies was determined via a Cochran's Q test and the I 2 statistic [54]. A fixed-effect model (the Mantel-Haenszel method) was used when the P-value was more than 0.10 and the I 2 was less than 40% [55]. Otherwise, a random-effects model (the DerSimonian and Laird method) was adopted [56]. Meta-regression analyses were conducted to explore the potential factors that contribute to heterogeneity. Furthermore, cumulative meta-analyses were conducted to observe whether the trend changed with the addition of studies. Sensitivity analyses were also conducted to evaluate the robustness of our results. Egger's linear regression and Begg's funnel plots were used to examine any potential publication bias [57,58]. All statistical calculations were performed with STATA version 14.0 (Stata Corporation, College Station, TX, USA). A two-sided P value < 0.05 was considered significant.

CONFLICTS OF INTEREST
The authors declare no competing financial interests.

FUNDING
This study was supported by the Foundations of the Science and Technology Department of Hubei Province (No. 2016CFB567, 2014CFB364) and Hubei Province health and family planning scientific research project (No. WJ2017F069, WJ2015Q041) and Bureau of Science and Technology of Shiyan City (17K67). The funders had no roles in study design, data collection and analysis, decision to publish, or preparation of the manuscript.