Ninjurin 2 rs118050317 gene polymorphism and endometrial cancer risk

Background Endometrial cancer is one of the most common female reproductive system tumors. Ninjurin2 (NINJ2) is a new adhesion factor. As a vascular susceptibility gene, it is highly expressed in other cancers and promotes the growth of cancer cells. We conducted an association analysis between NINJ2 gene polymorphism and endometrial cancer risk. Methods Five SNPs rs118050317, rs75750647, rs7307242, rs10849390 and rs11610368 of NINJ2 gene were genotyped in 351 endometrial cancer patients and 344 healthy controls. The clinical index difference between cases and controls were tested by one-way analysis of variance. The allele and genotype frequency of cases and controls were been compared by Chi square test. The odds ratios (OR) with 95% confidence interval (95% CI) were examined by logistic regression analysis. Results The SNP rs118050317 mutant allele C and homozygote CC genotype were significant increased the endometrial cancer risk (OR 1.46, 95% CI 1.04–2.06, p = 0.028; OR 8.43, 95% CI 1.05–67.89, p = 0.045). In the clinical index analysis, there were significant higher quantities of CEA, CA125 and AFP in cases serum than controls. Conclusion The NINJ2 gene polymorphism loci rs118050317 mutant allele C was associated with an increased risk of endometrial cancer. CEA, CA125 and AFP quantities were significant higher in endometrial cancer patients.


Introduction
Endometrial cancer is malignant tumor of endometrium epithelial, which also is one of the most common female reproductive system tumors [1]. In historically, endometrial cancer was been classified as type I (80-90%) and type II (10-20%), in histopathological classification divided tumors into endometrioid and non-endometrioid [2,3]. With the development of society and the improvement of economic conditions, the incidence of endometrial cancer has also increased year by year in China [4]. Increased estrogen exposure is a major risk factor for endometrial cancer, early menarche, infertility, obesity and late menopause are associated with endometrial cancer risk, too [5]. Moreover, women with a family history of endometrial cancer have a higher risk of the disease than those without a family history, suggesting that inherited genetic factors have an impact on the incidence of endometrial cancer [6].
Ninjurin2 (NINJ2) is a transmembrane protein that mediates cell-to-cell and cell-to-extracellular matrix interactions during development, differentiation, and regeneration of nervous system, the genetic polymorphisms of NINJ2 were associated with a decreased risk of Alzheimer's disease [13], ischemic stroke [14] and large artery atherosclerotic stroke [15]. Although there were significant association between NINJ2 gene polymorphisms and nervous system disease, but few studies were conducted between NINJ2 gene polymorphisms and cancer (Fig. 1).
The chronic inflammation may play an important role in endometrial carcinogenesis. IL-6 gene had an association with endometrial cancer risk [16]. NINJ2 can regulate the expression of IL-6 in human vascular endothelial cells. In this study we investigated whether alterations in the NINJ2 gene can influence the risk of endometrial cancer. We performed the case control study to investigate the relationship between five SNPs (rs118050317, rs75750647, rs7307242, rs10849390 and rs11610368) of NINJ2 gene and endometrial cancer risk in Chinese women. We take A as the allele of mutant, then, the dominant model is: AA + AG compared with GG. The recessive model was: AA compared with AG + GG. The co-dominant model was: AA, AG and GG were compared. The additive model is: AA ratio GG. We used these four genetic models to study the relationship between SNPs of different genotypes and clinical indicators in patients with endometrial cancer.

Study population
351 endometrial cancer patients (55.66 ± 8.46) were diagnosed and selected from Shaanxi Provincial Tumor Hospital. All patients were clinical diagnosed as endometrial cancer by pathology. All cases have no radiotherapy, chemotherapy and no other cancer history. Among them, 184 were smaller than 55 years old and 167 were greater than 55 years old. 344 healthy controls (55.60 ± 8.43) were selected from the physical examination center of Tang du physical examination center. All controls have no history of cancer and age matched with cases. Among them, 178 were smaller than 55 years old and 166 were greater than 55 years old ( Table 1).
We collected clinical indicators of participants as much as possible, including Carcinoembryonic antigen (CEA), Carbohydrate antigen 125 (CA125), CA199, Human epididymis protein 4 (HE4), Alpha fetoprotein (AFP), Serum ferritin (SF) and Tumor necrosis factor (TNF). These clinical indicators are female tumor markers, and their dynamic changes are related to the disease, which can provide an important basis for the judgment, prognosis and treatment of the disease [17,18]. All participators provided informed consent and the Ethical Committee approved by the study.

SNP selection and genotyping
Five SNPs rs118050317, rs75750647, rs7307242, rs10849390 and rs11610368 were selected from NINJ2 gene based on the minor allele frequency was greater than 0.05 in global population. The whole genome DNA was extracted from 5 ml peripheral blood using GoldMag-Mini whole blood genomic DNA purification kit (GoldMag Co. Ltd. Xi'an City, China). The primers of genotyping were designed by Agena on-line software (https ://agena cx.com/onlin e-tools /), the primer of this study were list in Table 2. Agena MassARRAY platform was used to detect the five SNPs in case and control group (Agena Bioscience, SanDiego, CA, USA).

Statistical analysis
The clinical index quantity in serum difference was tested by one-way analysis of variance by SPSS16.0 [19], see the following formula: (1) Establish test hypothesis and determine test level: H0: There was no difference between the two groups; H1: There are differences between the two groups, or they are not all equal; (2) Calculate the value of statistic F: SS 1 is the mean sum of squares between the groups; SS 2 is the sum of mean squares within the group. N is the number of groups. MS 1 is between the groups; MS 2 is within the group.
(3) Determine P value and make statistical inference. The Hardy-Weinberg equilibrium (HWE) p value of each SNP in controls was calculated by exact test, p value greater than 0.05 means the sample have reached . a, b, c, d, are the data in the contingency table, respectively. The allele and genotype distribution in cases and controls were compared by Chi square test. The odds ratios (OR) with 95% confidence interval (95% CI) were examined by logistic regression analysis before and after adjustment for age [20]. Suppose the dependent variable Z and a set of independent variables x1, x2, x3,…, xn, where Z is a continuous variable, we can fit a linear equation: The least square method is used to estimate the value of each β coefficient. odds = p/(1 − p), OR: Event occurrence probability of the experimental group (odds1)/Occurrence rate of events in control group (odds2).

Results
In this study, we recruited 351 endometrial cancer patients and 344 healthy controls, the age of cases and controls were matched (p = 0.923). There were significant differences in CEA, CA125 and AFP quantity in serum between cases and controls (p < 0.001; p = 0.048; p < 0.001). All of three clinical indicators were higher quantity in cases than controls (Table 1).
In Table 4, we calculated the genotype distribution in cases and controls under four different genotype model (co-dominant, dominant, recessive and log-additive) before and after adjustment the age. After adjusted the age, compared with GG genotype carriers, the rs118050317 CC genotype carriers were significant increased the endometrial cancer risk under co-dominant model (OR 8.43, 95% CI 1.05-67.89, p = 0.045), the results also significant under log-additive model (OR 1.47, 95% CI 1.04-2.07, p = 0.029) (Additional file 1: Table S2).
In the association analysis between different SNPs and different clinical index, we found there was significant difference between rs7307242 different genotype and CEA and AFP quantity (p = 0.021, p < 0.001), genotype AA corresponding to the highest quantity, followed by TT and AT genotype. rs75750647 different genotypes were significant associated with CA125 and CA199 quantity (p = 0.032, p = 0.033), the AA genotype carriers had the highest CA125 and CA199 quantity. SNP loci rs11610368 different genotypes were significant associated with HE4 quantity (p = 0.010), genotype AA corresponding to the highest quantity, followed by GG and AG (Table 5, Additional file 1: Table S1).

Discussion
In this study, we did an association study between NINJ2 gene polymorphism and endometrial cancer risk in 351 endometrial cancer patients and 344 healthy controls. We found the SNP rs118050317 mutant allele C and homozygote CC genotype were significant increased the endometrial cancer risk. In the clinical index analysis, there was significant higher quantity of CEA, CA125 and AFP in cases than controls. The gene encodes NINJ2 is located on chromosome 12p13. Toshiyuki et al. showed NINJ2 is a cell surface adhesion molecule [22]. As a vascular susceptibility gene, the SNPs in NINJ2 were genotyped to test for the association between variants of NINJ2 and dementia risk. It was found that the homozygosity of two SNPs rs11833579 and rs12425791 were associated with a decreased risk of Alzheimer's disease [13]. Moreover, the researchers also found the SNP rs12425791 of NINJ2 was significantly associated with ischemic stroke, and A allele increases the susceptibility of stroke [23]. In a family-based case control study, the A allele of rs11833579 may play a role in mediating susceptibility to ischemic stroke [14]. A functional polymorphism loci rs3809263 in the NINJ2 promoter was significant decreased the large artery atherosclerotic stroke risk. Moreover, the AA genotype carriers had significantly increased NINJ2 mRNA expression levels in Chinese population [15].
Previous study reported NINJ1, a homologue of NINJ2, can mediate inflammation processes [24]. In addition, NINJ2 is expressed in lymphocyte cell. Inflammation was likely to increase risk of developing endometrial cancer [25]. Wang et al. found NINJ2 can regulate the express of gene that associated with inflammation in human vascular endothelial cells, such as IL-6 [26]. In other study, a hypothesis about chronic inflammation may play an important role in endometrial carcinogenesis were investigated. The association between SNP in inflammatory pathway genes and endometrial cancer risk were conducted and found IL-6 SNP rs2069852 had an association with endometrial cancer risk [16]. So we speculate the rs118050317 C allele of NINJ2 through regulate the IL-6 expression to influence the ERK-NF-κB signaling pathway of endometrial cancer growth [27]. The underlying mechanism need to be further investigated.
In this study, there were some limitations. First, endometrial cancer is a high incidence of female reproductive system tumors but relatively low incidence in the population. So the example size of our study was relatively small, we will collect as many samples as we can and verify our results in a larger population in future. Second, the clinical information were limited in this study, to enrich our study, we would continue to collect the information of patients and prognosis information to analyze the association between NINJ2 gene and clinical information. The underlying mechanism of NINJ2 gene involving in endometrial cancer need to be researched next step.

Conclusion
In conclusion, the NINJ2 gene polymorphism loci rs118050317 mutant allele C was associated with an increased risk of endometrial cancer and there was significant higher quantity in serum of CEA, CA125 and AFP in cases than controls.