CASC15 polymorphisms are correlated with cervical cancer susceptibility in Chinese women

Abstract Background Cervical cancer is a frequent, common cancer in women, and causes high cancer‐related deaths among women in our world. Accumulating studies provided an important evidence for long noncoding RNA (lncRNA) polymorphisms in the susceptibility of various cancer. Here, we recruited 494 cervical cancer cases and 504 unrelated controls to assess the relationship between CASC15 (OMIM# 616610) polymorphisms and cervical cancer susceptibility. Methods Agena MassARRAY platform was conducted to genotype CASC15 polymorphisms. Odds ratios (ORs) and 95% confidence intervals (CIs) were analyzed through logistic regression to adjust for confounding factors, such as age and gender. Results Our study suggested that rs12212674 (NC_000006.12:g.22086845T>A) “A” allele was significantly associated with an increased risk of cervical cancer (OR = 1.31, 95% CI = 1.01–1.69, p = .041). The result was demonstrated in the log‐additive model (OR = 1.32, 95% CI = 1.02–1.72, p = .037). After age stratification, we also found that the “TT” genotype of rs4712653 (NC_000006.11:g.22125964T>C) in CASC15 was interaction with a higher cervical cancer risk in subjects aged ≤51 years in the co‐dominant model (OR = 2.08, 95% CI = 1.02–4.25, p = .044) and the recessive model (OR = 2.11, 95% CI = 1.05–4.24, p = .036). Whereas no significant correlation was found among other SNPs of CASC15 polymorphisms and the risk of cervical cancer. MDR analysis illustrated that the interaction between rs7740084 (NC_000006.11:g.21727531G>A), rs1555529 (NC_000006.11:g.21691704A>G), and rs12212674 had a certain effect on the progress of cervical cancer. Conclusion Our results revealed a potential interaction between CASC15 polymorphisms and cervical cancer susceptibility. The results provided important insights into CASC15 function in the development of cervical cancer.


| INTRODUCTION
Cervical cancer is the fourth common gynecological cancer, which causes high cancer-related deaths among women, especially in developing countries (McGuire, 2016). It is estimated that 266,000 deaths and 528,000 new cases occurred globally each year (Ferlay et al., 2015). The increasing researches have provided evidence that the human papillomavirus (HPV) is a primary risk factor of cervical cancer. However, most women are temporarily infected with HPV, and only a small portion of them will develop cervical cancer, indicating that other factors also played a crucial role in the development of cervical cancer (Hariri et al., 2011). Plenty of epidemiological studies suggested that environment stimulation and individual heritability were associated with the occurrence and development of cervical cancer (Liu, Zhou, Su, & Zhang, 2019;Liu, Lyu, Zhang, Sheng, & Tang, 2017). Single nucleotide polymorphisms (SNPs) are considered to be correlated with various cancers, such as lung cancer (Zhong et al., 2013), breast cancer (Xia et al., 2015), gastric cancer (Yuan et al., 2012), and cervical cancer (Gao et al., 2019). Thus, in order to develop effective personalized medicine, the detection of SNPs is very important for the susceptibility of cervical cancer.
Long noncoding RNAs (lncRNAs) are members of these ncRNA families. They are a type of RNA transcripts with more than 200 nucleotides and no protein-coding ability. Numerous studies have revealed that lncRNAs are involved in gene function via multiple mechanisms, such as transcriptional regulation, chromatin remodeling, and genetic imprinting (Glusman et al., 2006). Moreover, there are increasing studies indicated that lncRNA polymorphism was correlated with various cancer risks . Cancer susceptibility candidate 15 (CASC15, OMIM# 616610) is a highly active lncRNA, also known as LINC00340 (Yin, Zhao, Li, & Yin, 2018). Jing et al reported that the high expression of CASC15 affected the development of gastric cancer (Yao, Tang, Zhu, & Jing, 2017). A research consisting of 118 cases and 281 healthy controls confirmed that CASC15 polymorphisms significantly reduced the risk of neuroblastoma among Chinese children . Shan et al reported that patient with a higher expression of lncRNA CASC15 has a poor prognosis. And, CASC15 knockdown can inhibit cell proliferation, cell cycle progression, cell invasion ability, and epithelial-mesenchymal transition (EMT) signaling pathway. However, no study revealed that the correlation between CASC15 polymorphisms and cervical cancer susceptibility in the Chinese Han women.
Thus, we investigated the relationship between CASC15 polymorphisms and cervical cancer risk in the Chinese women. The results provided important insights into ln-cRNA CASC15 function in the development of cervical cancer.

| Study subjects
A total of 494 cases, newly diagnosed and histologically validated cervical cancer patients, were recruited from Shaanxi Provincial Cancer Hospital in the current study. Patients who have history of gynecologic diseases were excluded from our research. About 504 age and gender-matched unrelated controls were randomly recruited from the health care in the same hospital during the same time. The following are the exclusion criteria of controls: no gynecological neoplasm, no endometriosis, no other history of solid cancers, and no immune disorders. In addition, demographic and clinical factors, including age, histological types, tumor stage, carcinoembryonic antigen (CEA), serum ferritin (SF), tumor necrosis factor (TNF), sugar antigen (CA), alpha fetoprotein (AFP), and human epididymis protein (HE), were collected. The present study was approved by the clinical ethical committee of the Shaanxi Provincial Cancer Hospital. In addition, the detail researches of this study and the written informed were also signed.

| DNA extraction and genotyping
Peripheral blood samples (5 ml) were obtained from each individual. DNA was isolated from the blood samples by GoldMag DNA purification kit (GoldMag) following the manufacturer's instructions. Subsequently, NanoDrop 2000 platform (Thermo Fisher Scientific) was performed to evaluate DNA purity and concentration. Based on the minor allele frequency (MAF) > 5% and Hardy-Weinberg equilibrium (HWE) > 0.001%, CASC15 SNPs were selected in the global population from the 1,000 Genomes Project data (http://www.inter natio nalge nome.org/). When r 2 (the measure value of LD) > 0.8, the SNP can represent all the polymorphisms in a block. Ultimately, six SNPs including rs1555529 (NC_000006.11:g.21691704A>G), rs7740084 (NC_000006.11:g.21727531G>A), rs1928168 (NC_000006. 11:g.22017738T>C), rs12212674 (NC_000006.12:g.220868 45T>A), rs4712653 (NC_000006.11:g.22125964T>C), and rs9393266 (NC_000006.11:g.22220860C>T) were finally selected in the present study. Furthermore, the Agena Bioscience Assay Design Suite V2.0 software and the MassARRAY iPLEX platform (Agena Bioscience) designed the amplification and extension primers and conducted the SNPs' genotyping. In the end, all data were performed by Agena Bioscience TYPER version 4.0 software. by χ 2 tests among all individuals. We applied χ 2 test to evaluate if the candidate SNPs deviated from Hardy-Weinberg equilibrium. The relationship between CASC15 polymorphisms and the risk of cervical cancer was assesses through odds ratio (ORs) and 95% confidence interval (CI). We also detected the potential interaction between selected SNPs and cervical cancer risk using the MDR software package. All p-values were two-tailed and p < .05 was statistically significant.

| Characteristics of study individuals
All of 494 cervical cancer cases and 504 unrelated controls were involved in the research. The detail characteristics of the individuals are listed in Table 1. We found that the mean age of cases and controls were 51.65 ± 9.84 years and 51.37 ± 9.66 years, respectively. No difference between cervical cancer cases and healthy controls in age was observed (p = .645).

| CASC15 polymorphisms and cervical cancer risk
The detail information (such as the position, MAF, and HWE p value) of the candidate SNPs in participants is summarized in Table 2. The results confirmed that the genotype frequencies of the selected six SNPs were all in accord with Hardy-Weinberg equilibrium (p > .05).
The allele and genotype frequencies distribution were analyzed by χ 2 tests and odds ratios (ORs) to assess the correlation with cervical cancer risk. All results are displayed in Table 3. We validated that the "A" allele of rs12212674 increased the risk of cervical cancer (OR = 1.31, 95% CI = 1.01-1.69, p = .041), when compared with the "T" allele. In addition, the relationship between CASC15 polymorphisms and cervical cancer sensibility was examined in four genetic models. We observed that rs12212674 of CASC15 improved cervical cancer risk (OR = 1.32, 95% CI = 1.02-1.72, p = .037) in the log-additive model. However, no significant interaction was confirmed among the remaining SNPs of CASC15 polymorphisms and the risk of cervical cancer.

| Stratified analysis of CASC15 polymorphisms and cervical cancer risk
To control potential confounders, the participants were stratified by age. We demonstrated that rs4712653 "TT" genotype was associated with an increased risk of cervical cancer in aged ≤51 population (co-dominant: OR = 2.08, 95% CI = 1.02-4.25, p = .044; recessive: OR = 2.11, 95% CI = 1.05-4.24, p = .036, respectively). In terms of subjects with age >51 years, there was no statistically significant association between the candidate SNPs of CASC15 polymorphisms and cervical cancer risk (p > .05). The results are shown in Table 4.

| SNP-SNP interaction and cervical cancer risk
MDR analysis is summarized in Table 5. The interaction information analysis showed that the interaction between rs7740084, rs1555529, and rs12212674 had a certain effect on the progress of cervical cancer. The combination of high-risk and low-risk genotypes was determined according to the threshold, and we can see that the TT genotype of rs12212674, AA genotype of rs1555529, and AA genotype of rs7740084 had a higher risk of cervical cancer. And, the testing accuracy and cross-validation consistency of the two locus model (rs7740084 and rs1555529) and the three locus model (rs7740084, rs1555529, and rs12212674) was 54.39%, 4/10 and 56.70%, 4/10, respectively, which was significant.

| DISCUSSION
Cervical cancer is a common frequent cancer among women. It was reported that cervical cancer has a high mortality worldwide (McGuire, 2016). Recent evidences indicated that genetic factors played a crucial role in the development of cervical cancer . The case-control study indicated that rs12212674 of CASC15 polymorphisms was significantly associated with an increased risk of cervical cancer (p = .041). The "TT" genotype of rs4712653 was related to improved cervical cancer risk in aged ≤51 populations (p = .036).
Growing researches have confirmed that lncRNAs may have an effect on various malignant tumors, including cervical cancer, through regulating cell differentiation, proliferation, apoptosis, migration, and invasion (Camacho, Choudhari, & Gadad, 2018;Tang et al., 2017). Gao et al suggested that lncRNA PANDAR (OMIM# 617179) was significantly associated with cervical cancer, and upregulation of lncRNA PANDAR can promote cervical cancer cell proliferation (Huang, Xie, Ma, Zhao, & Gao, 2017). Recent study has shown that lncRNA HOTAIR (OMIM# 611400) was heightened in cervical cancer tissues and correlated with poor prognosis of cervical cancer patients (Li, Wang, Yu, Dong, & Qiu, 2015). In addition, a study including 1,209 patients and 1,348 healthy controls also demonstrated that rs7958904 of HOTAIR might affect cervical cancer susceptibility by modulation of cervical cancer cell proliferation (Jin et al., 2017). Wang et al suggested that rs11655237 (NC_000017.10:g.70400166C>T) of LINC00673 (OMIM# 617079) polymorphism was correlated with cervical cancer risk among Chinese population (Wang & Luo, 2018). Moreover, numerous lncRNAs are abnormally expressed in cervical cancer (Peng, Yuan, Jiang, Tang, & Li, 2016). To date, no studies have reported the correlation between lncRNA CASC15 polymorphisms and cervical cancer susceptibility in Chinese females.
Several studies have indicated that CASC15 expression was associated with different human diseases, including neuroblastoma (Russell et al., 2015), hepatocellular carcinoma (He, Zhang, et al., 2017), acute leukemia (Fernando et al., 2017), and gastric cancer (Yao et al., 2017;Yuan et al., 2012). Two genome-wide association studies (GWASs) have verified that CASC15 rs4712653 T>C was correlated with a decreased risk of neuroblastoma in Chinese children (He et  our best knowledge, we firstly demonstrated that CASC15 polymorphisms were interaction with cervical cancer susceptibility in Chinese women. We observed that CASC15 rs4712653 "TT" genotype was associated with the risk of cervical cancer in aged ≤51 people (p = .036).
In summary, the results revealed the role of CASC15 polymorphisms in cervical cancer sensibility among Chinese women. Besides, detail molecular mechanism studies to further explore the role of CASC15 variants in increasing cervical cancer risk were necessary to be performed.

ACKNOWLEDGMENT
We are grateful to the Shaanxi Provincial Cancer Hospital for providing samples. We also appreciate the reviewers and editors for their efforts and patience.

CONFLICT OF INTEREST
The authors have declared that they have no conflict of interest.

AUTHOR CONTRIBUTIONS
ZYG completed genotyping and performed the manuscript. ZCX and YS took part in genotyping. JM W, JF L, YW L, and HYL participated in the statistical analysis of the data and modified the manuscript. BL and TBJ designed the study, co-supervised the work, and finalized the manuscript. All the authors have read and approved the final manuscript. Abbreviation: MDR, multifactor dimensionality reduction.