Decreased Expression of CBX7 is an Independent Predictor of Poor Survival in Cervical Cancer


 Background: CBX7, a component of the PRC1, has been investigated as a potential biomarker in human malignant neoplasias. In present study, the value of CBX7 expression in the diagnostic and prognosis of cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) as examined via bioinformatics analysis of data obtained from Genotype-Tissue Expression (GTEx) database and The Cancer Genome Atlas (TCGA) database.Methods: Relationships between clinical factors and CBX7 were explored. The Kaplan-Meier method and Cox regression were used to identify the associations between clinicopathological characteristics and overall survival (OS) in CESC. Gene set enrichment analysis (GSEA) was performed using TCGA dataset. Results: Our results indicated the decreased expression of CBX7 in CESC, and difference in CBX7 expression was also identified according to age subgroups. CESC patients with decreased CBX7 expression had worse prognosis than those with high CBX7 expression. Multivariate analysis showed that CBX7 was an independent risk factor for OS. GSEA demonstrated pathways involved in the biosynthesis of unsaturated fatty acids, glycosaminoglycan biosynthesis-chondroitin sulfate, glyoxylate and dicarboxylate metabolism, nod-like receptor signaling pathway, O-glycan biosynthesis, one carbon pool by folate and protein export as differentially enriched in CESC with decreased CBX7 expression.Conclusion: We demonstrated that decreased CBX7 expression may be a potential independent biomarker for poor prognosis in CESC.


Introduction
Cervical cancer is an increasing global burden, both in developing and industrialized nations. In 2018, cervical cancer is the fourth leading cause of cancer death and the fourth diagnosed cancer in women globally, with approximately 570,000 cases and 311,000 deaths [1]. Numerous efforts, including implementation of human papillomavirus vaccine (HPV) and cervical screening, have been made to reduce incidence of cervical cancer [2]. But, the reduction the incidence is limited in many underdeveloped nations because of low-or middle-incomes [3]. Most women in early-stage tumors can be cured by surgery, however, women with advanced stage or recurrent disease need combination therapy such as radiotherapy or chemotherapy, the OS remains poor [4]. Therefore, it is an urgency to nd a novel and reliable biomarker for cervical cancer diagnosis and prognosis.
CBX7, a component of the PRC1, contains a conserved N-terminal PcG box and a CBX domain which binds methylated lysine residues [10]. Several studies showed that the reduced expression of CBX7 is involved in different human malignant neoplasias, such as gliomas and thyroid, breast, lung, pancreatic and and bladder carcinomas, which indicated that CBX7 may be a tumor suppressor [11][12][13][14][15][16]. In contrast, CBX7 mainly plays the role of oncogene in gastric cancer [17]. These results reveal the fact that the precise role of CBX7 in cancer progression may vary among different cancer types. However, the roles of CBX7 expression in the diagnostic and prognostic evaluation of patients with cervical cancer have not been well explored.
In this study, the expression of CBX7 mRNA in cervical cancer patients and healthy individuals was compared. In order to analyzed the diagnostic and prognostic value of CBX7, patients were divided into two groups according to the high and low CBX7 expression. We explored the relationship between the expression of CBX7 and clinical characteristics and OS of cervical cancer patients. Using GSEA to identi ed the signaling pathways which is related to the regulatory mechanisms of CBX7.

Materials And Methods
The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

Data Collection
Level 3 expression data and mRNA expression pro les (309 cases, including 3 normal cases, Work ow Type: HTseq-FPKM) were downloaded from TCGA database. Clinical characteristics regarding to survival time for cervical cancer patients was also obtained from TCGA database. Using the GTEx database, we obtained expression data for 10 normal cervical tissues (Table S1). Using R software (version 3.6.3) or Practical Extraction and Report Language (Perl) scripts on JAVA software to processed all data [18].

GSEA
To explore the potential correlations underlying the effection of CBX7 expression on cervical cancer prognosis, we applied GSEA to identi ed biological pathways pertaining to cervical cancer pathogenesisassociated CBX7 regulatory networks [19,20]. CBX7-high and CBX7-low expression were the phenotype labels. Each analysis of gene set permutations were conducted 1000 times.

Statistical Analysis
The correlations between expression of CBX7 and clinical features were evaluated by the logistic regression and Wilcoxon signed-rank test. The CBX7 expression of CESC cohort was obtained using box plots. Clinical factors associated with OS in CESC were identi ed using the Cox regression and Kaplan-Meier method. Univariate Cox analysis was applied to search for potential prognostic factors, and the correlations between CBX7 expression and survival along with clinical characteristics (age, grade, stage, lymph node status, and distant metastasis status) were examined using multivariate Cox analysis. The two groups of CBX7 expression was identi ed by the median values. Statistical signi cance was de ned as P value<0.05.

Characteristics of the Study Population
The clinical data pertaining to CESC were obtained from the TCGA database (Table S2), including patients' age, sex, clinical stage, histologic grade, tumor-node-metastasis (TNM) classi cation and survival status (Table 1).
Decreased Expression of CBX7 in CESC CBX7 expression in CESC tissues and normal cervical tissues was compared, as shown in Fig. 1. The results showed that expression of CBX7 was decreased in CESC (p = 4.07e-09). Moreover, difference in CBX7 expression was also obtained according to age (p = 0.044). The expression of CBX7 mRNA in various human tissues and different cancer types from Gene Expression Pro ling Interactive Analysis (GEPIA) was shown in Fig. 2, which computed the data from TCGA in the form of transcripts per million.
Identi cation of CBX7-Related Signaling Pathways by GSEA To identify the signaling pathways that are differentially enriched in cervical cancer, GSEA of gene expression pro les was performed to compare differentially enriched signaling pathways between CESC patients with high and low levels of CBX7 expression. GSEA revealed signi cant differences (NOM: normal; FDR: false discovery rate; FDR < 0.25 and NOM P-Value < 0.05) in the enrichment of the Molecular Signatures Database (MSigDB) Collection (c2.cp.kegg.v7.1.symbols.gmt), and the results were showed in Table 3. Gene sets related protein export, glycosaminoglycan biosynthesis-chondroitin sulfate, focal adhesion, proteasome, biosynthesis of unsaturated fatty acids, nod-like receptor signaling pathway, O-glycan biosynthesis and glyoxylate and dicarboxylate metabolism were various enriched with the decreased expression of CBX7 phenotype (Fig. 4).

Discussion
Currently, emerging evidence demonstrates that a number of cancer suppressors and oncogenes participates in CESC, some of which, including cyclin dependent kinase inhibitor 2A, DNA topoisomerase II alpha and minichromosome maintenance complex component 2 [21]. Nevertheless, novel biomarkers still needed for the diagnosis and prognosis of CESC. In present research, a gene expression pro le was identi ed based on bioinformatics analysis. CBX7 is rst reported to extend the lifespan of many normal human cells via the downregulation of Ink4a/Arf locus expression [22]. In the eld of oncology, CBX7 is rarely explored in cervical cancer, as yet. We exhaustively analyzed CBX7 expression and patients' clinical characteristics and identi ed its potential prognostic value in CESC.
In our study, we found that the decreased CBX7 expression was related to worse survival in CESC. Kaplan-Meier curves also indicated that the decreased expression of CBX7 was related to poor outcomes in CESC. Cox analyses showed that the CBX7 expression may be a potential biological marker for CESC prognosis.
CBX7 as a chromobox family protein, regulating several genes that are important for malignant neoplasias development and progression, such as drug resistance and epithelial-mesenchymal transition (EMT) [23,24]. It has been demonstrated that the loss of CBX7 enhances tumorigenicity and metastatic potential in epithelial ovarian cancer [25]. Accumulating evidence indicates that decreased CBX7 may also serve as an adverse prognostic biomarker for colon carcinoma patients [26]. The latest study reveals that CBX7 inhibits cell motility and cell growth and induces apoptosis in cervical cancer [27]. The present study showed that decreased CBX7 expression in CESC, this result is consistent with ndings for CBX7 expression in other types of tumors. We also found that age was associated with CBX7 expression.
Many studies have reported that CBX7 closely correlated with malignant transformation. Federico et al. found that CBX7 and Protein Arginine Methyltransferase 1 (PRMT1) complex is critical for PRMT1 and for the expression of E-cadherin, a crucial hallmark of EMT [28]. Jung et al. demonstrated that CBX7 act as a crucial regulator that induces human malignant hematopoietic stem and progenitor cells by noncanonical and canonical interactions and higher expression of CBX7 can also inhibit proliferation and promoted differentiation in acute myeloid leukemia, revealing therapeutic possibilities for leukemia [29]. Nawaz et al reported that CBX7 inhibit glioma cell migration through YAP/TAZ-CTGF-JNK signaling axis and identi ed the critical role of CBX7 in gliomagenesis [30]. Based on above reports, the decreased CBX7 expression indicates the progression of cell proliferation, which might promote poor outcomes in CESC. Pallante et al. explored CBX7 represents a critical prognostic factor in a wide range of cancer types [31].
Consistently, the present study revealed that CBX7 expression was related to OS in CESC, and the potential mechanism may be correlated with the biosynthesis of unsaturated fatty acids and O-glycan, protein export, glyoxylate and dicarboxylate metabolism, glycosaminoglycan biosynthesis-chondroitin sulfate, one carbon pool by folate and the nod-like receptor signaling pathway, as identi ed by GSEA. Moreover, we also identi ed the prognostic value of CBX7 expression in age subgroup of CESC and also explored that CBX7 expression correlated with G1/G2, G3/G4, stage I/II, T1, T4, N0 and M0 patients. Currently, surgery is the major treatment for CESC. The decreased CBX7 expression also adversely affected OS in patients with early stage I/II cancers, but not in those with advanced stage III/IV cancers, which further indicates the potential prognostic role of CBX7 expression in subgroup analyses and its application to precision therapy for CESC. However, the number of patients with advanced stage is decreased, which is a limitation in present research, future works need to increase the number of the samples to verify this result.

Conclusions
This study demonstrates that CBX7 is downregulated in CESC, and decreased CBX7 expression is associated with clinical progression and is an independent risk factor for OS in CESC. The nding suggests that CBX7 may be a potential biomarker in CESC.

Declarations Acknowledgments
The results of this study are based on data generated by the TCGA and GTEx database. We would like to thank "TCGA Research Network" for generating, curating, and providing the bladder cancer clinical data.
Authors' contributions LA analyzed the data and was the major contributor to performing the experiments and writing the manuscript. LA and JZ were involved in critically revising the manuscript for important intellectual content. ZJ, XY and PW made substantial contributions to conceiving the research and designing the draft of the research process. WO and HW contributed to writing the manuscript. All authors read and approved the nal manuscript. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The data for this research were downloaded from the TCGA and GTEx database, a public website. Our study did not require ethical board approval because it did not involve human or animal trials.

Funding
This study was funded by National Natural Science Foundation of China (NO. 81974409).

Availability of data and materials
The following information was supplied regarding data availability: The raw measurements are available in Supplemental Files. Table S1 showed CBX7 expression in cervical cancer tissues and normal cervical tissues. Table S2 showed the clinical data of cervical cancer patients. Table S3 showed the overall survival of cervical cancer.

Ethical statement and consent to participate
The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This research does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication
Not applicable.    Enrichment plots from GSEA. The GSEA results indicated that genes involved in biosynthesis of unsaturated fatty acids, glycosaminoglycan biosynthesis-chondroitin sulfate, glyoxylate and dicarboxylate metabolism, nod-like receptor signaling pathway, O-glycan biosynthesis, one carbon pool by folate and protein export were differentially enriched in cervical cancer patients with decreased CBX7 expression.