Bioinformatic Analysis of Prognostic Value and Immune Cell Inltration of Chromobox Family Proteins in Esophageal Cancer


 BackgroundChromobox (CBX) family proteins (CBXs) are important components of epigenetic regulation complexes. Abnormal expression of CBXs is related to the occurrence and development of various cancers. However, the role and mechanism of CBXs in esophageal cancer (ESCA) need further research. MethodsThe mRNA expression of CBXs, clinicopathological parameters, prognostic values, genetic alteration, enrichment analysis, and immune cell infiltration were analyzed with several databases including Oncomine, UALCAN, Kaplan-Meier plotter, cBioPortal, DAVID, and TIMER2.0.ResultsThe expression level of CBX1/2/3/4/8 was significantly upregulated in ESCA while that of CBX7 was down-regulated. Besides, mRNA expressions of CBX1/2/3/4/5/7/8 were correlated with patients’ individual cancer stages and the nodal metastatic status. The mRNA expression of CBX3/4/7/8 was significantly correlated with the overall survival (OS) in ESCA. What is more, high mutation rate of CBXs (49%) was also observed in ESCA patients. Then, CBXs were related to the infiltration of a variety of immune cells.Conclusions﻿This study may provide new ideas for the selection of prognostic biomarkers in the CBXs in ESCA.

CBXs was analyzed. The parameter settings that come with the tool were used, including log-rank p-value, HRs, and 95% CIs and the best cutoff value. p < 0.05 was considered as statically signi cant.
cBioPortal. cBioPortal (http://www.cbioportal.org) provides visualization tools for research and analysis of cancer genetic data 19,20 . Based on ESCA (TCGA, Firehose Legacy) database, the OS for CBX3/4/8, and CBX7 were explored, while gene mutation rate reached 49% in 184 ESCA patients. cBioPortal was also used to nd out the co-expressed genes of the CBXs. p < 0.05 was considered to be signi cant.

DAVID.
DAVID is a biological information database, mainly used for differential gene function and pathway enrichment analysis. Using the database, we obtained the related data pertaining to GO including biological processes (BP), cellular components (CC), and molecular functions (MF), and KEGG for CBXs and their co-expressed genes. TIMER2.0.
TIMER2.0(http://timer.cistrome.org/) uses RNA-Seq expression pro le data to detect the in ltration of immune cells in tumor tissues 21 . The correlations between CBXs members and Tumor-immune in ltrating cells (TIICs) were separately analyzed by TIMER2.0 platform.

Results
The mRNA expression of CBXs in ESCA.
Oncomine, TIMER2.0 and UALCAN databases were used to analyze mRNA expression levels of the eight CBX family proteins in ESCA. Based on the data obtained from Oncomine, the expression of CBXs in many kinds of cancers was signi cantly different from normal tissues (Fig. 1). As shown in Fig. 2, the expression level of CBX1/2/3/4/8 in tumor tissues was signi cantly higher than that in normal tissues, while the expression level of CBX7 was lower in the former than the latter with TIMER2.0 (Fig. 2). The expression of CBXs in ESCA was veri ed by UALCAN databases and the expression level of CBX1/2/3/4/8 was also signi cantly upregulated in ESCA (Fig. 3). These results were almost consistent with those from TIMER2.0.
Clinicopathological Parameters of CBXs in ESCA Patients.
Using the ESCA database in UALCAN, the correlations between the mRNA expression of CBXs and patients' individual cancer stages, and the nodal metastatic status were separately analyzed. As shown in Fig. 4a, mRNA expressions of CBX1/2/3/4/5/7/8 were correlated with patients' individual cancer stages while the expression of CBX6 had no correlation with that. Moreover, the mRNA expression of CBX7 was down-regulated in all cancer stages. Furthermore, the mRNA expression of CBX1/2/3/4/5/7/8 was related to the nodal metastatic status while the mRNA expression of CBX6 did not correlate with the nodal metastatic status (Fig. 4b).
Prognostic Value of CBXs in ESCA Patients.
In order to evaluate the value of differentially expressed CBXs in the progression of ESCA, Kaplan-Meier Plotter was used to analyze the correlation between different CBXs and clinical outcomes. As shown in Fig. 5a, the expression of CBX3 (HR = 3.12, p = 0.00028) and CBX8 (HR = 2.27, p = 0.035) was negatively correlated with the OS of the patient in EAC (Fig. 5a). However, the expression level of CBX7 (HR = 0.48, p = 0.039) was positively correlated with the OS. In ESCC, high mRNA expression of CBX4 (HR = 2.93, p = 0.008) was signi cantly correlated with poor OS (Fig. 5b).
Furthermore, cBioPortal was used to verify the relationships between CBX3/4/8, CBX7 and the OS of patients with ESCA. High mRNA expression of CBX3/4/8 was signi cantly correlated with poor OS (Fig. 6a), but CBX7 did not seem to have a signi cant effect on OS (Fig. 6b).
In the proliferation and progression of malignant tumors, the role of immune cells cannot be ignored. In our study, we used TIMER2.0 to analyze the relationship between eight members of CBXs and immune cell in ltration (Fig. 8). The expression of CBX1 was mainly involved in the in ltration of B cells, myeloid dendritic cells, macrophages and neutrophils in ESCA. CBX2 was in connection with the in ltration of B cells. Additionally, CBX3 was correlated with the in ltration of B cells and CD4 + T cells. CBX4 was correlated with the in ltration of macrophages, and neutrophils, while CBX5 was associated with the in ltration of CD4 + T cells, CD8 + T cells, myeloid dendritic cells, macrophages and neutrophils. Moreover, CBX6 was correlated with the in ltration of myeloid dendritic cells, macrophages and neutrophils, and CBX7 was connected with the in ltration of B cells, CD4 + T cells, CD8 + T cells, myeloid dendritic cells, macrophages and neutrophils. With regard to CBX8, the in ltration of CD4 + T cells, CD8 + T cells and myeloid dendritic cells was kept in with the expression.

Discussion
So far, many studies have reported that CBXs are related to the occurrence and development of a variety of tumors. However, the relationship between CBXs and the occurrence and development of ESCC urgently needs further elucidation. In this study, we analyzed the multiple levels of CBXs in ESCC including mRNA expression, mutation, prognostic value and Immune Cell In ltration through a variety of online databases.
High expression of CBX1 was related to poor differentiation of breast cancer and led to poor OS 22 . The expression of CBX1 was closely related to the staging of gastric cancer and lymph node metastasis 23 . In addition, CBX1 interacted with the transcription factor HMGA2, activates the Wnt/βCatenin signaling pathway and affected the prognosis of patients with liver cancer 24 . In this study, the expression of CBX1 in the tissues of patients with ESCA was signi cantly higher than that in normal tissues, which was basically consistent with the results of other researchers. However, our research showed that the expression of CBX1 was not signi cantly correlated with the OS of patients with ESCA. This might be because the sample size was too small and need further research with a larger sample size to con rm. Further, the mRNA expression of CBX1 was signi cantly correlated with individual cancer stage and nodal metastatic status.
Recent studies have shown that CBX2, a member of the CBXs family, is overexpressed in several tumors.
Loss of CBX2 impaired the proliferation of leukemia cells 25 . In ovarian, breast and lung tumors, the total rate of CBX2 ampli cation exceeded 30% and CBX2 was signi cantly associated with HER-2 positive status in breast cancer 26 . In addition, CBX2 was a critical regulator of the spread of ovarian cancer and chemoresistance 27 . In this study, the expression of CBX2 in esophageal cancer tissues was signi cantly increased, while it was also signi cantly correlated with individual cancer stage and nodal metastatic status. Although patients with high expression of CBX2 had lower OS in esophageal adenocarcinoma, the difference was not signi cant.
Up-regulation of CBX3 has been found in a variety of cancers, such as LUAD, colorectal cancer (CRC), gastric cancer and ESCC 6,28−30 . CBX3 directly inhibited the expression of transcription repressors NCOR2 and ZBTB7A, thereby affecting the proliferation, colony formation and migration of LUAD cells 28 . In addition, CBX3 controlled the development of CRC by directly regulating CDKN1A (p21Waf1/Cip1) 29 . CBX3 affected the prognosis of gastric cancer by regulating the cell cycle, mismatch repair and immunerelated pathways 30 . Down-regulation of miR-377 could promote the self-renewal of ESCC stem cells by promoting the expression of CBX3 and inhibiting the activation of the P53/P21 pathway 6 . In the present study, CBX3 was highly expressed in esophageal cancer tissues. The highly expressed CBX3 was signi cantly related to the poor OS of esophageal adenocarcinoma patients, which implied that CBX3 as an oncogene might take a signi cant part in the prognosis of EAC. Furthermore, CBX3 was also signi cantly correlated with individual cancer stage and nodal metastatic status, which further suggested that CBX3 might serve as a prognostic marker for EAC.
In HCC cells, after knocking out CBX4, proliferating cell nuclear antigen and cyclin E2 were downregulated, and p16 was up-regulated, resulting in decreased cell proliferation and impaired cell cycle progression 31 . CBX4 promoted the migration and invasion of breast cancer cells 32 . Moreover, CBX4 increased the expression and activity of P53, CDK2, Cyclin E, MMP2, MMP9 and CXCR4 by up-regulating the expression of BMI-1, and promoted the proliferation and metastasis of lung cancer in vitro 33 . In this study, the expression of CBX4 in the tissues of patients with ESCA was signi cantly higher than that in normal tissues. Furthermore, the mRNA expression was signi cantly correlated with individual cancer stage and nodal metastatic status, while higher CBX4 mRNA expression was correlated with worse OS and the differences were signi cant in ESCC. In summary, CBX4 might be a potential prognostic marker for ESCC.
Although the expression of CBX5 in esophageal cancer tissues had no signi cant difference compared with normal tissues in our study, the role of CBX5 in other malignant tumors deserved our attention. CBX5 was highly expressed in NSCLC and affected the growth of tumor cells 34 . The high expression of CBX5 was related to the poor prognosis of breast cancer patients and promoted tumor metastasis 35 ,which was different from our research results. Therefore, whether CBX5 is related to the occurrence and development of cancer still needs further research to con rm.
Similar to CBX5, CBX6 was con rmed to be upregulated in a variety of cancers including HCC 34 , glioblastoma multiforme 36 and breast cancer 37 . However, our report did not nd a signi cant correlation between CBX5 and the occurrence and development of ESCA. So the role of CBX5 in ESCA is still vague.
Surprisingly, the role of CBX7 in tumors seemed to be inconsistent with other CBXs members. CBX7 was found to be an important tumor suppressor in pancreatic cancer and inhibited the PTEN/Akt signaling pathway by increasing the level of PTEN transcription 38 . Moreover, CBX7 inhibited breast cancer tumorigenicity through epigenetic induction of DKK-1 mediated 39 . CBX7 expression in CRC tissue was signi cantly reduced or absent compared with normal colonic mucosa 40 . In our study, the expression of CBX7 in esophageal cancer tissues was signi cantly decreased. In EAC, patients with high expression of CBX7 had a higher OS, while patients with high expression of CBX7 had lower cancer stages and better lymph node metastasis status. Therefore, the data reported here suggested that the expression of CBX7 might re ect the prognosis of EAC and CBX7 might be an important tumor suppressor.
As an important member in the CBX family, CBX8 was de ned as a cancer-promoting factor in a variety of malignant tumors, such as colorectal cancer 13 , metastatic prostate cancer 41 , ESCC 42 .High CBX8 expression was associated with a low distant metastasis rate and good prognosis in patients with colorectal cancer 13 .CBX8 knockout inhibited cell proliferation, colony-forming ability, DNA repair and promoted apoptosis in ESCC 42 .In this study, the expression of CBX8 was signi cantly increased in ESCA and the mRNA expression was signi cantly correlated with individual cancer stage and nodal metastatic status. Moreover, high CBX8 expression was associated with poor OS in ESCC. The above results all indicated that CBX8 might have prognostic value in ESCC.
Then, GO analysis of CBXs and their co-expressed genes that may have prognostic value showed that the functions of these genes mainly involved DNA replication, methylation and mitosis. Changes in KEGG were mainly enriched in mismatch repair, DNA replication, pyrimidine metabolism, cell adhesion molecules (CAMs). These functions and pathways were all related to the occurrence and development of cancer.
Many studies had con rmed that immune cells in the tumor microenvironment played a great role in the occurrence and development of tumors [43][44][45] . Our research showed that CBXs were related to the in ltration of a variety of immune cells including B cells, CD4 + T cells, CD8 + T cells, myeloid dendritic cells, macrophages and neutrophils. This suggested that CBXs might become a new target for immunotherapy.
Our research has certain limitations. First, our data are all derived from online databases, lacking further veri cation by cell experiments, animal experiments and clinical experiments. Second, the number of samples in the online database was small, so a larger sample size is needed to verify our results. Finally, the speci c molecular mechanism of CBXs needs further research to verify our research and ndings.

Conclusion
In conclusion, we comprehensively analyzed the expression and prognostic value of the eight members of CBXs. The expression level of CBX1/2/3/4/8 in tumor tissues was signi cantly higher than that in normal tissues and the expression level of CBX7 was lower, while the expression level of CBX5/6 was different but not signi cant. Availability of data and materials All data generated from the analysis process of this study are available from the corresponding author on reasonable request.

Competing interests
All of the authors approved the publication of the paper and declared no con icts of interests.  The mRNA expression level of CBXs in ESCA (TIMER2.0, *p<0.05, **p<0.01, ***p<0.001.). The expression level of CBX1/2/3/4/8 in tumor tissues was signi cantly higher than that in normal tissues, while the expression level of CBX7 was lower in the former than the latter Page 18/23 The mRNA expression of CBXs in ESCA tissues and normal tissues (UALCAN, *p<0.05, **p<0.01, ***p<0.001.). The expression level of CBX1/2/3/4/8 was signi cantly upregulated in ESCA.