Bioinformatics analysis of predictive value of EZH2 in early breast cancer

Bioinformatics analysis of predictive value of EZH2 in early breast cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bioinformatics analysis of predictive value of EZH2 in early breast cancer Enwen Mao, Lei Sun This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4781872/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The breast cancer is still considered the second leading cause of cancer death in women, although in high-risk individuals through breast ultrasound, mammography and breast magnetic resonance tomography (such as the MRT) imaging screening in the early diagnosis of cancer, but still urgently needed for the diagnosis and treatment of early breast cancer, especially breast cancer to discover new biomarkers. Methods The gene chip data were retrieved from GEO database to screen the differentially expressed genes (DEG) in breast cancer.The expression of DEG was verified and the prognostic value was evaluated using GEPIA databases.The protein-protein interaction (PPI) network was plotted with STRING, and Cytoscape was used for cell map visualization. Timer database was used to analyze the correlation between DEGs and immune infiltration level in breast cancer, and Kaplan-Meier plotter was used to analyze the overall survival of the screened target gene in breast cancer patients. Results The functions of target genes were mainly in cell division, cell proliferation and signal transduction. These highly expressed genes are associated with poor prognosis in breast cancer patients, among which we found that EZH2 is of great significance for the diagnosis and prognosis of early breast cancer. The enrichment analysis of its functions and pathways includes PI3K-AKT signaling pathway, MAPK signaling pathway, protein binding, cell cycle, etc. EZH2 was positively correlated with immune infiltration cells in patients with breast cancer. Conclusion EZH2 in breast cancer may be a useful target for diagnosis and treatment of early breast cancer. Biological sciences/Computational biology and bioinformatics Health sciences/Medical research breast cancer EZH2 GEO database early diagnosis bioinformatics analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Breast cancer is the most common tumor in women all over the world, and it is also the main cause of cancer death in women 1 . About 11% of breast cancers worldwide occur in China, and the incidence has risen rapidly in recent decades, partly due to the rapid population growth and socio-economic development 2 . Although the incidence of breast cancer is not high, the absolute number of deaths in China is often high and the prognosis is poor 3 . One of the main reasons for the poor prognosis of breast cancer is the low diagnosis rate 4 . Since the 1980s, evidence from randomized controlled trials has shown that screening by mammography can detect small (< 15 mm) tumors with good results, especially before palpable and Preclinical 5 , therefore screening based on mammography makes early detection of breast cancer possible. Breast cancer can also be accompanied by abnormal tumor markers, including carcinoembryonic antigen and carbohydrate antigen 125, carbohydrate antigen 153, and carbohydrate antigen 50, which are all commonly used tumor markers for this disease, but the above-mentioned tumor markers There is no specificity 6 . For example, CEA is detectable in other types of cancer and even in non-cancer related diseases. The sensitivity of mammography is limited, and the tumor staging at the initial stage of breast cancer diagnosis has a great influence on the 5-year survival rate 7 . Therefore, further research is needed to develop and identify more specific and sensitive biomarkers for early diagnosis of breast cancer. Bioinformatics analysis is an interdisciplinary field, mainly involving molecular biology and genetics, computer science, mathematics and statistics. From a computational point of view, it solves data-intensive large-scale biological problems. The most common problem is to model biological processes at the molecular level and make inferences based on the collected data 8 . In our research, mature bioinformatics tools are used to screen potential biomarkers for early diagnosis of breast cancer. The Comprehensive Gene Expression (GEO) database is an international public repository that can archive and freely distribute high-throughput gene expression and other functional genomics data sets. The database not only provides data access rights for thousands of studies, but also provides various Web-based tools and strategies for selecting appropriate gene expression profiles 9 . Online analysis tools help to analyze the differentially expressed genes between the tumor group and the normal group. In our study, three gene matrix column data sets, namely GSE39004, GSE109169 and GSE139038 were downloaded from GEO. Use tools such as GEO2R, ​​STRING, GEPIA, TIMER, and Kaplan-Meier plotters. Finally, the modules related to breast cancer were screened for further development of the early breast cancer diagnosis system. Among them, EZH2 (Enhancer of zeste homolog 2) is a protein coding gene 10 . Diseases related to EZH2 include Weaver's syndrome and mantle cell lymphoma. The related pathways include lysine degradation and PKMTs methylation of histone lysine. Gene ontology (GO) annotations related to this gene include sequence-specific DNA binding and chromatin binding. The important paralog of this gene is EZH1 11 . In past reports, EZH2 has been up-regulated in various solid tumors including liver cancer 12 , lung cancer 13 and prostate cancer 14 , and has been mutated in various hematological malignancies (such as lymphoma) 15 . EZH2 inhibits the expression of excessive tumor suppressor genes in tumor cells, thereby promoting cell cycle, cell proliferation and cell invasion and promoting the development of cancer 16 . In addition, the executive molecular processes triggered by EZH2 , such as NF-κB activation, microRNA silencing, tumor immune evasion, and non-standard transcriptional regulation, seem to be the basic characteristics of each cancer 17 . Although there are related research reports on the expression of this gene in breast cancer 18 , there are few studies on the correlation between EZH2 gene and tumor infiltrating immune cells in the tumor microenvironment and the expression of EZH2 and screening early breast cancer. In the immune microenvironment of cancer cells, there are many immune cells and cytokines that interact with cancer cells to form a complex regulatory network. Through this research, we boldly imagine that targeting EZH2 may enhance breast cancer antigen presentation, anti-tumor immunity and bypass anti-PD-1 drug resistance, thus finding that the expression of EZH2 in cancer cells and immune cells can contribute to cancer immunity influential. 1. Materials and methods 1.1 Bioinformatics analysis data The subject and genetic information comes from the GEO database ( https://www.ncbi.nlm.nih.gov/geo/ ). In this study, three gene expression profiles (GSE39004, GSE109169 and GSE139038) were searched and selected from the GEO database. In short, in GSE39004, there are 124 early breast cancer samples and 56 non-cancerous samples. GSE109169 consists of 25 early breast cancer samples and 25 non-cancer samples from 25 patients. GSE139038 consists of 41 early breast cancer samples and 24 non-cancer samples. The characteristics of these patients are shown in Table 1 . A large number of high-throughput functional genomics studies have been collected in the GEO database. Use GEO2R to screen DEGs between breast cancer samples and non-cancer samples. Through online analysis of the GEPIA database ( http://gepia.cancer-pku.cn ), the expression of DEGs genes in breast cancer tissues and normal tissues was screened. The breast cancer data set of Kaplan-Meier Plotter database ( https://kmplot.com/analysis/ ) was used to verify the relationship between OS, PFS and the expression of 33 DEGs online. Finally, it is determined that the target gene of the study is EZH2 . Use STRING database ( https://string-db.org/ ) to search for EZH2 gene related information and related proteins, and construct a network diagram of EZH2 gene related proteins, and finally through the TIMER database ( https://cistrome.shinyapps.io/timer/ ) Used to analyze the correlation of EZH2 expression in B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells in the microenvironment of breast cancer. 1.2 GEPIA database search method GEPIA ( http://gepia2.cancer-pku.cn ) is a newly developed interactive web server for analyzing the RNA sequencing expression data of 9736 tumors and 8587 normal samples from the TCGA and the GTEx projects, using a standard processing pipeline. GEPIA provides customizable functions such as tumor/normal differential expression analysis, profiling according to cancer types or pathological stages, patient survival analysis, similar gene detection, correlation analysis, and dimensionality reduction analysis. This tool is developed by Zefang Tang, Chenwei Li, and Boxi Kang of Zhang Lab, Peking University, Beijing, China. Set the filter conditions as follows: select Expression on Box Plots; Gene: EZH2 , Log2 FC ≥ 1, P < 0.01, Cancer Type: BRCA. 1.3 Kaplan-Meier Plotter database analysis method Use an online tool, Kaplan-Meier plotter (KM plotter) to analyze the impact of selected DEGs on survival. This tool contains information on a large number of patients with different cancers. According to the individual expression of specific genes (high expression or low expression), breast cancer patients are divided into two groups. Obtain the operating system of breast cancer patients and draw it on the website, and display the hazard ratio and P value with 95% confidence interval on the graph. 1.4 STRING database analysis method STRING website can be used to analyze EZH2 gene related gene information and related protein functions, and construct EZH2 gene related protein network diagram through cytoscape. Use annotation, visualization and comprehensive discovery database (DAVID) to conduct GO and KEGG pathway enrichment analysis on the identified DEGs. Use p value < 0.05 to distinguish significantly enriched genes. 1.5 TIMER database analysis method TIMER web server ( https://cistrome.shinyapps.io/timer/ ) is a comprehensive resource for systematical analysis of immune infiltrates across diverse cancer types. The abundance of six immune infiltrates (B cells, CD4 + T cells, CD8 + T cells, neutrophils, macrophages, and dendritic cells) is estimated by TIMER algorithm. TIMER web server allows users to input function-specific parameters, with resulting figures dynamically displayed to conveniently access the immunological, clinical, and genomic features of the tumor. Timer database can be used to analyze the immune infiltration of different types of cancer, and the screening conditions are as follows: ( 1 ) gene symbol: EZH2 ; ( 2 ) cancer types: BRCA (breast invasive carcinoma); ( 3 ) immune infiltrates: B cell CD8 + T cell CD4 + T cell macrophage peripheral dendritic cell. To verify the results in Fig. 2 by TIMER2.0( http://timer.comp-genomics.org/ ), the screening conditions are as follows: ( 1 ) cancer exploration; ( 2 ) Gene_DE ( 3 ) Gene Expression: EZH2 . Gene_DE module allows users to study the differential expression between tumor and adjacent normal tissues for any gene of interest across all TCGA tumors. Distributions of gene expression levels are displayed using box plots. The statistical significance computed by the Wilcoxon test is annotated by the number of stars (*: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001). Users can identify genes that are up-regulated or down-regulated in the tumors compared to normal tissues for each cancer type, as displayed in gray columns when normal data are available. 1.6 Statistical methods SPSS22.0 software was used for statistical analysis, t-test was used for comparison between breast cancer tissues and normal tissues, kaplan-Meier and log-rank test were used for survival analysis, and P < 0.05 was considered statistically significant. 2. Results 2.1 DEGS Screening A total of 431, 842 and 2764 DEGS were identified from GSE39004, GSE109169 and GSE139038 data sets, respectively. A total of 189 genes were found in all three data sets. Interestingly, these gene expression trends were similar across the three data sets. Among them, 33 genes were up-regulated in breast cancer compared with non-cancer samples (Fig.1A), and 156 genes were down-regulated (Fig.1B).In all three data sets it is defined by |logFC|>2.5 and P<0.05. 2.2 Expression level of EZH2 mRNA in breast cancer GEPIA database analysis showed that compared with normal tissues, expression level of EZH2 mRNA in breast cancer tissues was significantly increased (P<0.01, Fig. 2).Moreover, the expression profile data were analyzed, and the differentially expressed genes were screened out according to the screening criteria, and the volcanogram of differentially expressed genes in breast cancer tissue samples and normal tissue samples was drawn (Fig.3). 2.3 Survival analysis results based on kaplan-Meierplotter database In order to further study the prognostic potential of EZH2 in breast cancer, we used Kaplan-Meier plotter database to evaluate the prognostic value of EZH2.Interestingly, poor prognosis in breast cancer was associated with EZH2 expression, and the differences were statistically significant (Fig.4;P=0.015). 2.4 EZH2 gene-related protein network diagram and Enrichment Analysis The EZH2 gene-related protein network diagram was obtained through STRING analysis of 25 major EZH2 gene-related proteins BUB1B, CDC45, CDK1, etc. Cytoscape was used to construct a visualization network (Fig.5).The GO analysis of EZH2 and the enrichment analysis of the KEGG pathway revealed that upregulated genes were mainly related to nuclear division, organel division and chromosome separation (Fig.6).In addition, up-regulated DEGs are related to KEGG pathways, including pi3K-Akt signaling pathway, MAPK signaling pathway, protein binding, cell cycle, etc. (Fig.7).Surprisingly, a wealth of pathways, such as extracellular matrix -receptor interactions, were observed in upregulated genes, leading to direct or indirect control of cellular activity, including adhesion, proliferation, and migration. 2.5 The correlation between the expression of EZH2 in breast cancer and the level of immune infiltration was studied using Timer database By analyzing the relevant data obtained from the Timer database, we were pleasantly surprised to find that in the immune microenvironment of breast cancer, EZH2mRNA was significantly positively correlated with tumor purity, and the expressions of B cells, CD8+T cells, CD4+T cells, neutrophils and dendritic cells were significantly positively correlated (P < 0.05, Fig.8).In other words, breast cancer patients with high EZH2 expression may be more prone to immune cell infiltration, and thus may become a population with high response to immunotherapy. Of course, this is just a guess, and we'll be testing this out in the real world with large clinical studies. In view of the differential expression of EZH2 between breast cancer tissue and normal tissue in Fig. 2, we used the timer database again to verify the conclusion, and the final conclusion was consistent with the previous one.(Fig.9) 3. Discussion Regardless of the progress of breast cancer treatment, breast cancer is still the leading cause of cancer-related death in women 20 . The high mortality rate of breast cancer is partly due to the lack of sufficient screening methods with high sensitivity and specificity for early breast cancer 21 . Therefore, it is imperative to identify potential biomarkers for early breast cancer screening. Fortunately, microarray technology and next-generation sequencing have become key tools in providing integrated genetic information on breast cancer samples and revealing detailed changes in disease progression. In the study, online bioinformatics methods were used to study biomarkers for early diagnosis of breast cancer. Three GEO datasets were downloaded from the website and identified 189 genomes, including 33 up-regulated genes. These upregulation genes are mainly involved in cell division, cell proliferation and signal transduction, and these pathways are closely related to cancer. In addition, of these DEGs, 37 genes showed the highest degree in the PPI network. Further survival analysis showed that EZH2 of these 37 modules was closely associated with shorter survival time in breast cancer patients. Studies have shown that EZH2 is overexpressed in various types of cancer, including gastric, hepatocellular and lung cancers. In one study, EZH2 inhibition led to significant upregulation of MHCI class expression in human and mouse human papillomavirus negative head and neck cancer strains in vitro and in vivo in mouse models. Enhanced antigen presentation of EZH2 inhibitors or CRISPR-mediated EZH2 defects in tumor cells increases antigen-specific CD8+T cell proliferation, IFN production, and cytotoxicity in tumor cells. Mechanistically, EZH2 inhibition reduces histone H3K27me3 modification on -2-microglobulin promotors. Finally, in an anti-PD-1 drug resistant model of head and neck cancer, combination therapy inhibits tumor surveillance 22 .Another study analysis of immunohistochemical and multiple immunofluorescence staining of EZH2 and PD - L1 expression in liver cancer tissue and its correlation, in the evaluation of EZH2 expression of PD - L1 adjustment, finally found EZH2 can through CD274 directly raise liver cancer cells and IRF1 promoter H3K27me3 level to suppress the immune checkpoint inhibitors PD - the expression of L1, and may be immune activation HCC potential therapeutic targets for the treatment of combination therapy. Clinical samples from HCC patients with immune-activated microenvironment showed a negative correlation between EZH2 and PD-L1 expression in HCC cells. Multivariate Cox analysis showed that the combination of EZH2 and PD-L1 was an independent prognostic factor for OS and RFS in liver cancer patients 23 . At present, for the treatment of breast cancer, PD-1/PD-L1 inhibitors have been in full blast in the study of breast cancer, especially TNBC, and the existing research results show that anti-PD-1 /PD-L1 therapy has a broad application prospect in neoadjuvant, adjuvant and combination therapy of TNBC. Based on the results of IMpassion130 study, FDA approved Atezolizumab combined with albumin binding paclitaxel for first-line treatment of metastatic or locally advanced PD-L1 positive TNBC 24 . At present, however, there is still a lack of effective immune markers to screen out the immune dominant population, and high, medium and low risk populations can be subdivided through the study of the threshold value of markers, so as to clarify the mechanism of immunotherapy. A particular concern of this study is that EZH2 expression is associated with the level of immune cell infiltration in breast cancer. By using the Timer database, we found that the expression level of EZH2 was positively correlated with the immune infiltration level of B cells, CD8+T cells, CD4+T cells, neutrophils and dendritic cells in breast cancer patients. With the in-depth study of tumor microenvironment, the presence of tumor infiltrating lymphocytes in breast cancer specimens has been proved to be an important predictive and prognostic factor. Emerging data also showed that MEK inhibition could up-regulate the expression of MHC and PD-L1 on the surface of TNBC cells in vivo and in vitro, so that the expression of PD-L1 could be higher in tumor cells and immune cells 25 . At present, EZH2 inhibitor “Tazemetostat” has shown excellent antitumor activity in the treatment of epithelial sarcoma and follicular lymphoma .Therefore, we have reason to believe that EZH2 will also become one of the major targets for the treatment of breast cancer. This may also indicate that EZH2 may be a potential target of immunotherapy for breast cancer, which is helpful to screen out the dominant population in clinical treatment. Overall, breast cancer is the most common malignancy in women. It is often characterized by a lack of early symptoms, resulting in less early detection of the disease, while testing late in the disease means treatment is more difficult and uncertain. By comparing the three gene profiles, we found that EZH2 may be the key gene associated with breast cancer. Bioinformatics comprehensive analysis is a powerful tool for biomarker screening. Interactions between selected proteins provide important information about breast cancer progression, as well as the underlying mechanisms of cancer occurrence, development, metastasis, and drug resistance. However, the predictive value of these markers should be confirmed in additional studies prior to clinical application. The underlying mechanisms of the activity of these genes also need to be elucidated, helping to map the predictive networks of these genes. We have optimized the parameters of the bioinformatics synthesis analysis to screen for potential biomarkers for early diagnosis of breast cancer and hope that our study will provide new methods for early diagnosis and prediction of breast cancer. In future studies, more attention should be paid to subclass analysis to elucidate the characteristics of each set of biomarkers. In our future study, we will recruit a group of breast cancer patients to study the sensitivity and specificity of these biomarkers in early screening of breast cancer patients, which may promote the clinical application of the biomarkers. Abbreviations EZH2, Enhancer of zeste homolog 2; OS, overall survival; DEG, differentially expressed genes; TNBC, triple negative breast cancer;PD-1, Programmed Death 1; PD-L1, Programmed Cell Death-Ligand 1;GO, gene ontology ;GEPIA, gene expression profiling interactive analysis ;TCGA, The Cancer Genome Atlas; TIMER, tumor immune evaluation resource Declarations Data Availability Data associated with the present paper can be obtained by contacting the corresponding author. Acknowledgments Not applicable. Disclosure The author declares no conflicts of interest. Funding This research has not been supported by relevant funds. Authors’ contributions Lei Sun contributed to the design and conception of the study. Lei Sun, Enwen Mao carried out data acquisition, analysis, and interpretation. Enwen Mao drafted the manuscript. Lei Sun oversaw critical revision of the manuscript for intellectual content. References Ahmad, A. Breast Cancer Statistics: Recent Trends. Adv Exp Med Biol 1152 , 1-7, doi:10.1007/978-3-030-20301-6_1 (2019). Li, T., Mello-Thoms, C. & Brennan, P. C. Descriptive epidemiology of breast cancer in China: incidence, mortality, survival and prevalence. Breast Cancer Res Treat 159 , 395-406, doi:10.1007/s10549-016-3947-0 (2016). Yang, G. et al. Rapid health transition in China, 1990–2010: findings from the Global Burden of Disease Study 2010. The Lancet 381 , 1987-2015, doi:10.1016/s0140-6736(13)61097-1 (2013). Waks, A. G. & Winer, E. P. Breast Cancer Treatment: A Review. JAMA 321 , 288-300, doi:10.1001/jama.2018.19323 (2019). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4781872","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":343134820,"identity":"edf19995-2b88-424d-9a18-2d8692f37d33","order_by":0,"name":"Enwen Mao","email":"","orcid":"","institution":"Jiaozhou Branch of Shanghai East Hospital, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Enwen","middleName":"","lastName":"Mao","suffix":""},{"id":343134821,"identity":"1fd49e17-2308-435a-8a61-ac9dd9b13bca","order_by":1,"name":"Lei Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYHACxgMMBjZybPyN7R8+GNjYEaUHqCXNmF/i8DHGGQVpyURqYTicKNmQlsbM8+EQYwMh5fIzcg8c+FBwOMHgwBmzxzYGB5gZ2A8f3YBPi8GNvISDMwzS8wwO95gb5xjc4WPgSUu7gVeLRI7BYR4D62KgLQbSOQbPmBkkeMzwapGfAdTyx4A5ccOBHANpC4PDjA2EtDDcAGphMHBOnAn0vjQDMVoMzrwxONgDCeTDhkBGMhshv8i35xg++PEHHJWNIIYdP/vhY/gdhgHYSFM+CkbBKBgFowAbAADez1CLCsAjFQAAAABJRU5ErkJggg==","orcid":"","institution":"Jiaozhou Branch of Shanghai East Hospital, Tongji University","correspondingAuthor":true,"prefix":"","firstName":"Lei","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2024-07-22 12:32:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4781872/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4781872/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63802210,"identity":"1233a881-26b2-4b3b-b547-060f72977bcb","added_by":"auto","created_at":"2024-09-02 13:17:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":676663,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of differentially expressed genes in mRNA expression profiling datasets (GSE39004, GSE109169, and GSE139038). Venn diagram of DEGs (P\u0026lt;0.05 and |Log FC| \u0026gt;1.5);A：Up regulation gene. B: Down regulation gene.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4781872/v1/4d2a63fd53c50bdd711c5177.png"},{"id":63802208,"identity":"682369db-13b6-49c5-b54c-fd55bc641ac6","added_by":"auto","created_at":"2024-09-02 13:17:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":468093,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent expression of \u003cem\u003eEZH2\u003c/em\u003emRNA in breast cancer and normal tissue by GEPIA P\u0026lt;0.01\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4781872/v1/63d2deb80c9ca995652545d2.png"},{"id":63802206,"identity":"59af2bfc-3ae1-44ea-b184-967ff67e9b19","added_by":"auto","created_at":"2024-09-02 13:17:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":506953,"visible":true,"origin":"","legend":"\u003cp\u003eVolcano map of differentially expressed genes between breast cancer and normal tissues\u003csup\u003e19\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4781872/v1/78b634a600de667582428585.png"},{"id":63802207,"identity":"1e8835fb-1cc1-41ae-8bfc-b92e47b02a0e","added_by":"auto","created_at":"2024-09-02 13:17:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":296489,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationship between\u003cem\u003eEZH2\u003c/em\u003e gene expression and OS in patients with breast cancer\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4781872/v1/8828b8dd0f5d9ab81f8dd5aa.png"},{"id":63802209,"identity":"2d29bab8-f5ed-4109-b3d1-95f05527320c","added_by":"auto","created_at":"2024-09-02 13:17:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1588252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEZH2 \u003c/em\u003egene related protein network\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4781872/v1/d0cd0ea683daf14622a1598e.png"},{"id":63802213,"identity":"a8745cda-d2fc-4f8e-9c90-45d03ce10158","added_by":"auto","created_at":"2024-09-02 13:17:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":431663,"visible":true,"origin":"","legend":"\u003cp\u003eHistogram of gene ontology analysis with \u003cem\u003eEZH2\u003c/em\u003e. Go, gene ontology; DEG, differentially expressed genes.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4781872/v1/b063c003e368b4e44c46bbc7.png"},{"id":63802214,"identity":"84ae2c9b-1062-4bf8-8910-1f7e3cccfec5","added_by":"auto","created_at":"2024-09-02 13:17:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":390677,"visible":true,"origin":"","legend":"\u003cp\u003eThe KEGG pathway with \u003cem\u003eEZH2\u003c/em\u003ewas used to analyze the bubble diagram. KEGG, Kyoto Encyclopedia of genes and genomes; DEG, differentially expressed genes.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4781872/v1/4c089be4673f3070f095f4c7.png"},{"id":63804010,"identity":"4ad71b51-6c32-4161-8eda-1d5b16e00b02","added_by":"auto","created_at":"2024-09-02 13:25:59","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1133622,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between \u003cem\u003eEZH2\u003c/em\u003egene expression and immune infiltration in breast cancer\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4781872/v1/77bda58de1750b21c85d021f.png"},{"id":63805062,"identity":"6edb6a1b-3e4b-4672-8b62-726d86050be1","added_by":"auto","created_at":"2024-09-02 13:33:59","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":630513,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent expression of \u003cem\u003eEZH2\u003c/em\u003emRNA in breast cancer and normal tissue by TIMER2.0 (*: p-value \u0026lt; 0.05; **: p-value \u0026lt;0.01; ***: p-value \u0026lt;0.001)\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4781872/v1/c559961b6d745b675d58ca21.png"},{"id":64805902,"identity":"7afc9005-a3b3-4635-8ea0-d71f432e6f45","added_by":"auto","created_at":"2024-09-19 04:25:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7154614,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4781872/v1/fddbcad6-772d-490d-8690-5271f57b3821.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bioinformatics analysis of predictive value of EZH2 in early breast cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer is the most common tumor in women all over the world, and it is also the main cause of cancer death in women \u003csup\u003e1\u003c/sup\u003e. About 11% of breast cancers worldwide occur in China, and the incidence has risen rapidly in recent decades, partly due to the rapid population growth and socio-economic development \u003csup\u003e2\u003c/sup\u003e. Although the incidence of breast cancer is not high, the absolute number of deaths in China is often high and the prognosis is poor\u003csup\u003e3\u003c/sup\u003e. One of the main reasons for the poor prognosis of breast cancer is the low diagnosis rate\u003csup\u003e4\u003c/sup\u003e. Since the 1980s, evidence from randomized controlled trials has shown that screening by mammography can detect small (\u0026lt;\u0026thinsp;15 mm) tumors with good results, especially before palpable and Preclinical\u003csup\u003e5\u003c/sup\u003e, therefore screening based on mammography makes early detection of breast cancer possible. Breast cancer can also be accompanied by abnormal tumor markers, including carcinoembryonic antigen and carbohydrate antigen 125, carbohydrate antigen 153, and carbohydrate antigen 50, which are all commonly used tumor markers for this disease, but the above-mentioned tumor markers There is no specificity\u003csup\u003e6\u003c/sup\u003e. For example, CEA is detectable in other types of cancer and even in non-cancer related diseases. The sensitivity of mammography is limited, and the tumor staging at the initial stage of breast cancer diagnosis has a great influence on the 5-year survival rate\u003csup\u003e7\u003c/sup\u003e. Therefore, further research is needed to develop and identify more specific and sensitive biomarkers for early diagnosis of breast cancer.\u003c/p\u003e \u003cp\u003eBioinformatics analysis is an interdisciplinary field, mainly involving molecular biology and genetics, computer science, mathematics and statistics. From a computational point of view, it solves data-intensive large-scale biological problems. The most common problem is to model biological processes at the molecular level and make inferences based on the collected data \u003csup\u003e8\u003c/sup\u003e. In our research, mature bioinformatics tools are used to screen potential biomarkers for early diagnosis of breast cancer. The Comprehensive Gene Expression (GEO) database is an international public repository that can archive and freely distribute high-throughput gene expression and other functional genomics data sets. The database not only provides data access rights for thousands of studies, but also provides various Web-based tools and strategies for selecting appropriate gene expression profiles\u003csup\u003e9\u003c/sup\u003e. Online analysis tools help to analyze the differentially expressed genes between the tumor group and the normal group. In our study, three gene matrix column data sets, namely GSE39004, GSE109169 and GSE139038 were downloaded from GEO. Use tools such as GEO2R, ​​STRING, GEPIA, TIMER, and Kaplan-Meier plotters. Finally, the modules related to breast cancer were screened for further development of the early breast cancer diagnosis system. Among them, \u003cem\u003eEZH2\u003c/em\u003e (Enhancer of zeste homolog 2) is a protein coding gene\u003csup\u003e10\u003c/sup\u003e. Diseases related to \u003cem\u003eEZH2\u003c/em\u003e include Weaver's syndrome and mantle cell lymphoma. The related pathways include lysine degradation and PKMTs methylation of histone lysine. Gene ontology (GO) annotations related to this gene include sequence-specific DNA binding and chromatin binding. The important paralog of this gene is EZH1\u003csup\u003e11\u003c/sup\u003e. In past reports, \u003cem\u003eEZH2\u003c/em\u003e has been up-regulated in various solid tumors including liver cancer\u003csup\u003e12\u003c/sup\u003e, lung cancer\u003csup\u003e13\u003c/sup\u003e and prostate cancer \u003csup\u003e14\u003c/sup\u003e, and has been mutated in various hematological malignancies (such as lymphoma)\u003csup\u003e15\u003c/sup\u003e. \u003cem\u003eEZH2\u003c/em\u003e inhibits the expression of excessive tumor suppressor genes in tumor cells, thereby promoting cell cycle, cell proliferation and cell invasion and promoting the development of cancer\u003csup\u003e16\u003c/sup\u003e. In addition, the executive molecular processes triggered by \u003cem\u003eEZH2\u003c/em\u003e, such as NF-κB activation, microRNA silencing, tumor immune evasion, and non-standard transcriptional regulation, seem to be the basic characteristics of each cancer\u003csup\u003e17\u003c/sup\u003e. Although there are related research reports on the expression of this gene in breast cancer\u003csup\u003e18\u003c/sup\u003e, there are few studies on the correlation between \u003cem\u003eEZH2\u003c/em\u003e gene and tumor infiltrating immune cells in the tumor microenvironment and the expression of \u003cem\u003eEZH2\u003c/em\u003e and screening early breast cancer. In the immune microenvironment of cancer cells, there are many immune cells and cytokines that interact with cancer cells to form a complex regulatory network. Through this research, we boldly imagine that targeting \u003cem\u003eEZH2\u003c/em\u003e may enhance breast cancer antigen presentation, anti-tumor immunity and bypass anti-PD-1 drug resistance, thus finding that the expression of \u003cem\u003eEZH2\u003c/em\u003e in cancer cells and immune cells can contribute to cancer immunity influential.\u003c/p\u003e"},{"header":"1. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e1.1 Bioinformatics analysis data\u003c/h2\u003e\n \u003cp\u003eThe subject and genetic information comes from the GEO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003c/span\u003e). In this study, three gene expression profiles (GSE39004, GSE109169 and GSE139038) were searched and selected from the GEO database. In short, in GSE39004, there are 124 early breast cancer samples and 56 non-cancerous samples. GSE109169 consists of 25 early breast cancer samples and 25 non-cancer samples from 25 patients. GSE139038 consists of 41 early breast cancer samples and 24 non-cancer samples. The characteristics of these patients are shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\" style=\"width: 991px;\" width=\"991\" height=\"654\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eA large number of high-throughput functional genomics studies have been collected in the GEO database. Use GEO2R to screen DEGs between breast cancer samples and non-cancer samples. Through online analysis of the GEPIA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn\u003c/span\u003e\u003c/span\u003e), the expression of DEGs genes in breast cancer tissues and normal tissues was screened. The breast cancer data set of Kaplan-Meier Plotter database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://kmplot.com/analysis/\u003c/span\u003e\u003c/span\u003e) was used to verify the relationship between OS, PFS and the expression of 33 DEGs online. Finally, it is determined that the target gene of the study is \u003cem\u003eEZH2\u003c/em\u003e. Use STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003c/span\u003e) to search for \u003cem\u003eEZH2\u003c/em\u003e gene related information and related proteins, and construct a network diagram of \u003cem\u003eEZH2\u003c/em\u003e gene related proteins, and finally through the TIMER database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cistrome.shinyapps.io/timer/\u003c/span\u003e\u003c/span\u003e) Used to analyze the correlation of \u003cem\u003eEZH2\u003c/em\u003e expression in B cells, CD8\u0026thinsp;+\u0026thinsp;T cells, CD4\u0026thinsp;+\u0026thinsp;T cells, macrophages, neutrophils, and dendritic cells in the microenvironment of breast cancer.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e1.2 GEPIA database search method\u003c/h2\u003e\n \u003cp\u003eGEPIA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia2.cancer-pku.cn\u003c/span\u003e\u003c/span\u003e) is a newly developed interactive web server for analyzing the RNA sequencing expression data of 9736 tumors and 8587 normal samples from the TCGA and the GTEx projects, using a standard processing pipeline. GEPIA provides customizable functions such as tumor/normal differential expression analysis, profiling according to cancer types or pathological stages, patient survival analysis, similar gene detection, correlation analysis, and dimensionality reduction analysis. This tool is developed by Zefang Tang, Chenwei Li, and Boxi Kang of Zhang Lab, Peking University, Beijing, China. Set the filter conditions as follows: select Expression on Box Plots; Gene: \u003cem\u003eEZH2\u003c/em\u003e, Log2 FC\u0026thinsp;\u0026ge;\u0026thinsp;1, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Cancer Type: BRCA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e1.3 Kaplan-Meier Plotter database analysis method\u003c/h2\u003e\n \u003cp\u003eUse an online tool, Kaplan-Meier plotter (KM plotter) to analyze the impact of selected DEGs on survival. This tool contains information on a large number of patients with different cancers. According to the individual expression of specific genes (high expression or low expression), breast cancer patients are divided into two groups. Obtain the operating system of breast cancer patients and draw it on the website, and display the hazard ratio and P value with 95% confidence interval on the graph.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e1.4 STRING database analysis method\u003c/h2\u003e\n \u003cp\u003eSTRING website can be used to analyze \u003cem\u003eEZH2\u003c/em\u003e gene related gene information and related protein functions, and construct \u003cem\u003eEZH2\u003c/em\u003e gene related protein network diagram through cytoscape. Use annotation, visualization and comprehensive discovery database (DAVID) to conduct GO and KEGG pathway enrichment analysis on the identified DEGs. Use p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 to distinguish significantly enriched genes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e1.5 TIMER database analysis method\u003c/h2\u003e\n \u003cp\u003eTIMER web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cistrome.shinyapps.io/timer/\u003c/span\u003e\u003c/span\u003e) is a comprehensive resource for systematical analysis of immune infiltrates across diverse cancer types. The abundance of six immune infiltrates (B cells, CD4\u0026thinsp;+\u0026thinsp;T cells, CD8\u0026thinsp;+\u0026thinsp;T cells, neutrophils, macrophages, and dendritic cells) is estimated by TIMER algorithm. TIMER web server allows users to input function-specific parameters, with resulting figures dynamically displayed to conveniently access the immunological, clinical, and genomic features of the tumor. Timer database can be used to analyze the immune infiltration of different types of cancer, and the screening conditions are as follows: (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e) gene symbol: \u003cem\u003eEZH2\u003c/em\u003e; (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e) cancer types: BRCA (breast invasive carcinoma); (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e) immune infiltrates: B cell CD8\u0026thinsp;+\u0026thinsp;T cell CD4\u0026thinsp;+\u0026thinsp;T cell macrophage peripheral dendritic cell. To verify the results in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e by TIMER2.0(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://timer.comp-genomics.org/\u003c/span\u003e\u003c/span\u003e), the screening conditions are as follows: (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e) cancer exploration; (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e) Gene_DE (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e) Gene Expression:\u003cem\u003eEZH2\u003c/em\u003e. Gene_DE module allows users to study the differential expression between tumor and adjacent normal tissues for any gene of interest across all TCGA tumors. Distributions of gene expression levels are displayed using box plots. The statistical significance computed by the Wilcoxon test is annotated by the number of stars (*: p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **: p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***: p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Users can identify genes that are up-regulated or down-regulated in the tumors compared to normal tissues for each cancer type, as displayed in gray columns when normal data are available.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e1.6 Statistical methods\u003c/h2\u003e\n \u003cp\u003eSPSS22.0 software was used for statistical analysis, t-test was used for comparison between breast cancer tissues and normal tissues, kaplan-Meier and log-rank test were used for survival analysis, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cstrong\u003e2.1 DEGS Screening\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 431, 842 and 2764 DEGS were identified from GSE39004, GSE109169 and GSE139038 data sets, respectively. A total of 189 genes were found in all three data sets. Interestingly, these gene expression trends were similar across the three data sets. \u0026nbsp;Among them, 33 genes were up-regulated in breast cancer compared with non-cancer samples (Fig.1A), and 156 genes were down-regulated (Fig.1B).In all three data sets it is defined by |logFC|\u0026gt;2.5 and P\u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Expression level of EZH2 mRNA in breast cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGEPIA database analysis showed that compared with normal tissues, expression level of EZH2 mRNA in breast cancer tissues was significantly increased (P\u0026lt;0.01, Fig. 2).Moreover, the expression profile data were analyzed, and the differentially expressed genes were screened out according to the screening criteria, and the volcanogram of differentially expressed genes in breast cancer tissue samples and normal tissue samples was drawn (Fig.3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Survival analysis results based on kaplan-Meierplotter database\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to further study the prognostic potential of\u0026nbsp;EZH2 in breast cancer, we used Kaplan-Meier plotter database to evaluate the prognostic value of EZH2.Interestingly, poor prognosis in breast cancer was associated with EZH2 expression, and the differences were statistically significant (Fig.4;P=0.015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 EZH2 gene-related protein network diagram and Enrichment Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe EZH2\u0026nbsp;gene-related protein network diagram was obtained through STRING analysis of 25 major EZH2 gene-related proteins BUB1B, CDC45, CDK1, etc. Cytoscape was used to construct a visualization network (Fig.5).The GO analysis of\u0026nbsp;EZH2 and the enrichment analysis of the KEGG pathway revealed that upregulated genes were mainly related to nuclear division, organel division and chromosome separation (Fig.6).In addition, up-regulated DEGs are related to KEGG pathways, including pi3K-Akt signaling pathway, MAPK signaling pathway, protein binding, cell cycle, etc. (Fig.7).Surprisingly, a wealth of pathways, such as extracellular matrix -receptor interactions, were observed in upregulated genes, leading to direct or indirect control of cellular activity, including adhesion, proliferation, and migration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 The correlation between the expression of EZH2 in breast cancer and the level of immune infiltration was studied using Timer database\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBy analyzing the relevant data obtained from the Timer database, we were pleasantly surprised to find that in the immune microenvironment of breast cancer, EZH2mRNA was significantly positively correlated with tumor purity, and the expressions of B cells, CD8+T cells, CD4+T cells, neutrophils and dendritic cells were significantly positively correlated (P \u0026lt; 0.05, Fig.8).In other words, breast cancer patients with high EZH2 expression may be more prone to immune cell infiltration, and thus may become a population with high response to immunotherapy. Of course, this is just a guess, and we\u0026apos;ll be testing this out in the real world with large clinical studies. In view of the differential expression of EZH2 between breast cancer tissue and normal tissue in Fig. 2, we used the timer database again to verify the conclusion, and the final conclusion was consistent with the previous one.(Fig.9)\u003c/p\u003e"},{"header":"3. Discussion ","content":"\u003cp\u003eRegardless of the progress of breast cancer treatment, breast cancer is still the leading cause of cancer-related death in women\u003csup\u003e20\u003c/sup\u003e . The high mortality rate of breast cancer is partly due to the lack of sufficient screening methods with high sensitivity and specificity for early breast cancer\u003csup\u003e21\u003c/sup\u003e. Therefore, it is imperative to identify potential biomarkers for early breast cancer screening. Fortunately, microarray technology and next-generation sequencing have become key tools in providing integrated genetic information on breast cancer samples and revealing detailed changes in disease progression. In the study, online bioinformatics methods were used to study biomarkers for early diagnosis of breast cancer. Three GEO datasets were downloaded from the website and identified 189 genomes, including 33 up-regulated genes. These upregulation genes are mainly involved in cell division, cell proliferation and signal transduction, and these pathways are closely related to cancer. In addition, of these DEGs, 37 genes showed the highest degree in the PPI network. Further survival analysis showed that \u003cem\u003eEZH2\u003c/em\u003e of these 37 modules was closely associated with shorter survival time in breast cancer patients.\u003c/p\u003e\n\u003cp\u003eStudies have shown that \u003cem\u003eEZH2\u003c/em\u003e is overexpressed in various types of cancer, including gastric, hepatocellular and lung cancers. In one study, \u003cem\u003eEZH2\u003c/em\u003e inhibition led to significant upregulation of MHCI class expression in human and mouse human papillomavirus negative head and neck cancer strains in vitro and in vivo in mouse models. Enhanced antigen presentation of \u003cem\u003eEZH2\u003c/em\u003e inhibitors or CRISPR-mediated \u003cem\u003eEZH2\u003c/em\u003e defects in tumor cells increases antigen-specific CD8+T cell proliferation, IFN production, and cytotoxicity in tumor cells. Mechanistically, \u003cem\u003eEZH2\u003c/em\u003e inhibition reduces histone H3K27me3 modification on -2-microglobulin promotors. Finally, in an anti-PD-1 drug resistant model of head and neck cancer, combination therapy inhibits tumor surveillance\u003csup\u003e22\u003c/sup\u003e.Another study analysis of immunohistochemical and multiple immunofluorescence staining of \u003cem\u003eEZH2\u003c/em\u003e and PD - L1 expression in liver cancer tissue and its correlation, in the evaluation of \u003cem\u003eEZH2\u003c/em\u003e expression of PD - L1 adjustment, finally found \u003cem\u003eEZH2\u003c/em\u003e can through CD274 directly raise liver cancer cells and IRF1 promoter H3K27me3 level to suppress the immune checkpoint inhibitors PD - the expression of L1, and may be immune activation HCC potential therapeutic targets for the treatment of combination therapy. Clinical samples from HCC patients with immune-activated microenvironment showed a negative correlation between \u003cem\u003eEZH2\u003c/em\u003e and PD-L1 expression in HCC cells. Multivariate Cox analysis showed that the combination of \u003cem\u003eEZH2\u003c/em\u003e and PD-L1 was an independent prognostic factor for OS and RFS in liver cancer patients\u003csup\u003e23\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAt present, for the treatment of breast cancer, PD-1/PD-L1 inhibitors have been in full blast in the study of breast cancer, especially TNBC, and the existing research results show that anti-PD-1 /PD-L1 therapy has a broad application prospect in neoadjuvant, adjuvant and combination therapy of TNBC. Based on the results of IMpassion130 study, FDA approved Atezolizumab combined with albumin binding paclitaxel for first-line treatment of metastatic or locally advanced PD-L1 positive TNBC\u003csup\u003e24\u003c/sup\u003e. At present, however, there is still a lack of effective immune markers to screen out the immune dominant population, and high, medium and low risk populations can be subdivided through the study of the threshold value of markers, so as to clarify the mechanism of immunotherapy. A particular concern of this study is that \u003cem\u003eEZH2\u003c/em\u003e expression is associated with the level of immune cell infiltration in breast cancer. By using the Timer database, we found that the expression level of \u003cem\u003eEZH2\u003c/em\u003e was positively correlated with the immune infiltration level of B cells, CD8+T cells, CD4+T cells, neutrophils and dendritic cells in breast cancer patients. With the in-depth study of tumor microenvironment, the presence of tumor infiltrating lymphocytes in breast cancer specimens has been proved to be an important predictive and prognostic factor. Emerging data also showed that MEK inhibition could up-regulate the expression of MHC and PD-L1 on the surface of TNBC cells in vivo and in vitro, so that the expression of PD-L1 could be higher in tumor cells and immune cells\u003csup\u003e25\u003c/sup\u003e. At present, \u003cem\u003eEZH2\u003c/em\u003e inhibitor \u0026ldquo;Tazemetostat\u0026rdquo; has shown excellent antitumor activity in the treatment of epithelial sarcoma and follicular lymphoma .Therefore, we have reason to believe that \u003cem\u003eEZH2\u003c/em\u003e will also become one of the major targets for the treatment of breast cancer. This may also indicate that \u003cem\u003eEZH2\u003c/em\u003e may be a potential target of immunotherapy for breast cancer, which is helpful to screen out the dominant population in clinical treatment.\u003c/p\u003e\n\u003cp\u003eOverall, breast cancer is the most common malignancy in women. It is often characterized by a lack of early symptoms, resulting in less early detection of the disease, while testing late in the disease means treatment is more difficult and uncertain. By comparing the three gene profiles, we found that \u003cem\u003eEZH2\u0026nbsp;\u003c/em\u003emay be the key gene associated with breast cancer. Bioinformatics comprehensive analysis is a powerful tool for biomarker screening. Interactions between selected proteins provide important information about breast cancer progression, as well as the underlying mechanisms of cancer occurrence, development, metastasis, and drug resistance. However, the predictive value of these markers should be confirmed in additional studies prior to clinical application. The underlying mechanisms of the activity of these genes also need to be elucidated, helping to map the predictive networks of these genes. We have optimized the parameters of the bioinformatics synthesis analysis to screen for potential biomarkers for early diagnosis of breast cancer and hope that our study will provide new methods for early diagnosis and prediction of breast cancer. In future studies, more attention should be paid to subclass analysis to elucidate the characteristics of each set of biomarkers. In our future study, we will recruit a group of breast cancer patients to study the sensitivity and specificity of these biomarkers in early screening of breast cancer patients, which may promote the clinical application of the biomarkers.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eEZH2, Enhancer of zeste homolog 2; OS, overall survival; DEG, differentially expressed genes; TNBC, triple negative breast cancer;PD-1, Programmed Death 1; PD-L1, Programmed Cell Death-Ligand 1;GO, gene ontology ;GEPIA, gene expression profiling interactive analysis ;TCGA, The Cancer Genome Atlas; TIMER, tumor immune evaluation resource\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData associated with the present paper can be obtained by contacting the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declares no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research has not been supported by relevant funds.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLei Sun contributed to the design and conception of the study. Lei Sun, Enwen Mao carried out data acquisition, analysis, and interpretation. Enwen Mao drafted the manuscript. Lei Sun oversaw critical revision of the manuscript for intellectual content.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmad, A. Breast Cancer Statistics: Recent Trends. \u003cem\u003eAdv Exp Med Biol\u003c/em\u003e \u003cstrong\u003e1152\u003c/strong\u003e, 1-7, doi:10.1007/978-3-030-20301-6_1 (2019).\u003c/li\u003e\n\u003cli\u003eLi, T., Mello-Thoms, C. \u0026amp; Brennan, P. C. 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Targeting EZH2 Enhances Antigen Presentation, Antitumor Immunity, and Circumvents Anti-PD-1 Resistance in Head and Neck Cancer. \u003cem\u003eClin Cancer Res\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 290-300, doi:10.1158/1078-0432.CCR-19-1351 (2020).\u003c/li\u003e\n\u003cli\u003eXiao, G.\u003cem\u003e et al.\u003c/em\u003e EZH2 negatively regulates PD-L1 expression in hepatocellular carcinoma. \u003cem\u003eJ Immunother Cancer\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 300, doi:10.1186/s40425-019-0784-9 (2019).\u003c/li\u003e\n\u003cli\u003eSchmid, P.\u003cem\u003e et al.\u003c/em\u003e Atezolizumab and Nab-Paclitaxel in Advanced Triple-Negative Breast Cancer. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e379\u003c/strong\u003e, 2108-2121, doi:10.1056/NEJMoa1809615 (2018).\u003c/li\u003e\n\u003cli\u003eLoi, S.\u003cem\u003e et al.\u003c/em\u003e RAS/MAPK Activation Is Associated with Reduced Tumor-Infiltrating Lymphocytes in Triple-Negative Breast Cancer: Therapeutic Cooperation Between MEK and PD-1/PD-L1 Immune Checkpoint Inhibitors. \u003cem\u003eClin Cancer Res\u003c/em\u003e\u003cstrong\u003e22\u003c/strong\u003e, 1499-1509, doi:10.1158/1078-0432.CCR-15-1125 (2016).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"breast cancer, EZH2, GEO database, early diagnosis, bioinformatics analysis","lastPublishedDoi":"10.21203/rs.3.rs-4781872/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4781872/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe breast cancer is still considered the second leading cause of cancer death in women, although in high-risk individuals through breast ultrasound, mammography and breast magnetic resonance tomography (such as the MRT) imaging screening in the early diagnosis of cancer, but still urgently needed for the diagnosis and treatment of early breast cancer, especially breast cancer to discover new biomarkers.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe gene chip data were retrieved from GEO database to screen the differentially expressed genes (DEG) in breast cancer.The expression of DEG was verified and the prognostic value was evaluated using GEPIA databases.The protein-protein interaction (PPI) network was plotted with STRING, and Cytoscape was used for cell map visualization. Timer database was used to analyze the correlation between DEGs and immune infiltration level in breast cancer, and Kaplan-Meier plotter was used to analyze the overall survival of the screened target gene in breast cancer patients.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe functions of target genes were mainly in cell division, cell proliferation and signal transduction. These highly expressed genes are associated with poor prognosis in breast cancer patients, among which we found that \u003cem\u003eEZH2\u003c/em\u003e is of great significance for the diagnosis and prognosis of early breast cancer. The enrichment analysis of its functions and pathways includes PI3K-AKT signaling pathway, MAPK signaling pathway, protein binding, cell cycle, etc.\u003cem\u003eEZH2\u003c/em\u003e was positively correlated with immune infiltration cells in patients with breast cancer.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEZH2\u003c/em\u003e in breast cancer may be a useful target for diagnosis and treatment of early breast cancer.\u003c/p\u003e","manuscriptTitle":"Bioinformatics analysis of predictive value of EZH2 in early breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-02 13:17:54","doi":"10.21203/rs.3.rs-4781872/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aaf54868-153a-4ce1-a846-81becee6fffe","owner":[],"postedDate":"September 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":36354937,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":36354938,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2024-09-19T04:17:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-02 13:17:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4781872","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4781872","identity":"rs-4781872","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}