Differential expression of COL6 family is a potential tumor marker for predicting gastric cancer

Differential expression of COL6 family is a potential tumor marker for predicting gastric 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 Research Article Differential expression of COL6 family is a potential tumor marker for predicting gastric cancer Lei Ma, Yanfeng Wang, Tao Li, Jun Ling, Bao-Zheng Wang, Wei-Ji Cai, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2739455/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 Gastric cancer (GC) is one of the most common cancers in the worldwide and is the leading cause of death in patients. Despite significant advances in treatment, the 5-year survival rate remains low. Therefore, the discovery of tumor markers that can predict gastric cancer and estimate its prognosis will improve this situation. Methods In this study, TIMER2, Kaplan–Meier plotter and GEPIA databases were used to analyze the expression of COL6 family gene in gastric cancer tissues, and analyzed their relationship with different pathological stages and tumor prognosis. Meanwhile, we analyzed the Genetic Alteration, Co-expression, Neighbor Gene Network, Interaction Analyses, enrichment analysis and immune infiltration analysis of COL6 in Patients with GC in cBioPortal, STRING databases and XIANTAO platform. Results We found that the COL6 family (COL6A1, COL1A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5 and COL6A6) genes were significantly up-regulated in gastric adenocarcinoma relative to normal tissue adjacent to the cancer. A significant correlation was found between the expression of COL6A1/2/3 and the pathological stage of GC patients. GC patients with high COL6A1/2/3/5/6 expression were all associated with poor survival, while COL6A4P1/P2/6 were significantly associated with metastasis or recurrence in patients. Subsequently, we performed functional enrichment analysis and found that its cellular function was associated with Focal adhesion, ECM-receptor interaction. Our data also showed that COL6A1/2/3/5/6 were positively with immune infiltration, while a negative correlation was found between COL6A/4P1/4P2 and immune cells infiltration, which may be associated with poorer relapse-free survival (RFS). Conclusion Our findings suggest that the COL6 family (COL6A1/2/3/4P1/4P2/5/6) is a potential biomarker for determining the prognosis of gastric cancer and It also provides theoretical basis for immunotherapy. COL6 family gastric cancer biomarker prognosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Gastric cancer (GC) is one of the most common malignant tumors of the digestive tract, and according to the latest statistics, it ranks fifth in the number of new cases and fourth in mortality worldwide[ 1 ]. The main reason for the short overall survival (OS) and the early occurrence of recurrence or metastasis in gastric cancer patients is that patients do not have specific early clinical manifestations are easily missed diagnosis[ 2 ]. In addition, the lack of sensitive and specific predictors for gastric cancer diagnosis is the fundamental reason why patients with gastric cancer cannot be diagnosed early. For patients with early gastric cancer, early diagnosis is an extremely important means to reduce the mortality rate. So far, the diagnosis of gastric cancer mainly relies on gastroscopic pathological biopsy, which is an invasive examination and it is difficult to perform large-scale pathological biopsy on the population. However, there are no specific clinical manifestations and sensitive diagnostic methods in the early stage of gastric cancer, most patients are already in the advanced stage when they are diagnosed, and the optimal treatment time is missed. As a result, the 5-year survival rate of gastric cancer patients remains poor [ 3 – 5 ]. However, early diagnosis and treatment of gastric cancer can extend the survival time and improve the quality of life of patients[ 6 , 7 ]. Although patients with progressive gastric cancer still account for a large proportion of patients, some progress has been made in treatments such as radiotherapy, chemotherapy, and neoadjuvant chemotherapy, surgical resection remains the best treatment for gastric cancer at present[ 8 ]. Therefore, finding biomarkers with high specificity and sensitivity is important for the early diagnosis and prognosis of gastric cancer. By utilizing bioinformatics techniques, we can screen for key genes associated with diseases that are now widely used in clinical disease research [ 9 , 10 ]. With the rapid development of second-generation gene sequencing technology and the establishment of various databases, using bioinformatics to further analyze the available data resources has become a hot research topic in cancer and bioinformatics, based on which novel clinical indicators that can predict the prognostic survival of patients are provided. In this study, we analyzed the expression of the COL6 family (COL6A1/2/3/4P1/4P2/5/6) in gastric cancer with several large public databases and evaluated its potential application as a prognostic marker and therapeutic target. Additional data for clinicians to determine the prognostic survival of patients with gastric cancer. Methods TIMER2 TIMER2 (tumor immune estimation resource, version 2, timer.cistrome.org ) is a reliable, intuitive tool that provides systematic evaluations of the infiltration of different immune cells and their clinical impact[ 11 ]. In our study, we used TIMER2 to analyze COL6 (COL6A1/2/3/4P1/4P2/5/6) in 33 types of tumor tissues and adjacent normal tissues gene expression, and we used the TCGA database to analyze genes that were not found expressed in tumor tissue and adjacent normal tissues. GEPIA2 The GEPIA2 (Gene Expression Profiling Interactive Analysis, version 2) tool ( http://gepia.cancer-pku.cn/index.html ) is a web-based tool that provides key interactive and customizable features based on cancer genome atlas (TCGA) and genotypic tissue expression (GTEx) data [ 12 ]. In this study, we analyzed the expression of COL6A1, COL6A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5 and COL6A6A6 across different pathological stages of TCGA cancer using the HEPIA2 tool. Apply the expression data of the log2 [TPM (+ 1 per million transcripts)] conversion to a box diagram or violin diagram. Survival Analysis Kaplan-Meier plotter ( http://kmplot.com/analysis/ ) was used to analyze the effects of key genes screened for this study on [ 13 ] overall survival (OS) and recurrence-free survival (RFS) in patients with gastric cancer. Patient samples were divided into high-expression and low-gene-expressed groups and assessed using the Kaplan-Meier survival chart, showing a 95% confidence interval for risk ratio (HR) and significance difference value (log rankP) based on time series testing (log rank test). cBioPortal cBioPortal ( www.cbioportal.org ) was a comprehensive web resource, can visualize and analyze multidimensional cancer genomics data [ 14 ]. Based on the TCGA database, the gene modification, co-expression, and network modules of COL6A1, COL6A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5, and COL6A6 were obtained from cBioPortal. Four hundred and seventy-eight stomach carcinoma samples (TCGA, Firehose Legacy) were analyzed. STRING The purpose of String ( https://string-db.org/ ) is to collect, score, and integrate all publicly available sources of protein interaction (PPI) data, supplementing these data with computational predictions of potential functions [ 15 ]. We performed PPI network analysis of differentially expressed COL6A1, COL6A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5, and COL6A6 to explore their interactions with STRING. XIANTAO Gene enrichment analysis of Kyoto Encyclopedia of Genes was performed on the entire transcriptomic gene expression dataset (407 GC patients) of the TCGA project using the XIANTAO platform ( www.xiantao.love ), followed by an assessment of tumor immune infiltration. A total of 24 immune markers were used to distinguish between different immune cells. Results Up-Regulated Expression of COL6A1/2/3/4P1/4P2/5/6 in STAD We first examined the expression levels of COL6A1/2/3/4P2/6 in several tumor tissues in TIMER2. We subsequently analyzed the expression of COL6A4P1 and COL6A5 in gastric cancer patients using the TCGA database. The analysis showed that the expression of COL6A1/2/3/4P1/4P2/5/6 was significantly higher in GC than in non-tumor tissues (Figure 1) . The Prognostic Value of COL6A1/2/3/4P1/4P2/5/6 and Related Clinic-Pathological Characters We then assessed the correlation between the expression of differentially expressed COL6A1, COL6A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5, and COL6A6 and the pathological stage of GC patients and found a significant correlation between the expression of COL6A1 (p = 0.000367), COL6A2 (p = 0.00017), COL6A3 (p = 3.44e-06), COL6A4P1 (p = 0.864), COL6A4P2 (p = 0.355), COL6A5 (p = 0.187) and COL6A6 (p = 0.321), and pathological stage (Figure 2) . As the tumor progressed, the expression of COL6A1, COL6A2, and COL6A3 increased. These data suggest that COL6A1, COL6A2, and COL6A3 play a significant role in the tumorigenesis and progression of GC. The Prognostic Value of COL6 in Patients With GC To assess the value of differentially expressed COL6 genes in gastric cell carcinoma progression, we evaluated the correlation between differentially expressed COL6 genes and clinical outcomes using Kaplan-Meier plotter. Based on our data, it can be seen that patients with gastric carcinoma presented with lower transcript levels of COL6A1 (p=0.047), COL6A2 (p=0.017), COL6A3 (p=0.05), COL6A5 (p=0.028) and COL6A6 (p=0.025) were significantly associated with longer overall survival (Figure 3) . We also evaluated the value of differentially expressed COL6 in the progression-free survival of STAD patients. We found that patients with STAD with lower levels of COL6A4P1 (p=0.006), COL6A4P2 (p=0.0091), and COL6A6 (p=0.002) transcripts were significantly associated with longer recurrence-free survival (Figure 4) . Genetic Alteration, Co-expression, Neighbor Gene Network, and Interaction Analyses of COL6 in Patients With GC We performed a comprehensive analysis of the molecular characterization of differentially expressed COL6A1/2/3/4P1/4P2/5/6. The provisional dataset of TCGA was used to analyze the genetic alterations of differentially expressed COL6A1/2/3/4P1/4P2/5/6. Query GC samples for COL6 members were altered in 4,7,11,0.7,0.9,2.9, and 8% of samples, respectively (Figure 5A) . An enhancement of mRNA expression was the most prevalent alteration in these samples. Next, we explored the potential co-expression of differentially expressed COL6A1, COL1A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5, and COL6A6. COL6A1, COL6A2, COL6A3, COL6A5, and COL6A6 expression all showed moderate to high correlation (Figure 5B). Then, we performed PPI network analysis of differentially expressed COL6A1, COL6A2, COL6A3, COL6A5, and COL6A6 (Figure 5C-D) to explore their potential interactions. As we anticipated, there is some kind of correlation between them. Functional Enrichment Analysis of COL6 in Patients With GC The functions of differentially expressed COL6A1/2/3/4P1/4P2/5/6 were analyzed using the XIANTAO platform. The figure (Figure 6) shows the 9 most abundant GO items. Among the 3 most abundant functions in the BP class, chondrocyte morphogenesis, growth plate cartilage chondrocyte morphogenesis, chondrocyte morphogenesis involved in sarcolemma, collagen-containing extracellular matrix, and collagen trimer were the three most abundant items in the CC category. Among the molecular functional MF categories, differentially expressed COL6A1/2/3/4P1/4P2/5/6 were mainly enriched in platelet-derived growth and extracellular matrix structural constituent, extracellular matrix structural constituent. KEGG pathway analysis was also performed, and focal adhesion, protein digestion and absorption, ECM-receptor interaction were significantly associated with the development of gastric cell carcinoma. Immune Cell Infiltration of COL6 in Patients With GC To investigate whether COL6 members are involved in inflammatory response and immune cell infiltration, thus affecting the clinical prognosis of gastric cancer patients. Therefore, we explored the correlation between differentially expressed COL6A1/2/3/4P1/4P2/5/6 and immune cell infiltration using the XIANTAO platform. It can be seen that COL6A1/2/3/5/6 showed a positive correlation with most immune cells, while a negative correlation with a few immune cells. Interestingly, COL6A4P1/P2, on the other hand, showed a negative correlation with the majority of immune cells and a positive correlation with a minority of immune cells (Figure 7) . Discussion In recent years, efforts have been made to better understand the early diagnosis and early treatment of gastric cancer, thus helping to further improve the survival time and prognosis of patients. At present, the treatment of gastric cancer is mainly surgical, but many patients are found to have already reached in the middle or late stages, and there is little improvement in patient OS, especially in developing countries like China, where there is an urgent need to discover substances that can be highly expressed in GC to predict tumorigenesis, and development. According to existing reports, collagen genes are involved in tumor extracellular matrix (ECM) receptor interactions and adhesion plaque pathways and played a key role in tumor invasion and metastasis [ 16 , 17 ], which is consistent with our present study. And the ECM is closely related to the tumor microenvironment (TME), which further enhances the progression and / or resistance to the treatment [ 18 ]. The prognostic value of COL genes for a variety of malignancies has been confirmed by several authors. The results of RanAo showed that COL1A2, COL6A3 and THBS2 gene silencing inhibited the proliferation, migration and invasion of gastric cancer cells [ 19 ]. Svoronos found that COL6A3 could be independently used as a prognostic indicator for pancreatic cancer[ 20 ]. Hanlin demonstrated that COL6A1 was identified as a key gene associated with anti-VEGF therapy and may provide new therapeutic ideas for targeted treatment of glioblastoma [ 21 ]. Dang’s study firstly found that COL6A4P2 gene polymorphism was associated with lung cancer risk in Chinese population[ 22 ]. USP3 promotes gastric cancer progression and metastasis by deubiquitinating COL9A3 and COL6A5 [ 23 ]. COL6A6 has also been found to be a promoter gene in the lung [ 24 ], breast [ 25 ], and ovarian cancers [ 26 ]. However, there are few reports on the progress of studies on COL6A4P1, COL6A4P2, COL6A6 in gastric cancer. Therefore, we performed a systematic analysis of the genes in the COL6 family this time to identify the driver genes among them that can predict tumorigenesis. We first explored the expression of COL6 members and their correlation with the pathological stage of gastric cell carcinoma. We found that all members of the COL6 family were highly expressed in gastric cancer tissues, expression of COL6A1, COL6A2, COL6A3 and COL6A6 increased with increasing tumor number. High expression of COL6A1, COL6A2, COL6A3, COL6A5 and COL6A6 in GC patients was significantly associated with poorer overall survival, while low expression of COL6A4P1, COL6A4P2 and COL6A6 was associated with better progression-free survival in GC patients. These data suggest that the differentially expressed COL6 family may play an important role in gastric cell carcinoma. GAO found that high expression of COL6A1, COL6A2, and COL6A3 was associated with poorer prognosis in GC patients[ 27 ], but this time we found that COL6A1, COL6A2, COL6A3, COL6A5, and COL6A6 were associated with each other closely, but how they interact with each other needs to be verified in the next experiments. The development and progression of gastric cancer are complex and multifaceted, and based on currently known reports genetic alterations have been found to play a crucial role in this process [ 27 ]. Since COL6 members are significantly differentially expressed in gastric cancer, we subsequently explored their genetic mutations in gastric cancer. It was found that the differentially expressed COL6A1, COL6A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5 and COL6A6 had a certain mutation rate in gastric cancer tissues. Subsequently, we found different degrees of correlation among the COL6 family, which predicts that these genes play a certain synergistic role in the development of gastric cell carcinoma. We then investigated the function of the differentially expressed COL6 family using graphene oxide enrichment analysis and KEGG pathway enrichment analysis. As the results demonstrate, we found that the functions of these genes were mainly related to extracellular matrix structure, focal adhesion, and ECM-receptor interactions. Previous studies have shown that ECM had multiple effects on lymphocytes [ 28 ] exerting some influence on lymphocyte migration, recognition, activation and differentiation, and thus multiple roles in the function of the immune system in vivo, and we speculated that the COL6 family may influence tumor development by regulating immune cells. Whereas, the importance of focal adhesion in cancer cell adhesion, motility, proliferation, and survival and overexpression in cancer cells [ 29 ], and the combination of Focal adhesion kinase (FAK) inhibitors with chemotherapy, radiotherapy, and immunotherapy is expected to prolong the survival of patients [ 30 , 31 ]. These data suggest that the COL6 family, which is differentially expressed in gastric cell carcinoma, is a potential target for drug therapy. Current known evidence suggests that immune cell infiltration can influence tumor progression and recurrence, which in turn affects overall patient survival and progression-free survival [ 32 , 33 ]. In this study, we found that COL6 family expression was correlated to varying degrees for all 24 immune cells, with COL6A1/2/3/5/6 positively regulating most immune cells and COL6A4P1/P2 negatively regulating most immune cells, which also suggest that high COL6A4P1/P2 expression decreases immune This suggests that the COL6 family can not only be used as a prognostic indicator, but may also reflect the immune status. However, other independent cohort study and in vitro or in vivo studies are needed to validate our results. Conclusion In this study, we used bioinformatics analysis to explore the role of COL6 family genes in gastric cancer. We performed a major tumor informatics analysis to explore the expression profile of COL6 gene in GC and various clinicopathological parameters. Extensive data mining from various publicly available databases revealed that COL6 expression was upregulated in various GC corresponding to their normal tissues. In addition, survival map analysis, PPIs, co-expression analysis, functional analysis, and immune infiltration analysis all suggest that COL6 has significant prognostic and clinicopathological value in gastric cancer. Therefore, the COL6 family may have potential functions as prognostic biomarkers or tumor markers, and also provide theoretical basis for immunotherapy of tumors. Declarations Ethics approval Ethical approval is not required for the current study due to the nature of the current study. Consent for publication Not applicable. Availability of data and material The data that support the findings of this study are openly available in TIMER2(tumor immune estimation resource, version 2) at timer.cistrome.org ; Kaplan–Meier plotter at http://kmplot.com/analysis/ ; the GEPIA2 at http://gepia.cancer-pku.cn/index.html; cBioPortal at www.cbioportal.org ; String at https://string-db.org/; the XIANTAO platform at www.xiantao.love. Competing interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Acknowledgements The authors would like to thank Dr. Qilun Liu, Dr. Jing Chen, and Dr. Tao Li for critical reading of the manuscript. We would particularly like to acknowledge my group team members for their wonderful collaboration and patient support. Authors' contributions Q.L.Liu , B.Z.Wang and W.J.Cai provised of study materials or patients. J.Chen, T.Li and Q.L.Liu administrated the support. L.Ma, J.Chen, Y.F.Wang, and J.Lin collected and assembled of data. L.Ma, J.Chen Q.L.Liu and L.H.Qi analysed the data and interpretation. L.Ma and Y.F.Wang wrote the main manuscript text. J.Chen, T.Li and Q.L.Liu reviewed, supervised and administrated this project. All authors reviewed the manuscript. Funding This work was funded by Key Research and Development Program of Ningxia (2021BEGO3087), Natural Science Foundation of Ningxia Province (2020AAC03403, 2020AAC03178). References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-49.http://dx.doi.org/10.3322/caac.21660 Necula L, Matei L, Dragu D, Neagu AI, Mambet C, Nedeianu S, et al. Recent advances in gastric cancer early diagnosis. 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The prognostic landscape of tumor-infiltrating immune cell and immunomodulators in lung cancer. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2017;95:55-61.http://dx.doi.org/10.1016/j.biopha.2017.08.003 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-2739455","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":189398695,"identity":"1aa3b5ca-dda2-4e2a-bf61-e21ed6005d9e","order_by":0,"name":"Lei Ma","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Ma","suffix":""},{"id":189398696,"identity":"b569f4e6-6f90-4188-a7e3-08ebb690e13a","order_by":1,"name":"Yanfeng Wang","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yanfeng","middleName":"","lastName":"Wang","suffix":""},{"id":189398699,"identity":"4233daca-87df-447f-8a30-6115275cee7b","order_by":2,"name":"Tao Li","email":"","orcid":"","institution":"The General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Li","suffix":""},{"id":189398700,"identity":"05762179-583b-41ed-a203-6b4b755cc958","order_by":3,"name":"Jun Ling","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Ling","suffix":""},{"id":189398702,"identity":"1c3952f8-5e89-4723-aed1-b28dfea0bd56","order_by":4,"name":"Bao-Zheng Wang","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bao-Zheng","middleName":"","lastName":"Wang","suffix":""},{"id":189398704,"identity":"4a70b73e-72f2-417e-a953-8c09a766b933","order_by":5,"name":"Wei-Ji Cai","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei-Ji","middleName":"","lastName":"Cai","suffix":""},{"id":189398706,"identity":"143d6aef-7c6b-40f9-850a-74f3fd6d80c1","order_by":6,"name":"Lihua Qi","email":"","orcid":"","institution":"The General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lihua","middleName":"","lastName":"Qi","suffix":""},{"id":189398707,"identity":"dd112e1f-aec8-4906-9866-ed446f7f5a87","order_by":7,"name":"Jing Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYDACCRA2YGBgY+//+OCDgY0d8Vr4eA4YG84oSEsmTgsIyEkkmEnzfDjE2EBIB//s5mMPLAru2LVJJKRJ2xgcYGZgP3x0A15L7hxLN5AweJbcxvPgsHWOwR0+Bp60tBv4tBhI5JhJSBgcTmZjT2y8nWPwjJlBgseMgJb8bxAtDMkM0hYGhxkbCGvJYQNpsWPjSGOSZiBGi8SNNLDDEth4zjAb9hikJbMR8gv/jORn0hJ/DtvLt/cwPvjxx8aOn/3wMbxaQIAZGDeJDTAeGyHlIMD4gYHBnhiFo2AUjIJRMEIBAOnqRZ6u4d3GAAAAAElFTkSuQmCC","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jing","middleName":"","lastName":"Chen","suffix":""},{"id":189398709,"identity":"12c135a7-9f19-4ffe-98e9-c4c8d31aaf39","order_by":8,"name":"Qilun Liu","email":"","orcid":"","institution":"The General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qilun","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2023-03-27 00:44:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2739455/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2739455/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":35451280,"identity":"27a97e1a-098b-4e19-9de5-3904c3c7e856","added_by":"auto","created_at":"2023-04-07 14:41:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":978844,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression levels of COL6 members in several tumor tissues （TIMER2）. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05；**\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01；***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001. (A) COL6A1, (B) COL6A2, (C) COL6A3, (D) COL6A4P1, (E) COL6A4P2, (F) COL6A5, (G) COL6A6.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2739455/v1/f4dd96ee2c8d7ceab470c23c.jpg"},{"id":35449939,"identity":"5802f712-2825-4caf-ab2a-92f65fc27677","added_by":"auto","created_at":"2023-04-07 14:33:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":555500,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between different expressed COL6 members and the pathological stage of GC patients (GEPIA). (A) COL6A1, (B) COL6A2, (C) COL6A3, (D) COL6A4P1, (E) COL6A4P2, (F) COL6A5, (G) COL6A6.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2739455/v1/de47f18626c515451010ce8c.jpg"},{"id":35449945,"identity":"2ac94e97-cbd2-40cc-b6cd-7c6e87588322","added_by":"auto","created_at":"2023-04-07 14:33:36","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":562018,"visible":true,"origin":"","legend":"\u003cp\u003eThe prognostic value of COL6 members in GC patients in the overall survival curve (Kaplan–Meier plotter). The overall survival curve of (A) COL6A1, (B) COL6A2, (C) COL6A3, (D) COL6A4P1, (E) COL6A4P2, (F) COL6A5, (G) COL6A6 in GC.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2739455/v1/babb7dc2fdab4c732650379e.jpg"},{"id":35451279,"identity":"faeb2efc-08cf-473b-8e9c-0b6f8a16b8bb","added_by":"auto","created_at":"2023-04-07 14:41:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":544965,"visible":true,"origin":"","legend":"\u003cp\u003eThe prognostic value of different expressed COL6 members in GC patients in the disease-free survival curve (Kaplan–Meier plotter). The disease-free survival curve of (A) COL6A1, (B) COL6A2, (C) COL6A3, (D) COL6A4P1, (E) COL6A4P2, (F) COL6A5, (G) COL6A6 in GC.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2739455/v1/bf5be90d437bb7c0e9f40436.jpg"},{"id":35449943,"identity":"486f5afe-c885-4a70-967c-618669d449f6","added_by":"auto","created_at":"2023-04-07 14:33:36","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":649543,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic alteration, neighbor gene network, and interaction analyses of different expressed COL6 members in GC patients. (A) Summary of alterations in different expressed COL6 members in GC. (B) Correlation heat map of different expressed COL6 members in GC. (C, D) Protein–protein interaction network of different expressed COL6 members.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2739455/v1/3fac692e864bddca85c29077.jpg"},{"id":35449942,"identity":"eabce430-1a77-47cd-b66e-f2dde9bbe7ce","added_by":"auto","created_at":"2023-04-07 14:33:36","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":619559,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG bubble of mutant genes of COL6 members. KEGG, Kyoto Encyclopedia of Genes and Genomes.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2739455/v1/4a0b26e1c08e61b50a6eaf92.jpg"},{"id":35451281,"identity":"50ee881e-e7a7-4454-b0a0-544a05d619be","added_by":"auto","created_at":"2023-04-07 14:41:36","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":863540,"visible":true,"origin":"","legend":"\u003cp\u003eThe correlation between different expressed COL6 members and immune cell infiltration (XIANTAO). The correlation between the abundance of immune cell and the expression of (A) COL6A1, (B) COL6A2, (C) COL6A3, (D) COL6A4P1, (E) COL6A4P2, (F) COL6A5, (G) COL6A6 in GC.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2739455/v1/f9dedcaebc0173733df19478.jpg"},{"id":39290779,"identity":"4a261842-344a-4275-8c3b-b88506474791","added_by":"auto","created_at":"2023-06-29 10:59:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1135252,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2739455/v1/a4187bd0-bdd6-4031-85df-ca5b7a70a4ea.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Differential expression of COL6 family is a potential tumor marker for predicting gastric cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGastric cancer (GC) is one of the most common malignant tumors of the digestive tract, and according to the latest statistics, it ranks fifth in the number of new cases and fourth in mortality worldwide[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The main reason for the short overall survival (OS) and the early occurrence of recurrence or metastasis in gastric cancer patients is that patients do not have specific early clinical manifestations are easily missed diagnosis[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In addition, the lack of sensitive and specific predictors for gastric cancer diagnosis is the fundamental reason why patients with gastric cancer cannot be diagnosed early. For patients with early gastric cancer, early diagnosis is an extremely important means to reduce the mortality rate. So far, the diagnosis of gastric cancer mainly relies on gastroscopic pathological biopsy, which is an invasive examination and it is difficult to perform large-scale pathological biopsy on the population. However, there are no specific clinical manifestations and sensitive diagnostic methods in the early stage of gastric cancer, most patients are already in the advanced stage when they are diagnosed, and the optimal treatment time is missed. As a result, the 5-year survival rate of gastric cancer patients remains poor [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, early diagnosis and treatment of gastric cancer can extend the survival time and improve the quality of life of patients[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Although patients with progressive gastric cancer still account for a large proportion of patients, some progress has been made in treatments such as radiotherapy, chemotherapy, and neoadjuvant chemotherapy, surgical resection remains the best treatment for gastric cancer at present[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, finding biomarkers with high specificity and sensitivity is important for the early diagnosis and prognosis of gastric cancer.\u003c/p\u003e \u003cp\u003eBy utilizing bioinformatics techniques, we can screen for key genes associated with diseases that are now widely used in clinical disease research [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. With the rapid development of second-generation gene sequencing technology and the establishment of various databases, using bioinformatics to further analyze the available data resources has become a hot research topic in cancer and bioinformatics, based on which novel clinical indicators that can predict the prognostic survival of patients are provided. In this study, we analyzed the expression of the COL6 family (COL6A1/2/3/4P1/4P2/5/6) in gastric cancer with several large public databases and evaluated its potential application as a prognostic marker and therapeutic target. Additional data for clinicians to determine the prognostic survival of patients with gastric cancer.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eTIMER2\u003c/h2\u003e\n\u003cp\u003eTIMER2 (tumor immune estimation resource, version 2, timer.cistrome.org ) is a reliable, intuitive tool that provides systematic evaluations of the infiltration of different immune cells and their clinical impact[\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. In our study, we used TIMER2 to analyze COL6 (COL6A1/2/3/4P1/4P2/5/6) in 33 types of tumor tissues and adjacent normal tissues gene expression, and we used the TCGA database to analyze genes that were not found expressed in tumor tissue and adjacent normal tissues.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eGEPIA2\u003c/h2\u003e\n\u003cp\u003eThe GEPIA2 (Gene Expression Profiling Interactive Analysis, version 2) tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/index.html\u003c/span\u003e\u003c/span\u003e) is a web-based tool that provides key interactive and customizable features based on cancer genome atlas (TCGA) and genotypic tissue expression (GTEx) data [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. In this study, we analyzed the expression of COL6A1, COL6A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5 and COL6A6A6 across different pathological stages of TCGA cancer using the HEPIA2 tool. Apply the expression data of the log2 [TPM (+\u0026thinsp;1 per million transcripts)] conversion to a box diagram or violin diagram.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch2\u003eSurvival Analysis\u003c/h2\u003e\n\u003cp\u003eKaplan-Meier plotter (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://kmplot.com/analysis/\u003c/span\u003e\u003c/span\u003e) was used to analyze the effects of key genes screened for this study on [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e] overall survival (OS) and recurrence-free survival (RFS) in patients with gastric cancer. Patient samples were divided into high-expression and low-gene-expressed groups and assessed using the Kaplan-Meier survival chart, showing a 95% confidence interval for risk ratio (HR) and significance difference value (log rankP) based on time series testing (log rank test).\u003c/p\u003e\n\u003ch2\u003ecBioPortal\u003c/h2\u003e\n\u003cp\u003ecBioPortal (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://gepia.cancer-pku.cn/index.html\" target=\"_blank\"\u003ewww.cbioportal.org\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e) was a comprehensive web resource, can visualize and analyze multidimensional cancer genomics data [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. Based on the TCGA database, the gene modification, co-expression, and network modules of COL6A1, COL6A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5, and COL6A6 were obtained from cBioPortal. Four hundred and seventy-eight stomach carcinoma samples (TCGA, Firehose Legacy) were analyzed.\u003c/p\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eSTRING\u003c/h2\u003e\n\u003cp\u003eThe purpose of String (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003c/span\u003e) is to collect, score, and integrate all publicly available sources of protein interaction (PPI) data, supplementing these data with computational predictions of potential functions [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. We performed PPI network analysis of differentially expressed COL6A1, COL6A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5, and COL6A6 to explore their interactions with STRING.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eXIANTAO\u003c/h2\u003e\n\u003cp\u003eGene enrichment analysis of Kyoto Encyclopedia of Genes was performed on the entire transcriptomic gene expression dataset (407 GC patients) of the TCGA project using the XIANTAO platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://gepia.cancer-pku.cn/index.html\" target=\"_blank\"\u003ewww.xiantao.love\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e), followed by an assessment of tumor immune infiltration. A total of 24 immune markers were used to distinguish between different immune cells.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eUp-Regulated Expression of COL6A1/2/3/4P1/4P2/5/6 in STAD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe first examined the expression levels of COL6A1/2/3/4P2/6 in several tumor tissues in TIMER2. We subsequently analyzed the expression of COL6A4P1 and COL6A5 in gastric cancer patients using the TCGA database. The analysis showed that the expression of COL6A1/2/3/4P1/4P2/5/6 was significantly higher in GC than in non-tumor tissues \u003cstrong\u003e(Figure 1)\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Prognostic Value of COL6A1/2/3/4P1/4P2/5/6 and Related Clinic-Pathological Characters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then assessed the correlation between the expression of differentially expressed COL6A1, COL6A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5, and COL6A6 and the pathological stage of GC patients and found a significant correlation between the expression of COL6A1 (p = 0.000367), COL6A2 (p = 0.00017), COL6A3 (p = 3.44e-06), COL6A4P1 (p = 0.864), COL6A4P2 (p = 0.355), COL6A5 (p = 0.187) and COL6A6 (p = 0.321), and pathological stage \u003cstrong\u003e(Figure 2)\u003c/strong\u003e. As the tumor progressed, the expression of COL6A1, COL6A2, and COL6A3 increased. These data suggest that COL6A1, COL6A2, and COL6A3 play a significant role in the tumorigenesis and progression of GC. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Prognostic Value of COL6 in Patients With GC \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the value of differentially expressed COL6 genes in gastric cell carcinoma progression, we evaluated the correlation between differentially expressed COL6 genes and clinical outcomes using Kaplan-Meier plotter. Based on our data, it can be seen that patients with gastric carcinoma presented with lower transcript levels of COL6A1 (p=0.047), COL6A2 (p=0.017), COL6A3 (p=0.05), COL6A5 (p=0.028) and COL6A6 (p=0.025) were significantly associated with longer overall survival \u003cstrong\u003e(Figure 3)\u003c/strong\u003e. We also evaluated the value of differentially expressed COL6 in the progression-free survival of STAD patients. We found that patients with STAD with lower levels of COL6A4P1 (p=0.006), COL6A4P2 (p=0.0091), and COL6A6 (p=0.002) transcripts were significantly associated with longer recurrence-free survival \u003cstrong\u003e(Figure 4)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic Alteration, Co-expression, Neighbor Gene Network, and Interaction Analyses of COL6 in Patients With GC \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe performed a comprehensive analysis of the molecular characterization of differentially expressed COL6A1/2/3/4P1/4P2/5/6. The provisional dataset of TCGA was used to analyze the genetic alterations of differentially expressed COL6A1/2/3/4P1/4P2/5/6. Query GC samples for COL6 members were altered in 4,7,11,0.7,0.9,2.9, and 8% of samples, respectively \u003cstrong\u003e(Figure 5A)\u003c/strong\u003e. An enhancement of mRNA expression was the most prevalent alteration in these samples. Next, we explored the potential co-expression of differentially expressed COL6A1, COL1A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5, and COL6A6. COL6A1, COL6A2, COL6A3, COL6A5, and COL6A6 expression all showed moderate to high correlation \u003cstrong\u003e(Figure 5B).\u003c/strong\u003e Then, we performed PPI network analysis of differentially expressed COL6A1, COL6A2, COL6A3, COL6A5, and COL6A6\u003cstrong\u003e (Figure 5C-D)\u003c/strong\u003e to explore their potential interactions. As we anticipated, there is some kind of correlation between them.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional Enrichment Analysis of COL6 in Patients With GC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe functions of differentially expressed COL6A1/2/3/4P1/4P2/5/6 were analyzed using the XIANTAO platform. The figure \u003cstrong\u003e(Figure 6) \u003c/strong\u003eshows the 9 most abundant GO items. Among the 3 most abundant functions in the BP class, chondrocyte morphogenesis, growth plate cartilage chondrocyte morphogenesis, chondrocyte morphogenesis involved in sarcolemma, collagen-containing extracellular matrix, and collagen trimer were the three most abundant items in the CC category. Among the molecular functional MF categories, differentially expressed COL6A1/2/3/4P1/4P2/5/6 were mainly enriched in platelet-derived growth and extracellular matrix structural constituent, extracellular matrix structural constituent. KEGG pathway analysis was also performed, and focal adhesion, protein digestion and absorption, ECM-receptor interaction were significantly associated with the development of gastric cell carcinoma.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmune Cell Infiltration of COL6 in Patients With GC \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether COL6 members are involved in inflammatory response and immune cell infiltration, thus affecting the clinical prognosis of gastric cancer patients. Therefore, we explored the correlation between differentially expressed COL6A1/2/3/4P1/4P2/5/6 and immune cell infiltration using the XIANTAO platform. It can be seen that COL6A1/2/3/5/6 showed a positive correlation with most immune cells, while a negative correlation with a few immune cells. Interestingly, COL6A4P1/P2, on the other hand, showed a negative correlation with the majority of immune cells and a positive correlation with a minority of immune cells \u003cstrong\u003e(Figure 7)\u003c/strong\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, efforts have been made to better understand the early diagnosis and early treatment of gastric cancer, thus helping to further improve the survival time and prognosis of patients. At present, the treatment of gastric cancer is mainly surgical, but many patients are found to have already reached in the middle or late stages, and there is little improvement in patient OS, especially in developing countries like China, where there is an urgent need to discover substances that can be highly expressed in GC to predict tumorigenesis, and development. According to existing reports, collagen genes are involved in tumor extracellular matrix (ECM) receptor interactions and adhesion plaque pathways and played a key role in tumor invasion and metastasis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], which is consistent with our present study. And the ECM is closely related to the tumor microenvironment (TME), which further enhances the progression and / or resistance to the treatment [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The prognostic value of COL genes for a variety of malignancies has been confirmed by several authors. The results of RanAo showed that COL1A2, COL6A3 and THBS2 gene silencing inhibited the proliferation, migration and invasion of gastric cancer cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Svoronos found that COL6A3 could be independently used as a prognostic indicator for pancreatic cancer[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Hanlin demonstrated that COL6A1 was identified as a key gene associated with anti-VEGF therapy and may provide new therapeutic ideas for targeted treatment of glioblastoma [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Dang\u0026rsquo;s study firstly found that COL6A4P2 gene polymorphism was associated with lung cancer risk in Chinese population[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. USP3 promotes gastric cancer progression and metastasis by deubiquitinating COL9A3 and COL6A5 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. COL6A6 has also been found to be a promoter gene in the lung [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], breast [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and ovarian cancers [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, there are few reports on the progress of studies on COL6A4P1, COL6A4P2, COL6A6 in gastric cancer. Therefore, we performed a systematic analysis of the genes in the COL6 family this time to identify the driver genes among them that can predict tumorigenesis.\u003c/p\u003e \u003cp\u003eWe first explored the expression of COL6 members and their correlation with the pathological stage of gastric cell carcinoma. We found that all members of the COL6 family were highly expressed in gastric cancer tissues, expression of COL6A1, COL6A2, COL6A3 and COL6A6 increased with increasing tumor number. High expression of COL6A1, COL6A2, COL6A3, COL6A5 and COL6A6 in GC patients was significantly associated with poorer overall survival, while low expression of COL6A4P1, COL6A4P2 and COL6A6 was associated with better progression-free survival in GC patients. These data suggest that the differentially expressed COL6 family may play an important role in gastric cell carcinoma. GAO found that high expression of COL6A1, COL6A2, and COL6A3 was associated with poorer prognosis in GC patients[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], but this time we found that COL6A1, COL6A2, COL6A3, COL6A5, and COL6A6 were associated with each other closely, but how they interact with each other needs to be verified in the next experiments.\u003c/p\u003e \u003cp\u003eThe development and progression of gastric cancer are complex and multifaceted, and based on currently known reports genetic alterations have been found to play a crucial role in this process [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Since COL6 members are significantly differentially expressed in gastric cancer, we subsequently explored their genetic mutations in gastric cancer. It was found that the differentially expressed COL6A1, COL6A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5 and COL6A6 had a certain mutation rate in gastric cancer tissues. Subsequently, we found different degrees of correlation among the COL6 family, which predicts that these genes play a certain synergistic role in the development of gastric cell carcinoma.\u003c/p\u003e \u003cp\u003eWe then investigated the function of the differentially expressed COL6 family using graphene oxide enrichment analysis and KEGG pathway enrichment analysis. As the results demonstrate, we found that the functions of these genes were mainly related to extracellular matrix structure, focal adhesion, and ECM-receptor interactions. Previous studies have shown that ECM had multiple effects on lymphocytes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] exerting some influence on lymphocyte migration, recognition, activation and differentiation, and thus multiple roles in the function of the immune system in vivo, and we speculated that the COL6 family may influence tumor development by regulating immune cells. Whereas, the importance of focal adhesion in cancer cell adhesion, motility, proliferation, and survival and overexpression in cancer cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and the combination of Focal adhesion kinase (FAK) inhibitors with chemotherapy, radiotherapy, and immunotherapy is expected to prolong the survival of patients [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These data suggest that the COL6 family, which is differentially expressed in gastric cell carcinoma, is a potential target for drug therapy.\u003c/p\u003e \u003cp\u003eCurrent known evidence suggests that immune cell infiltration can influence tumor progression and recurrence, which in turn affects overall patient survival and progression-free survival [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this study, we found that COL6 family expression was correlated to varying degrees for all 24 immune cells, with COL6A1/2/3/5/6 positively regulating most immune cells and COL6A4P1/P2 negatively regulating most immune cells, which also suggest that high COL6A4P1/P2 expression decreases immune This suggests that the COL6 family can not only be used as a prognostic indicator, but may also reflect the immune status. However, other independent cohort study and in vitro or in vivo studies are needed to validate our results.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we used bioinformatics analysis to explore the role of COL6 family genes in gastric cancer. We performed a major tumor informatics analysis to explore the expression profile of COL6 gene in GC and various clinicopathological parameters. Extensive data mining from various publicly available databases revealed that COL6 expression was upregulated in various GC corresponding to their normal tissues. In addition, survival map analysis, PPIs, co-expression analysis, functional analysis, and immune infiltration analysis all suggest that COL6 has significant prognostic and clinicopathological value in gastric cancer. Therefore, the COL6 family may have potential functions as prognostic biomarkers or tumor markers, and also provide theoretical basis for immunotherapy of tumors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval is not required for the current study due to the nature of the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are openly available in TIMER2(tumor immune estimation resource, version 2) at timer.cistrome.org ; Kaplan\u0026ndash;Meier plotter at http://kmplot.com/analysis/ ; the GEPIA2 at http://gepia.cancer-pku.cn/index.html; cBioPortal at www.cbioportal.org ; String at https://string-db.org/; the XIANTAO platform at www.xiantao.love.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Dr. Qilun Liu, Dr. Jing Chen, and Dr. Tao Li for critical reading of the manuscript. We would particularly like to acknowledge my group team members for their wonderful collaboration and patient support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQ.L.Liu , B.Z.Wang and W.J.Cai provised of study materials or patients. J.Chen, T.Li and Q.L.Liu administrated the support. L.Ma, J.Chen, Y.F.Wang, and J.Lin collected and assembled of data. L.Ma, J.Chen Q.L.Liu and L.H.Qi analysed the data and interpretation. L.Ma and Y.F.Wang wrote the main manuscript text. J.Chen, T.Li and Q.L.Liu reviewed, supervised and administrated this project. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by Key Research and Development Program of Ningxia (2021BEGO3087), Natural Science Foundation of Ningxia Province (2020AAC03403, 2020AAC03178).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-49.http://dx.doi.org/10.3322/caac.21660\u003c/li\u003e\n\u003cli\u003eNecula L, Matei L, Dragu D, Neagu AI, Mambet C, Nedeianu S, et al. Recent advances in gastric cancer early diagnosis. World journal of gastroenterology. 2019;25(17):2029-44.http://dx.doi.org/10.3748/wjg.v25.i17.2029\u003c/li\u003e\n\u003cli\u003eBray F, Jemal A, Grey N, Ferlay J, Forman D. 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Stromal Protein-Mediated Immune Regulation in Digestive Cancers. Cancers. 2021;13(1).http://dx.doi.org/10.3390/cancers13010146\u003c/li\u003e\n\u003cli\u003eKhalaf K, Hana D, Chou JT, Singh C, Mackiewicz A, Kaczmarek M. Aspects of the Tumor Microenvironment Involved in Immune Resistance and Drug Resistance. Frontiers in immunology. 2021;12:656364.http://dx.doi.org/10.3389/fimmu.2021.656364\u003c/li\u003e\n\u003cli\u003eAo R, Guan L, Wang Y, Wang JN. Silencing of COL1A2, COL6A3, and THBS2 inhibits gastric cancer cell proliferation, migration, and invasion while promoting apoptosis through the PI3k-Akt signaling pathway. Journal of cellular biochemistry. 2018;119(6):4420-34.http://dx.doi.org/10.1002/jcb.26524\u003c/li\u003e\n\u003cli\u003eSvoronos C, Tsoulfas G, Souvatzi M, Chatzitheoklitos E. Prognostic value of COL6A3 in pancreatic adenocarcinoma. 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Biomedicine \u0026amp; pharmacotherapy = Biomedecine \u0026amp; pharmacotherapie. 2017;95:55-61.http://dx.doi.org/10.1016/j.biopha.2017.08.003\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":"COL6 family, gastric cancer, biomarker, prognosis","lastPublishedDoi":"10.21203/rs.3.rs-2739455/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2739455/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eGastric cancer (GC) is one of the most common cancers in the worldwide and is the leading cause of death in patients. Despite significant advances in treatment, the 5-year survival rate remains low. Therefore, the discovery of tumor markers that can predict gastric cancer and estimate its prognosis will improve this situation.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this study, TIMER2, Kaplan\u0026ndash;Meier plotter and GEPIA databases were used to analyze the expression of COL6 family gene in gastric cancer tissues, and analyzed their relationship with different pathological stages and tumor prognosis. Meanwhile, we analyzed the Genetic Alteration, Co-expression, Neighbor Gene Network, Interaction Analyses, enrichment analysis and immune infiltration analysis of COL6 in Patients with GC in cBioPortal, STRING databases and XIANTAO platform.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that the COL6 family (COL6A1, COL1A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5 and COL6A6) genes were significantly up-regulated in gastric adenocarcinoma relative to normal tissue adjacent to the cancer. A significant correlation was found between the expression of COL6A1/2/3 and the pathological stage of GC patients. GC patients with high COL6A1/2/3/5/6 expression were all associated with poor survival, while COL6A4P1/P2/6 were significantly associated with metastasis or recurrence in patients. Subsequently, we performed functional enrichment analysis and found that its cellular function was associated with Focal adhesion, ECM-receptor interaction. Our data also showed that COL6A1/2/3/5/6 were positively with immune infiltration, while a negative correlation was found between COL6A/4P1/4P2 and immune cells infiltration, which may be associated with poorer relapse-free survival (RFS).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur findings suggest that the COL6 family (COL6A1/2/3/4P1/4P2/5/6) is a potential biomarker for determining the prognosis of gastric cancer and It also provides theoretical basis for immunotherapy.\u003c/p\u003e","manuscriptTitle":"Differential expression of COL6 family is a potential tumor marker for predicting gastric cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-07 14:33:31","doi":"10.21203/rs.3.rs-2739455/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":"736a6ee2-74e7-4315-97ec-88840ecdd25a","owner":[],"postedDate":"April 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-06-29T10:59:43+00:00","versionOfRecord":[],"versionCreatedAt":"2023-04-07 14:33:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2739455","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2739455","identity":"rs-2739455","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}