The Molecular Mechanism of ALG-2 in Regulating Proliferation, Migration, and Epithelial- Mesenchymal Transition in Oral Squamous Cell Carcinoma

The Molecular Mechanism of ALG-2 in Regulating Proliferation, Migration, and Epithelial- Mesenchymal Transition in Oral Squamous Cell Carcinoma | 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 The Molecular Mechanism of ALG-2 in Regulating Proliferation, Migration, and Epithelial- Mesenchymal Transition in Oral Squamous Cell Carcinoma Chengxian Jiang, Yifan Li, Haiyue Chen, Yinzhu Huang, Zhezhu Han, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7828746/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 Objective: This study aimed to investigate the expression characteristics of apoptosis-linked gene-2 (ALG-2) in oral squamous cell carcinoma (OSCC) tissues and cells, its impact on malignant cell behaviors, and its potential molecular mechanisms by integrating bioinformatics and experimental approaches. The goal was to assess whether ALG-2 could provide a theoretical basis for the early diagnosis, prognosis evaluation, and targeted therapy of OSCC. Methods: 1.The Cancer Genome Atlas (TCGA) database was analyzed to determine ALG-2 expression in head and neck squamous cell carcinoma (HNSC) and evaluate its correlation with patient overall survival (OS) and disease-free survival (DFS). Genetic alteration and immune infiltration analyses were also performed to explore the potential role of ALG-2 in HNSC. 2.Immunohistochemistry (IHC) was used to detect ALG-2 protein expression in a tissue microarray containing 50 OSCC tissues and 20 matched adjacent non-cancerous tissues, and its relationship with clinicopathological parameters was analyzed. 3.Western blot (WB) was performed to assess ALG-2 protein levels in the normal human oral epithelial cell line HOK and the OSCC cell lines CAL-27 and HSC-3. ALG-2 expression was knocked down in CAL-27 and HSC-3 cells via liposome-mediated transient siRNA transfection. Cells were divided into Con (untreated), si-NC (negative control), and si-ALG-2 (experimental) groups. The effects of ALG-2 knockdown on OSCC cell proliferation and migration were evaluated using CCK-8, colony formation, wound healing, and Transwell migration assays. 4.Western blot was used to detect changes in the expression of key epithelial-mesenchymal transition (EMT) marker proteins (E-cadherin and Vimentin) after ALG-2 knockdown to explore its potential regulatory role in the EMT process of OSCC. Results: 1.Bioinformatic analysis revealed that ALG-2 expression was significantly higher in HNSC tissues than in normal tissues (P < 0.001). Patients with high ALG-2 expression had significantly shorter OS (P = 0.0048, HR=1.5) and DFS (P = 0.017, HR=1.5) compared to those with low expression. The total genetic alteration frequency of ALG-2 in HNSC was less than 0.8%, primarily comprising mutations and amplifications. No significant differences were found in DFS, disease-specific survival (DSS), progression-free survival (PFS), or OS between patients with and without ALG-2 genetic alterations (P > 0.05). ALG-2 expression levels positively correlated with cancer-associated fibroblast (CAF) infiltration (P < 0.01) and negatively correlated with CD8⁺ T cell infiltration (P < 0.05). ALG-2 co-expressed genes were enriched in biological pathways including the Fanconi anemia pathway, biosynthesis of unsaturated fatty acids, viral life cycle - HIV-1, fatty acid elongation, and amyotrophic lateral sclerosis. These co-expressed genes were primarily clustered in core processes related to DNA maintenance and genomic integrity. 2.IHC staining results showed that ALG-2 protein was primarily localized in the cytoplasm and cell membrane of OSCC cells. Its expression was significantly higher in OSCC tissues than in adjacent tissues (P = 0.007) and correlated with tumor size (P=0.002), lymph node metastasis (P=0.028), T category (P=0.041), and TNM stage (P=0.039). 3.Western blot results indicated that ALG-2 protein levels were significantly higher in CAL-27 and HSC-3 OSCC cell lines compared to the HOK normal oral epithelial cell line (P < 0.01). Knockdown of ALG-2 in CAL-27 and HSC-3 cells significantly up-regulated the EMT marker E-cadherin (P < 0.05, P < 0.01) and down-regulated the marker Vimentin (P < 0.01 for both). 4.Functional assays demonstrated that ALG-2 knockdown significantly inhibited the proliferative viability of CAL-27 and HSC-3 cells in both CCK-8 and colony formation assays (P < 0.01). It also significantly reduced the migratory capacity of both cell lines in wound healing and Transwell migration assays (P < 0.01). Conclusion :ALG-2 is significantly overexpressed in OSCC tissues and cells and is closely associated with poor patient prognosis. Silencing ALG-2 effectively inhibits the proliferation and migration of OSCC cells, potentially by regulating EMT-related proteins. Therefore, ALG-2 shows promise as a novel molecular marker for OSCC diagnosis and prognosis and may provide a potential target for developing targeted therapies. Health sciences/Biomarkers Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Computational biology and bioinformatics Health sciences/Oncology Oral squamous cell carcinoma ALG-2 Proliferation Migration Epithelial-mesenchymal transition Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure.docx 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-7828746","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":549863110,"identity":"da4f668c-b8c0-420f-be01-995f34991499","order_by":0,"name":"Chengxian Jiang","email":"","orcid":"","institution":"Yanbian University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chengxian","middleName":"","lastName":"Jiang","suffix":""},{"id":549863111,"identity":"ff1f6146-e77d-4c14-8aa6-783fcc8cbbc6","order_by":1,"name":"Yifan Li","email":"","orcid":"","institution":"Yanbian University 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Carcinoma","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"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":"Oral squamous cell carcinoma, ALG-2, Proliferation, Migration, Epithelial-mesenchymal transition","lastPublishedDoi":"10.21203/rs.3.rs-7828746/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7828746/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003eThis study aimed to investigate the expression characteristics of apoptosis-linked gene-2 (ALG-2) in oral squamous cell carcinoma (OSCC) tissues and cells, its impact on malignant cell behaviors, and its potential molecular mechanisms by integrating bioinformatics and experimental approaches. The goal was to assess whether ALG-2 could provide a theoretical basis for the early diagnosis, prognosis evaluation, and targeted therapy of OSCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003e1.The Cancer Genome Atlas (TCGA) database was analyzed to determine ALG-2 expression in head and neck squamous cell carcinoma (HNSC) and evaluate its correlation with patient overall survival (OS) and disease-free survival (DFS). Genetic alteration and immune infiltration analyses were also performed to explore the potential role of ALG-2 in HNSC.\u003c/p\u003e\n\u003cp\u003e2.Immunohistochemistry (IHC) was used to detect ALG-2 protein expression in a tissue microarray containing 50 OSCC tissues and 20 matched adjacent non-cancerous tissues, and its relationship with clinicopathological parameters was analyzed.\u003c/p\u003e\n\u003cp\u003e3.Western blot (WB) was performed to assess ALG-2 protein levels in the normal human oral epithelial cell line HOK and the OSCC cell lines CAL-27 and HSC-3. ALG-2 expression was knocked down in CAL-27 and HSC-3 cells via liposome-mediated transient siRNA transfection. Cells were divided into Con (untreated), si-NC (negative control), and si-ALG-2 (experimental) groups. The effects of ALG-2 knockdown on OSCC cell proliferation and migration were evaluated using CCK-8, colony formation, wound healing, and Transwell migration assays.\u003c/p\u003e\n\u003cp\u003e4.Western blot was used to detect changes in the expression of key epithelial-mesenchymal transition (EMT) marker proteins (E-cadherin and Vimentin) after ALG-2 knockdown to explore its potential regulatory role in the EMT process of OSCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003e1.Bioinformatic analysis revealed that ALG-2 expression was significantly higher in HNSC tissues than in normal tissues (P \u0026lt; 0.001). Patients with high ALG-2 expression had significantly shorter OS (P = 0.0048, HR=1.5) and DFS (P = 0.017, HR=1.5) compared to those with low expression. The total genetic alteration frequency of ALG-2 in HNSC was less than 0.8%, primarily comprising mutations and amplifications. No significant differences were found in DFS, disease-specific survival (DSS), progression-free survival (PFS), or OS between patients with and without ALG-2 genetic alterations (P \u0026gt; 0.05). ALG-2 expression levels positively correlated with cancer-associated fibroblast (CAF) infiltration (P \u0026lt; 0.01) and negatively correlated with CD8⁺ T cell infiltration (P \u0026lt; 0.05). ALG-2 co-expressed genes were enriched in biological pathways including the Fanconi anemia pathway, biosynthesis of unsaturated fatty acids, viral life cycle - HIV-1, fatty acid elongation, and amyotrophic lateral sclerosis. These co-expressed genes were primarily clustered in core processes related to DNA maintenance and genomic integrity.\u003c/p\u003e\n\u003cp\u003e2.IHC staining results showed that ALG-2 protein was primarily localized in the cytoplasm and cell membrane of OSCC cells. Its expression was significantly higher in OSCC tissues than in adjacent tissues (P = 0.007) and correlated with tumor size (P=0.002), lymph node metastasis (P=0.028), T category (P=0.041), and TNM stage (P=0.039).\u003c/p\u003e\n\u003cp\u003e3.Western blot results indicated that ALG-2 protein levels were significantly higher in CAL-27 and HSC-3 OSCC cell lines compared to the HOK normal oral epithelial cell line (P \u0026lt; 0.01). Knockdown of ALG-2 in CAL-27 and HSC-3 cells significantly up-regulated the EMT marker E-cadherin (P \u0026lt; 0.05, P \u0026lt; 0.01) and down-regulated the marker Vimentin (P \u0026lt; 0.01 for both).\u003c/p\u003e\n\u003cp\u003e4.Functional assays demonstrated that ALG-2 knockdown significantly inhibited the proliferative viability of CAL-27 and HSC-3 cells in both CCK-8 and colony formation assays (P \u0026lt; 0.01). It also significantly reduced the migratory capacity of both cell lines in wound healing and Transwell migration assays (P \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e:ALG-2 is significantly overexpressed in OSCC tissues and cells and is closely associated with poor patient prognosis. Silencing ALG-2 effectively inhibits the proliferation and migration of OSCC cells, potentially by regulating EMT-related proteins. Therefore, ALG-2 shows promise as a novel molecular marker for OSCC diagnosis and prognosis and may provide a potential target for developing targeted therapies.\u003c/p\u003e","manuscriptTitle":"The Molecular Mechanism of ALG-2 in Regulating Proliferation, Migration, and Epithelial- Mesenchymal Transition in Oral Squamous Cell Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-27 11:58:32","doi":"10.21203/rs.3.rs-7828746/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":"b1658ec6-141a-4c03-84a3-9ad10b8ffa34","owner":[],"postedDate":"November 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58502882,"name":"Health sciences/Biomarkers"},{"id":58502883,"name":"Biological sciences/Cancer"},{"id":58502884,"name":"Biological sciences/Cell biology"},{"id":58502885,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":58502886,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2025-11-27T11:58:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-27 11:58:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7828746","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7828746","identity":"rs-7828746","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}