P16INK4a affects the malignant biological behavior of gallbladder cancer cells by regulating the Wnt/β-catenin pathway

P16INK4a affects the malignant biological behavior of gallbladder cancer cells by regulating the Wnt/β-catenin pathway | 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 P16INK4a affects the malignant biological behavior of gallbladder cancer cells by regulating the Wnt/β-catenin pathway Wei Guo, Xu Guo, Yunlong Bai, Yamu Li, Xiaoshuang Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8647099/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 To investigate the effect of cyclin-dependent kinase inhibitor 2A (p16INK4a) on the malignant biological behavior of gallbladder carcinoma (GBC) cells and analyze the underlying mechanism. Methods GBC-SD cells were assigned into four groups: control, pc-NC, pc-p16INK4a, and pc-p16INK4a+SKL2001. GBC-SD cells overexpressing p16INK4a were constructed by lentiviral vector infection and treated with Wnt/β-catenin pathway agonist SKL2001. CCK-8 experiment and colony formation experiment were used to detect cell proliferation activity. Flow cytometry was used to detect cell cycle distribution. Transwell chamber was used to detect the migration and invasion abilities of cells. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the mRNA level of p16INK4a in cells. Western Blot was used to detect the expression of p16INK4a and Wnt/β-catenin pathway-related proteins in cells. The in vivo effect of overexpression of p16INK4a on the growth of transplanted tumors was analyzed using a nude mouse subcutaneous transplant tumor model. Results p16INK4a showed low expression in GBC cell lines. Compared with the control group and the pc-NC group, the mRNA and protein expressions of p16INK4a in pc-p16INK4a group were significantly higher with higher proportion of G0/G1 phase cells, while the OD value of cell proliferation, colony number, proportions of S and G2/M phase cells, numbers of cell migration and invasion, and protein levels of β-catenin, c-Myc, and Cyclin D1 were much lower ( P < 0.05). SKL2001 could conspicuously weaken the effect of overexpression of p16INK4a on GBC-SD cells ( P < 0.05). The results of in vivo experiments showed that compared with the control group and the pc-NC group, the volume and weight of transplanted tumors, and positive expression of β-catenin protein in the pc-p16INK4a group were lower, while the positive expression of p16INK4a protein was higher ( P < 0.05), indicating that SKL2001 can attenuate the inhibitory effect of overexpression of p16INK4a on the growth of transplanted tumors in nude mice (P < 0.05). Conclusion Overexpression of p16INK4a may inhibit the malignant biological behavior of GBC-SD cells by suppressing the activation of the Wnt/β-catenin signaling pathway. Biological sciences/Cancer Biological sciences/Cell biology Health sciences/Oncology Gallbladder carcinoma Cyclin-dependent kinase inhibitor 2A Proliferation Cell cycle Wnt/β-catenin pathway Full Text Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation.pdf 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. 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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-8647099","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":614911562,"identity":"0c81efec-06e4-4390-beb8-bafe53c7538f","order_by":0,"name":"Wei Guo","email":"","orcid":"","institution":"the First Affiliated Hospital of Henan University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Guo","suffix":""},{"id":614911563,"identity":"326cdec7-1f1a-4f4b-9ba6-2ee95d4974bb","order_by":1,"name":"Xu Guo","email":"","orcid":"","institution":"the First Affiliated Hospital of Henan 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pathway","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":"Gallbladder carcinoma, Cyclin-dependent kinase inhibitor 2A, Proliferation, Cell cycle, Wnt/β-catenin pathway","lastPublishedDoi":"10.21203/rs.3.rs-8647099/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8647099/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo investigate the effect of cyclin-dependent kinase inhibitor 2A (p16INK4a) on the malignant biological behavior of gallbladder carcinoma (GBC) cells and analyze the underlying mechanism.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eGBC-SD cells were assigned into four groups: control, pc-NC, pc-p16INK4a, and pc-p16INK4a+SKL2001. GBC-SD cells overexpressing p16INK4a were constructed by lentiviral vector infection and treated with Wnt/β-catenin pathway agonist SKL2001. CCK-8 experiment and colony formation experiment were used to detect cell proliferation activity. Flow cytometry was used to detect cell cycle distribution. Transwell chamber was used to detect the migration and invasion abilities of cells. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the mRNA level of p16INK4a in cells. Western Blot was used to detect the expression of p16INK4a and Wnt/β-catenin pathway-related proteins in cells. The in vivo effect of overexpression of p16INK4a on the growth of transplanted tumors was analyzed using a nude mouse subcutaneous transplant tumor model.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ep16INK4a showed low expression in GBC cell lines. Compared with the control group and the pc-NC group, the mRNA and protein expressions of p16INK4a in pc-p16INK4a group were significantly higher with higher proportion of G0/G1 phase cells, while the OD value of cell proliferation, colony number, proportions of S and G2/M phase cells, numbers of cell migration and invasion, and protein levels of β-catenin, c-Myc, and Cyclin D1 were much lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). SKL2001 could conspicuously weaken the effect of overexpression of p16INK4a on GBC-SD cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The results of in vivo experiments showed that compared with the control group and the pc-NC group, the volume and weight of transplanted tumors, and positive expression of β-catenin protein in the pc-p16INK4a group were lower, while the positive expression of p16INK4a protein was higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that SKL2001 can attenuate the inhibitory effect of overexpression of p16INK4a on the growth of transplanted tumors in nude mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOverexpression of p16INK4a may inhibit the malignant biological behavior of GBC-SD cells by suppressing the activation of the Wnt/β-catenin signaling pathway.\u003c/p\u003e","manuscriptTitle":"P16INK4a affects the malignant biological behavior of gallbladder cancer cells by regulating the Wnt/β-catenin pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 14:44:24","doi":"10.21203/rs.3.rs-8647099/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":"05706729-15ac-44d5-9377-5d2ae4d3753c","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65428764,"name":"Biological sciences/Cancer"},{"id":65428765,"name":"Biological sciences/Cell biology"},{"id":65428766,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-04-03T05:25:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 14:44:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8647099","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8647099","identity":"rs-8647099","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}