Cinobufagin targets POU6F1 to downregulate LINC01410-mediated proliferation and metastasis in osteosarcoma

Cinobufagin targets POU6F1 to downregulate LINC01410-mediated proliferation and metastasis in osteosarcoma | 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 Cinobufagin targets POU6F1 to downregulate LINC01410-mediated proliferation and metastasis in osteosarcoma Bingxia Zhang, Liqin Yang, Yang Liu, Zidong Ma, Mridul Roy, Yufei Ma, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7976755/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 Venenum bufonis (VB) is a traditional Chinese medicine (TCM) extracted from toad venom that has been used to treat various tumours. Cinobufagin (CBG), a major bioactive compound derived from the VB, has demonstrated clinical efficacy against metastatic bone tumours, but its LncRNA-modulating mechanisms in osteosarcoma (OS) remain unknown.This study investigates a novel mechanism by which CBG exerts its anti-tumor effects through targeting long non-coding RNAs (LncRNAs). Methods OS cell lines were treated with CBG. A series of cell biological experiments were performed to detect the effects of CBG on the proliferation, migration and invasion of osteosarcoma cells. RNA sequencing identified CBG-regulated lncRNAs. Proteome microarray analysis was used to screen for transcription factors that bind to CBG. Molecular docking and microscale thermophoresis experiments confirmed the binding of CBG to POU6F1. Chromatin immunoprecipitation and dual-luciferase reporter assays validated POU6F1 binding and the transcriptional regulation of LINC01410. In vivo , the efficacy of CBG in the treatment of OS proliferation and pulmonary metastasis was evaluated in subcutaneous xenograft and lung metastasis models. Results The experimental results show that CBG inhibits the malignant progression of OS, including proliferation and lung metastasis. RNA-seq revealed that LINC01410 is a key oncogenic lncRNA downregulated by CBG. Mechanistically, CBG directly binds to the transcription factor POU6F1 and inhibits its occupancy of the LINC01410 promoter, thereby inhibiting PI3K/AKT/mTOR signalling. Our study provides the first evidence for the mechanism by which CBGs target lncRNAs. Conclusions CBG exerts anti-OS effects by disrupting POU6F1-mediated transcription of LINC01410, thereby inactivating PI3K/AKT/mTOR signalling. This study supports the development of CBG as a therapeutic agent derived from TCM. Cinobufagin Osteosarcoma LINC01410 POU6F1 PI3K/AKT/mTOR pathway Full Text Additional Declarations No competing interests reported. Supplementary Files Supplementaryprimerssequence.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-7976755","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":542948297,"identity":"a2ab02f4-b504-48f1-aa3c-8902a83049a5","order_by":0,"name":"Bingxia Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYBACNvb2gw8+VPy3Y2NmPkCcFj6eM8mGM84wJ/OztyUQp0VOIsFMmLONmXFmzxkDIh3GcyCNmYGNjdngRs7HG28Y7OR0GwhpYW889riAh4fP4EbuZss5DMnGZgcI25JuPENCAmhL7jZpHoYDidsIagH6RZrHwIBxw42cZ6RoSUgAeZ+NSC3gQD5wABTIxpZzDIjwi3w7MCo//jsAisqHN95U2MkR1IICJHiIjBpkLaTqGAWjYBSMghEBAE9AQYyjD0lIAAAAAElFTkSuQmCC","orcid":"","institution":"The First Affiliated Hospital of Henan Medical University","correspondingAuthor":true,"prefix":"","firstName":"Bingxia","middleName":"","lastName":"Zhang","suffix":""},{"id":542948298,"identity":"d410ee9c-5332-4c32-888a-367c11e8fc61","order_by":1,"name":"Liqin Yang","email":"","orcid":"","institution":"The First Affiliated Hospital of Henan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liqin","middleName":"","lastName":"Yang","suffix":""},{"id":542948300,"identity":"d295d7c0-26db-460b-8f33-b8033c68bd3f","order_by":2,"name":"Yang Liu","email":"","orcid":"","institution":"The First Affiliated Hospital of Henan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Liu","suffix":""},{"id":542948301,"identity":"fcc04c65-1daa-4216-a27f-74d7d1fac67b","order_by":3,"name":"Zidong Ma","email":"","orcid":"","institution":"The First Affiliated Hospital of Henan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zidong","middleName":"","lastName":"Ma","suffix":""},{"id":542948302,"identity":"5a28cb75-f4e1-4f18-bb62-252db65c5578","order_by":4,"name":"Mridul Roy","email":"","orcid":"","institution":"The First Affiliated Hospital of Henan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mridul","middleName":"","lastName":"Roy","suffix":""},{"id":542948303,"identity":"0a21091e-ae26-42cb-8994-9d79fc1cef1e","order_by":5,"name":"Yufei Ma","email":"","orcid":"","institution":"The First Affiliated Hospital of Henan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yufei","middleName":"","lastName":"Ma","suffix":""},{"id":542948304,"identity":"d856e43e-e076-4573-972b-16e8e57cf95d","order_by":6,"name":"Jiaqi Lu","email":"","orcid":"","institution":"The First Affiliated Hospital of Henan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiaqi","middleName":"","lastName":"Lu","suffix":""},{"id":542948305,"identity":"58738905-35a0-45a2-a7ee-7e4c88f09963","order_by":7,"name":"Xiaoxia Yan","email":"","orcid":"","institution":"Luoyang Orthopedic-Traumatological Hospital of Henan Province","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxia","middleName":"","lastName":"Yan","suffix":""},{"id":542948309,"identity":"e228e7ff-7e38-45d6-b75a-325f3b59faf3","order_by":8,"name":"Fei Cao","email":"","orcid":"","institution":"The First Affiliated Hospital of Henan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Cao","suffix":""},{"id":542948312,"identity":"81b2f644-c74a-457a-96f9-5242a6c7804c","order_by":9,"name":"Xiaohong Kang","email":"","orcid":"","institution":"The First Affiliated Hospital of Henan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaohong","middleName":"","lastName":"Kang","suffix":""}],"badges":[],"createdAt":"2025-10-29 07:23:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7976755/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7976755/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96244886,"identity":"32b3b8b6-472b-4c65-94ed-eac89a7cb731","added_by":"auto","created_at":"2025-11-19 07:19:30","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8099965,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript2025.10.31.docx","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/77298c5c5e41abc21757b00c.docx"},{"id":96244723,"identity":"1c262a5b-361f-4722-84cf-44415c6a39b0","added_by":"auto","created_at":"2025-11-19 07:19:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17190,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryprimerssequence.docx","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/7b9c91bfe65f161be78b81e7.docx"},{"id":95949712,"identity":"12d6fc72-b5f9-45a3-b28a-1ce635cd71e8","added_by":"auto","created_at":"2025-11-14 18:56:18","extension":"json","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10917,"visible":true,"origin":"","legend":"","description":"","filename":"ee80a88fca144200aac9a12b13a03cc1.json","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/4b9a14f91a7d219f35575282.json"},{"id":95949727,"identity":"90fbf418-e356-420d-8e1a-7955f2ca3e9f","added_by":"auto","created_at":"2025-11-14 18:56:19","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":20851228,"visible":true,"origin":"","legend":"","description":"","filename":"Figures1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/e731fd1579fc3318789db16c.tif"},{"id":95949715,"identity":"07116108-e76e-4d2a-b755-f5d4878b27b7","added_by":"auto","created_at":"2025-11-14 18:56:18","extension":"xml","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":135227,"visible":true,"origin":"","legend":"","description":"","filename":"ee80a88fca144200aac9a12b13a03cc11enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/8c4130b8b2d2cf0da44591b3.xml"},{"id":95949726,"identity":"b10b8881-f239-4316-adec-bbde89e1d295","added_by":"auto","created_at":"2025-11-14 18:56:18","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5474260,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/c6e8083eb2234741a5a46fc5.jpeg"},{"id":96245461,"identity":"950e947f-9467-46ce-85de-c8a28d9520eb","added_by":"auto","created_at":"2025-11-19 07:20:42","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8378644,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/d9cf4e8bb5f6f91dcd290f1a.jpeg"},{"id":96245468,"identity":"4848f31a-bc6c-44e2-a445-37b78771c1e9","added_by":"auto","created_at":"2025-11-19 07:20:42","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4384600,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/a7e484041cbf581849c87aa7.jpeg"},{"id":95949720,"identity":"4dbbf03e-dfb1-4ff1-aa0c-416bc29cfa2b","added_by":"auto","created_at":"2025-11-14 18:56:18","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8148176,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/e2dba9942d8903aad8bdbd07.jpeg"},{"id":96244940,"identity":"cd840781-6f09-447d-b8b3-f4512b981c60","added_by":"auto","created_at":"2025-11-19 07:19:35","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4835956,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/7c8c3d4cc30788203a6468de.jpeg"},{"id":95949730,"identity":"8b60eccb-995c-4ab6-8238-8d48952e151c","added_by":"auto","created_at":"2025-11-14 18:56:19","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2516674,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/facee9e79f94442a06b0404c.jpeg"},{"id":95949717,"identity":"bcbcb169-5e9d-49a1-bde8-e9f83bf21a39","added_by":"auto","created_at":"2025-11-14 18:56:18","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":187356,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/8325b441f01a13aabaed97ac.png"},{"id":96244894,"identity":"cf4e7f1e-eeed-4c73-911c-d693e1aab68f","added_by":"auto","created_at":"2025-11-19 07:19:30","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":311540,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/269f747c6570e5965cdb1213.png"},{"id":96243843,"identity":"f83ea6f1-ba47-41d2-a540-d1ff7ea6cd7f","added_by":"auto","created_at":"2025-11-19 07:17:09","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":362720,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/aa3d32e77b960783a7bfc0b9.png"},{"id":96245755,"identity":"f8b66df8-f7ab-4f40-a09c-bca7354ea832","added_by":"auto","created_at":"2025-11-19 07:22:21","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":148662,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/22d389e5089318028f22b11e.png"},{"id":96244245,"identity":"a0727517-baa3-4261-9295-fdc5657b354d","added_by":"auto","created_at":"2025-11-19 07:18:00","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":420346,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/5b16105cbc719700529413b0.png"},{"id":96244798,"identity":"9c487b56-0b8b-4572-98dc-6ba6f34033ff","added_by":"auto","created_at":"2025-11-19 07:19:16","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115269,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/25fd55675544c6c4d7ff8e32.png"},{"id":96244754,"identity":"534399b8-d50f-4fdc-af87-df47ac0e4524","added_by":"auto","created_at":"2025-11-19 07:19:09","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":33420,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/b4ded14c3f978796956d06e2.png"},{"id":95949713,"identity":"9e6e3c5a-22d6-433a-87b7-52e140c77375","added_by":"auto","created_at":"2025-11-14 18:56:18","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":31937,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/c573b4bbd4f339daa6907cc6.png"},{"id":96244659,"identity":"f4eb36ce-d2cf-4575-8901-2f9c91c0d4e6","added_by":"auto","created_at":"2025-11-19 07:19:01","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":135817,"visible":true,"origin":"","legend":"","description":"","filename":"ee80a88fca144200aac9a12b13a03cc11structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/42a065d699f158a2f3ae965d.xml"},{"id":95949731,"identity":"bc0b0e16-f8a7-45a9-af1a-023859fb72de","added_by":"auto","created_at":"2025-11-14 18:56:19","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":145314,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/7e7c343154e78a36539d7229.html"},{"id":99319721,"identity":"0a7d288d-9409-44ab-afe3-04c82afdb491","added_by":"auto","created_at":"2025-12-31 16:37:42","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2174068,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript2025.10.31.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1_covered_4e2e0f3b-f901-489e-b400-0f62488473bb.pdf"},{"id":95949710,"identity":"e91a3328-bd8b-4d1b-81a2-d03e6cb25b6a","added_by":"auto","created_at":"2025-11-14 18:56:18","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":17190,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryprimerssequence.docx","url":"https://assets-eu.researchsquare.com/files/rs-7976755/v1/1e31762c9ab72c55e99dd1c7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cinobufagin targets POU6F1 to downregulate LINC01410-mediated proliferation and metastasis in osteosarcoma","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":"Cinobufagin, Osteosarcoma, LINC01410, POU6F1, PI3K/AKT/mTOR pathway","lastPublishedDoi":"10.21203/rs.3.rs-7976755/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7976755/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eVenenum bufonis (VB) is a traditional Chinese medicine (TCM) extracted from toad venom that has been used to treat various tumours. Cinobufagin (CBG), a major bioactive compound derived from the VB, has demonstrated clinical efficacy against metastatic bone tumours, but its LncRNA-modulating mechanisms in osteosarcoma (OS) remain unknown.This study investigates a novel mechanism by which CBG exerts its anti-tumor effects through targeting long non-coding RNAs (LncRNAs).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eOS cell lines were treated with CBG. A series of cell biological experiments were performed to detect the effects of CBG on the proliferation, migration and invasion of osteosarcoma cells. RNA sequencing identified CBG-regulated lncRNAs. Proteome microarray analysis was used to screen for transcription factors that bind to CBG. Molecular docking and microscale thermophoresis experiments confirmed the binding of CBG to POU6F1. Chromatin immunoprecipitation and dual-luciferase reporter assays validated POU6F1 binding and the transcriptional regulation of LINC01410. \u003cem\u003eIn vivo\u003c/em\u003e, the efficacy of CBG in the treatment of OS proliferation and pulmonary metastasis was evaluated in subcutaneous xenograft and lung metastasis models.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe experimental results show that CBG inhibits the malignant progression of OS, including proliferation and lung metastasis. RNA-seq revealed that LINC01410 is a key oncogenic lncRNA downregulated by CBG. Mechanistically, CBG directly binds to the transcription factor POU6F1 and inhibits its occupancy of the LINC01410 promoter, thereby inhibiting PI3K/AKT/mTOR signalling. Our study provides the first evidence for the mechanism by which CBGs target lncRNAs.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eCBG exerts anti-OS effects by disrupting POU6F1-mediated transcription of LINC01410, thereby inactivating PI3K/AKT/mTOR signalling. This study supports the development of CBG as a therapeutic agent derived from TCM.\u003c/p\u003e","manuscriptTitle":"Cinobufagin targets POU6F1 to downregulate LINC01410-mediated proliferation and metastasis in osteosarcoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-14 18:56:13","doi":"10.21203/rs.3.rs-7976755/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":"cc56f25d-c181-476f-b2f3-153e85988530","owner":[],"postedDate":"November 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-30T19:08:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-14 18:56:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7976755","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7976755","identity":"rs-7976755","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}