m1A-regulated DIAPH3 promotes the invasiveness of colorectal cancer via stabilization of KRT19

m1A-regulated DIAPH3 promotes the invasiveness of colorectal cancer via stabilization of KRT19 | 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 m1A-regulated DIAPH3 promotes the invasiveness of colorectal cancer via stabilization of KRT19 Shuyi Mi, Jie Hu, Wenwen Chen, Jingyu Chen, Zhipeng Xu, Meng Xue This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3350465/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Background : In recent years, the emphasis has shifted to understanding the role of N1-methyladenosine (m1A) in tumor progression as little is known about its regulatory effect on mRNA and its role in the metastasis of colorectal cancer (CRC). Methods : We performed methylated RNA immunoprecipitation sequencing of tumor tissues and tumor-adjacent normal tissues from three patients with CRC to determine the m1A profile of mRNA in CRC. The expression of diaphanous-related formin 3 (DIAPH3) and its correlation with clinicopathological characteristics of CRC were evaluated using immunohistochemistry and online datasets. The role of DIAPH3 in the migration and invasion of CRC cells was evaluated using wound healing assay, Transwell assay and xenograft metastatic model. The downstream targets of DIAPH3 were screened using mass spectrometry. By co-transfecting DIAPH3 siRNA and a keratin 19 (KRT19) ectopic plasmid into CRC cells, the role of DIAPH3-KRT19 signaling axis was confirmed. Results : The mRNA level of DIAHP3 and its m1A modifications increased simultaneously in the CRC tissues. In addition, high DIAPH3 expression in CRC tissues is significantly associated with metastasis and progression to an advanced stage. After the knockdown of DIAPH3, the migration and invasion capabilities of CRC cells suffered a notable decline, which could be rescued by overexpressing KRT19. In addition, the proteasome inhibitor MG132 could block the degradation of KRT19 induced by DIAPH3 silencing. Conclusions : Our study reveals that DIAPH3 mRNA was modified in CRC cells by m1A methylation. Silencing DIAPH3 suppresses the migration and invasion of CRC cells, potentially through the proteasome-dependent degradation of downstream KRT19. CRC m1A DIAPH3 migration invasion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Full Text Additional Declarations The authors declare no competing interests. Table 1 is available in the supplementary files section. Supplementary Files supplementaryFigures.pptx Table1.docx TableS1.docx TableS2.docx TableS3.docx TableS4.docx Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions 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-3350465","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":237000464,"identity":"3059d11a-cefc-4bf6-ae42-243eb4b9a363","order_by":0,"name":"Shuyi Mi","email":"","orcid":"","institution":"Zhejiang University School of Medicine Second Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shuyi","middleName":"","lastName":"Mi","suffix":""},{"id":237000465,"identity":"6a0ee5f0-8275-4255-b25a-e553eabe28ed","order_by":1,"name":"Jie Hu","email":"","orcid":"","institution":"Zhejiang University School of Medicine Second Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Hu","suffix":""},{"id":237000466,"identity":"55aad6b4-ff57-4243-ae0a-0adc093ebacf","order_by":2,"name":"Wenwen Chen","email":"","orcid":"","institution":"Zhejiang University School of Medicine Second Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenwen","middleName":"","lastName":"Chen","suffix":""},{"id":237000467,"identity":"3735a36e-ac0f-4c7c-a18b-fde2a9f11396","order_by":3,"name":"Jingyu Chen","email":"","orcid":"","institution":"Zhejiang University School of Medicine Second Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jingyu","middleName":"","lastName":"Chen","suffix":""},{"id":237000468,"identity":"52edb46c-b4ca-4ca3-9692-3a244f194b36","order_by":4,"name":"Zhipeng Xu","email":"","orcid":"","institution":"Zhejiang University School of Medicine Second Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhipeng","middleName":"","lastName":"Xu","suffix":""},{"id":237000469,"identity":"75966be9-8d40-4ecd-afdd-061e830fc72f","order_by":5,"name":"Meng Xue","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYBADOQaGA0CKjQQtxqRrSWwAU8RoMbiRfHTDxx216fMbzxgwfCg7zMA/uwG/FskZaWk3Z545ntvYcMaAcca5wwwSdw7g18IvkWN2m7ftWG4zwxkDZt62wwwGEgn4tbBJ5H+7/bftWDobSMtfYrQAbWG7zdhWk8AD0sJIjBbJnmdmN3vbDhjOYDhWcLDnXDqPxA0CWgyOJz+78bOtTl5+xuGND36UWcvxzyCgBQoOMzBIHABHJg9R6oGgDuirBmIVj4JRMApGwUgDADiNRvXszgBoAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-8647-6197","institution":"Zhejiang University School of Medicine Second Affiliated Hospital","correspondingAuthor":true,"prefix":"","firstName":"Meng","middleName":"","lastName":"Xue","suffix":""}],"badges":[],"createdAt":"2023-09-13 04:25:02","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-3350465/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-3350465/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57800550,"identity":"a953aa23-7b19-47b8-b523-858cbde04fa6","added_by":"auto","created_at":"2024-06-05 21:42:13","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":317644,"visible":true,"origin":"","legend":"\u003cp\u003em1A upregulated the expression of DIAPH3 in CRC cells.\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eA\u003c/strong\u003e The star plot shows the distribution of genes with both differential (hyper or hypo) m1A peaks and differential mRNA (up or down) expression (n = 3). The fold change of either expression or m1A methylation of mRNA \u0026lt;5 is labeled as gray dots, while the others are marked with dots of different colors. The red dots highlighted by a circle represent up-regulated transcripts with an increased abundance of m1A in CRC tissues. \u003cstrong\u003eB\u003c/strong\u003e qPCR validation of the genes with the FPKM values increased most markedly in colorectal tumor (CT), when compared to adjacent normal tissues (CN) (n = 3). \u003cstrong\u003eC\u003c/strong\u003e A visualized m1A-peak map along the mRNA of DIAPH3 in colorectal tumor (CT) and adjacent normal tissues (CN). \u003cstrong\u003eD\u003c/strong\u003e A significant positive correlation (r = 0.34, p = 6e-13) was found between DIAPH3 mRNA and TRMT6 mRNA in CRC according to the GEPIA database. \u003cstrong\u003eE\u003c/strong\u003e The protein expression level of TRMT6 after transfecting TRMT6 siRNA in HCT116 and RKO cells (n = 3). \u003cstrong\u003eF\u003c/strong\u003e Relative abundance of m1A methylated DIAPH3 mRNA in HCT116 and RKO cells after silencing TRMT6 (n = 3). \u003cstrong\u003eG\u003c/strong\u003e The relative expression of DIAPH3 mRNA in HCT116 and RKO cells after silencing TRMT6 siRNA (n = 3). T6, TRMT6; Neg, Negative; si, si-RNA; CN, adjacent normal tissues; CT, colorectal tumor; RIP, RNA immunoprecipitation; \u003csup\u003e**\u003c/sup\u003ep \u0026lt;0.01, \u003csup\u003e*\u003c/sup\u003ep \u0026lt;0.05\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3350465/v2/4f7e09343247d90d199e1e75.jpg"},{"id":57800547,"identity":"e9d75304-06fe-4a13-9722-6e7e3d124b36","added_by":"auto","created_at":"2024-06-05 21:42:13","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":434793,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of DIAPH3 was upregulated in CRC.\u003cstrong\u003e A\u003c/strong\u003e The mRNA expression level of DIAPH3 in patients with rectum adenocarcinoma (READ) and colon adenocarcinoma (COAD) in the GEPIA database. Left (green): normal (N = 318 in READ, N = 349 in COAD); right (red): tumor (T = 92 in READ, T = 275 in COAD), error bars denote mean ± SEM. \u003cstrong\u003eB\u003c/strong\u003e Survival analysis based on the level of DIAPH3 expression in patients with CRC in GenomicScape database. \u003cstrong\u003eC\u003c/strong\u003e Representative images of DIAPH3 IHC staining in three pairs of CRC samples are shown (original magnification ×200). \u003cstrong\u003eD \u003c/strong\u003eIHC scores of DIAPH3 staining in matched CRC and normal tissues (n = 173). \u003cstrong\u003eE\u003c/strong\u003e Kaplan-Meier analysis of overall survival in CRC patients whose tumors display ‘Low DIAPH3’ versus ‘High DIAPH3’ expression. Log rank test, p = 0.022 (n = 89). N, adjacent normal tissues; T, CRC tissues; READ, rectum adenocarcinoma; COAD, colon adenocarcinoma; OS, overall survival; \u003csup\u003e**\u003c/sup\u003ep \u0026lt;0.01, \u003csup\u003e*\u003c/sup\u003ep \u0026lt;0.05\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3350465/v2/329214e3cff62b7adca79d9b.jpg"},{"id":57800551,"identity":"59d1c9b8-db3b-4c92-9df6-fab775672d21","added_by":"auto","created_at":"2024-06-05 21:42:13","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":608482,"visible":true,"origin":"","legend":"\u003cp\u003eDIAPH3 regulates the migration and invasion of human CRC cell lines. \u003cstrong\u003eA \u003c/strong\u003eThe transfection efficiency of DIAPH3 siRNAs in HCT116 and RKO cells was validated by western blotting. \u003cstrong\u003eB\u003c/strong\u003e After seeding in the upper Transwell chamber and incubation for 48 h, HCT116 and RKO cells that migrated to the bottom of the membrane were stained with crystal violet. The representative images were captured under the microscope at a magnification of 200×. \u003cstrong\u003eC \u003c/strong\u003eThe average number of migratory cells was counted in five random fields (n = 5). \u003cstrong\u003eD\u003c/strong\u003e After seeding in the Matrigel-coated chamber and incubation for 48 h, HCT116 and RKO cells that invaded the bottom of the membrane were stained with crystal violet. The representative images were captured under the microscope at a magnification of 200×. \u003cstrong\u003eE\u003c/strong\u003e The average number of invaded cells was counted in five random fields (n = 5). \u003cstrong\u003eF\u003c/strong\u003e Wound healing assays were conducted to compare the migration capabilities of CRC cells after knocking down DIAPH3 (n = 3). The difference in cell margin between 0 h and 24 h showed moving track of cells. \u003cstrong\u003eG\u003c/strong\u003e Relative closure of the wound was quantified. D3, DIAPH3; Neg, Negative; si, siRNA; \u003csup\u003e**\u003c/sup\u003ep \u0026lt;0.01; \u003csup\u003e*\u003c/sup\u003ep \u0026lt;0.05\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3350465/v2/f2fe82948f5dd7a4a6017132.jpg"},{"id":57800558,"identity":"9480b720-cc20-4aec-8898-1840dba2f5ec","added_by":"auto","created_at":"2024-06-05 21:42:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":498983,"visible":true,"origin":"","legend":"\u003cp\u003eKnocking out DIAPH3 inhibited the migration and invasion of CRC cells. \u003cstrong\u003eA\u003c/strong\u003e Genomic DNA sequences of DIAPH3 targeting site in HCT116 KO and RKO KO cell clones. \u003cstrong\u003eB\u003c/strong\u003e Knocking-out efficiency of DIAPH3 confirmed by western blotting. \u003cstrong\u003eC\u003c/strong\u003e After seeding in the upper Transwell chamber and incubation for 48 h, HCT116 and RKO cells that migrated to the bottom of the membrane were stained with crystal violet. The representative images were captured under the microscope at a magnification of 200×. \u003cstrong\u003eD\u003c/strong\u003e The average number of migratory cells was counted in five random fields (n = 5). \u003cstrong\u003eE\u003c/strong\u003e After seeding in the Matrigel-coated chamber and incubation for 48 h, HCT116 and RKO cells that invaded the bottom of the membrane were stained with crystal violet. The representative images were captured under the microscope at a magnification of 200×. \u003cstrong\u003eF\u003c/strong\u003e The average number of invaded cells was counted in five random fields (n = 5). \u003cstrong\u003eG\u003c/strong\u003e Wound healing assays were conducted to compare the migration capabilities of two CRC cells after knocking out DIAPH3 (n = 3). The difference in cell margin between 0 h and 24 h showed moving track of cells. \u003cstrong\u003eH\u003c/strong\u003e Relative closure of the wound was quantified. \u003cstrong\u003eI\u003c/strong\u003e In vivo bioluminescence imaging of the mice injected with negative-KO and DIAPH3-KO HCT116 cells at the fifth and sixth week. \u003cstrong\u003eJ\u003c/strong\u003e Quantitative analysis of bioluminescence imaging intensity on day 42 (n =\u0026nbsp;5). D3, DIAPH3; Neg, Negative; KO, knock out; \u003csup\u003e**\u003c/sup\u003ep \u0026lt;0.01; \u003csup\u003e*\u003c/sup\u003ep \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3350465/v2/353b8812db2cc7e0de0825c6.png"},{"id":57800553,"identity":"ac5364dd-f7e6-419a-a3b5-6a4ccbc23447","added_by":"auto","created_at":"2024-06-05 21:42:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":537401,"visible":true,"origin":"","legend":"\u003cp\u003eSuppression of DIAPH3 induced the degradation of KRT19. \u003cstrong\u003eA\u003c/strong\u003e The Volcano Plots showed the distribution of genes with differential (up or down) expression after silencing DIAPH3 (n = 3). The fold change of expression of protein \u0026lt;1.3 or p value \u0026gt;0.1 is labeled as gray dots, while the others are marked with colorful dots. \u003cstrong\u003eB\u003c/strong\u003e Seven potential downstream targets of DIAPH3 were validated by western blotting after DIAPH3 was knocked out in HCT116 and RKO cells. \u003cstrong\u003eC\u003c/strong\u003e The HCT116 and RKO cells were transfected with KRT19 ectopic plasmids and the transfection efficiency was validated by western blotting. \u003cstrong\u003eD\u003c/strong\u003e After seeding in the upper Transwell chamber and incubation for 48 h, HCT116 and RKO cells that migrated to the bottom of the membrane were stained with crystal violet. The representative images were captured under the microscope at a magnification of 200×. \u003cstrong\u003eE\u003c/strong\u003e The average number of migratory cells was counted in five random fields (n = 5). \u003cstrong\u003eF\u003c/strong\u003e After seeding in the Matrigel-coated chamber and incubation for 48 h, HCT116 and RKO cells that invaded the bottom of the membrane were stained with crystal violet. The representative images were captured under the microscope at a magnification of 200×. \u003cstrong\u003eG\u003c/strong\u003e The average number of invaded cells was counted in five random fields (n = 5). D3, DIAPH3; Neg, Negative; KO, knock out; \u003csup\u003e**\u003c/sup\u003ep \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-3350465/v2/eb7995a4383192c95fceefff.png"},{"id":57800605,"identity":"33291506-9697-4a50-9f17-6607b7c3829a","added_by":"auto","created_at":"2024-06-05 21:50:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":502260,"visible":true,"origin":"","legend":"\u003cp\u003eDIAPH3 regulated the invasiveness of CRC cells through KRT19. \u003cstrong\u003eA\u003c/strong\u003e HCT116 and RKO cells were transfected with negative control siRNA or DIAPH3 siRNA, and then transfected with empty vector or KRT19 ectopic plasmid 48 h later. The transfection efficiency was assessed by western blotting. \u003cstrong\u003eB\u003c/strong\u003e After seeding in the upper Transwell chamber and incubation for 48 h, HCT116 and RKO cells that migrated to the bottom of the membrane were stained with crystal violet. The representative images were captured under the microscope at a magnification of 200×. \u003cstrong\u003eC\u003c/strong\u003e The average number of migratory cells was counted in five random fields (n = 5). \u003cstrong\u003eD\u003c/strong\u003e After seeding in the Matrigel-coated chamber and incubation for 48 h, HCT116 and RKO cells that invaded the bottom of the membrane were stained with crystal violet. The representative images were captured under the microscope at a magnification of 200×. \u003cstrong\u003eE\u003c/strong\u003e The average number of invaded cells was counted in five random fields (n = 5). \u003cstrong\u003eF\u003c/strong\u003e CRC cells with or without DIAPH3 silencing were treated with 100 mg/mL CHX and protein lysates were collected at indicated time points for western blot analysis. \u003cstrong\u003eG\u003c/strong\u003e KRT19 was quantified and plotted relative to the 0-time point. \u003cstrong\u003eH\u003c/strong\u003e MG132 (20 \u003cimg width=\"9\" height=\"19\" src=\"file:///C:/Users/maha15/AppData/Local/Temp/msohtmlclip1/01/clip_image002.png\"/\u003eM, 24 h) treatment on HCT116 and RKO cells with or without DIAPH3 silencing. D3, DIAPH3; Neg, Negative; si, siRNA; KO, knock out;\u003csup\u003e **\u003c/sup\u003ep \u0026lt;0.01 versus Neg si + Empty Vector; \u003csup\u003e##\u003c/sup\u003ep \u0026lt;0.01 versus DIAPH3 si + Empty Vector.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-3350465/v2/9b43ed2a0e50d3fd8daae86f.png"},{"id":57800559,"identity":"2e387db0-6be5-41e2-8cbd-85f28b5f3837","added_by":"auto","created_at":"2024-06-05 21:42:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":309813,"visible":true,"origin":"","legend":"\u003cp\u003eWorking model of DIAPH3 promoting CRC metastasis through KRT19. In CRC, m1A enhances the expression of DIAPH3 mRNA. Increased DIAPH3 suppresses the degradation of CLK3 in a proteasome-dependent manner, which accelerates CRC metastasis.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-3350465/v2/b577d11a53e1718600de24d0.png"},{"id":57801205,"identity":"01987786-43a3-4522-8277-28eec011fca5","added_by":"auto","created_at":"2024-06-05 22:14:18","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2419749,"visible":true,"origin":"","legend":"","description":"","filename":"manucriptunmarked.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3350465/v2_covered_92b49e44-6667-44df-91a6-cae403d5c7f3.pdf"},{"id":57800789,"identity":"c5ca2904-de41-4ab7-9280-b865dc52dac0","added_by":"auto","created_at":"2024-06-05 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21:50:13","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":12291,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-3350465/v2/0d066bb4bf68bd4536ce1df1.docx"},{"id":57800604,"identity":"cb4b3146-d1ee-41d0-a450-4b254541c544","added_by":"auto","created_at":"2024-06-05 21:50:13","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":11746,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-3350465/v2/8810588b343c291d4b9c5856.docx"}],"financialInterests":"\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eTable 1 is available in the supplementary files section.\u003c/p\u003e","formattedTitle":"m1A-regulated DIAPH3 promotes the invasiveness of colorectal cancer via stabilization of KRT19","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":"CRC, m1A, DIAPH3, migration, invasion","lastPublishedDoi":"10.21203/rs.3.rs-3350465/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3350465/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/em\u003e: In recent years, the emphasis has shifted to understanding the role of N1-methyladenosine (m1A) in tumor progression as little is known about its regulatory effect on mRNA and its role in the metastasis of colorectal cancer (CRC).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/em\u003e: We performed methylated RNA immunoprecipitation sequencing of tumor tissues and tumor-adjacent normal tissues from three patients with CRC to determine the m1A profile of mRNA in CRC. The expression of diaphanous-related formin 3 (DIAPH3) and its correlation with clinicopathological characteristics of CRC were evaluated using immunohistochemistry and online datasets. The role of DIAPH3 in the migration and invasion of CRC cells was evaluated using wound healing assay, Transwell assay and xenograft metastatic model. The downstream targets of DIAPH3 were screened using mass spectrometry. By co-transfecting DIAPH3 siRNA and a keratin 19 (KRT19) ectopic plasmid into CRC cells, the role of DIAPH3-KRT19 signaling axis was confirmed.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/em\u003e: The mRNA level of DIAHP3 and its m1A modifications increased simultaneously in the CRC tissues. In addition, high DIAPH3 expression in CRC tissues is significantly associated with metastasis and progression to an advanced stage. After the knockdown of DIAPH3, the migration and invasion capabilities of CRC cells suffered a notable decline, which could be rescued by overexpressing KRT19. In addition, the proteasome inhibitor MG132 could block the degradation of KRT19 induced by DIAPH3 silencing.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/em\u003e: Our study reveals that DIAPH3 mRNA was modified in CRC cells by m1A methylation. Silencing DIAPH3 suppresses the migration and invasion of CRC cells, potentially through the proteasome-dependent degradation of downstream KRT19.\u003c/p\u003e","manuscriptTitle":"m1A-regulated DIAPH3 promotes the invasiveness of colorectal cancer via stabilization of KRT19","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2024-06-05 21:42:08","doi":"10.21203/rs.3.rs-3350465/v2","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}},{"code":1,"date":"2023-09-29 22:10:45","doi":"10.21203/rs.3.rs-3350465/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":"4d9338a7-55b2-420a-9c7f-b6cc22091406","owner":[],"postedDate":"June 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-13T00:35:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-05 21:42:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-3350465","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3350465","identity":"rs-3350465","version":["v2"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}