Honeysuckle-Derived MicroRNA2911 Inhibits Tumor Growth by Targeting TGF-β1

Honeysuckle-Derived MicroRNA2911 Inhibits Tumor Growth by Targeting TGF-β1 | 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 Honeysuckle-Derived MicroRNA2911 Inhibits Tumor Growth by Targeting TGF-β1 Chunyan Liu, Mengzhen Xu, Luocheng Yan, Yulian Wang, Zhen Zhou, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-498240/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background: Honeysuckle is a time‐honored herb with anticancer activity in traditional Chinese medicine. Recently, accumulating reports are suggesting that the microRNAs in this medicinal plant not only play a physiological role in its original system, but also can be transmitted to another species as potential therapeutic components. In the numerous bioactive investigations, the anti-tumor effects of these microRNAs in the magical herb are rarely studied, especially the special miR2911, a honeysuckle-encoded atypical microRNA, with high stability during the boiling process and unique biological activity to target TGF-β1 mRNA. Methods: Luciferase assay was conducted to test the ability of miR2911 to target TGF-β1 mRNA. ELISA was performed to determine the expression level of TGF-β1 of mouse colorectal adenocarcinoma cells CT26 when treated with miR2911 and tumor tissue in Sidt1 +/+ and Sidt1 -/- mice. qRT-PCR was performed to examine the level of expression of miR2911. Tumor-bearing wild and nude mice were employed to evaluate the anti-tumor effect of honeysuckle and miR2911 in vivo . Tumor tissue necrosis was observed by H&E staining. Besides, the infiltration of T lymphocytes across solid tumors was tested by immunostaining staining. Results: Our results showed that honeysuckle slowed the development of colon cancers down. Further research showed that miR2911 could bind strongly to TGF-β1 mRNA and down-regulated the expression of TGF-β1 and had a high stability under boiling and acid condition. Moreover, SIDT1 mediated dietary miR2911 inter-species absorption. And we found that miR2911 had a similar anticancer effect as honeysuckle. Mechanistically, miR2911 reversed the tumor-promoting effect of TGF-β1 by an increase of T lymphocytes infiltration, resulting in slowing the colon cancer process in immunocompetent mice. Consistent with this inference, the anti-tumor effect of miR2911 was revealed to be abolished in T cell immune deficiency mice. Conclusion: Taken together, honeysuckle-derived miR2911 showed an anti-tumor effect in colon cancers though targeting TGF-β1 mRNA. The down-regulation of TGF-β1 promoted T lymphocytes infiltration, and accordingly impeded the colon tumor development. Clinical Pharmacology Toxicology honeysuckle miR2911 colon cancer TGF-β1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 19 May, 2021 Review # 1 received at journal 09 May, 2021 Reviews received at journal 07 May, 2021 Reviewer # 2 agreed at journal 07 May, 2021 Reviewer # 1 agreed at journal 07 May, 2021 Editor assigned by journal 06 May, 2021 Reviewers invited by journal 06 May, 2021 Submission checks completed at journal 06 May, 2021 Editor invited by journal 06 May, 2021 First submitted to journal 05 May, 2021 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. 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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-498240","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":25790524,"identity":"32c5a888-b9fd-4ef6-949f-88bbc9c31c29","order_by":0,"name":"Chunyan Liu","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chunyan","middleName":"","lastName":"Liu","suffix":""},{"id":25790525,"identity":"a49e8aca-57ff-4a69-94d0-f3bb7d3fd281","order_by":1,"name":"Mengzhen Xu","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mengzhen","middleName":"","lastName":"Xu","suffix":""},{"id":25790526,"identity":"349435d6-9079-4868-af3a-1d93a808a5b5","order_by":2,"name":"Luocheng Yan","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Luocheng","middleName":"","lastName":"Yan","suffix":""},{"id":25790527,"identity":"b42bf402-fe9f-4986-8b1a-7ca8254434f7","order_by":3,"name":"Yulian Wang","email":"","orcid":"","institution":"Nanjing University - Xianlin Campus","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yulian","middleName":"","lastName":"Wang","suffix":""},{"id":25790528,"identity":"c883b413-414e-453c-8791-67962a8e25be","order_by":4,"name":"Zhen Zhou","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Zhou","suffix":""},{"id":25790529,"identity":"d222afa9-d1a9-46db-8f9e-3c21274da629","order_by":5,"name":"Shaocong Wang","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shaocong","middleName":"","lastName":"Wang","suffix":""},{"id":25790530,"identity":"cb2d3b21-f350-4db4-9ae6-4e4a36babee0","order_by":6,"name":"Yajie Sun","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yajie","middleName":"","lastName":"Sun","suffix":""},{"id":25790531,"identity":"f7c1e240-278f-4409-8ef6-12f68df9dd01","order_by":7,"name":"Junfeng Zhang","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Junfeng","middleName":"","lastName":"Zhang","suffix":""},{"id":25790532,"identity":"da359e9d-d679-47fd-b7a8-1ccdd11b8239","order_by":8,"name":"Lei Dong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYBACPgbGBgkgLcfAcABIsRGhhQ2qxZgULQwMIC2JDXAuQS3szY03Pu6oTd/OeMaA4UPZYQb+2Q0EtPAcbLaceeZ47s6GMwaMM84dZpC4c4CAFonENmnetmO5Gw6cMWDmbTvMYCCRQECL/EOwlnQDkJa/RGmRYARpqUkAa2EkSgtPItAvbQcMNxw4VnCw51w6j8QNAlr42Y8/vPGxrU7e4MbhjQ9+lFnL8c8goAUKDgNj5wA4MnmIUg8EdUD7GohVPApGwSgYBSMNAABuhkWTHSFDzgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2013-4191","institution":"Nanjing University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Dong","suffix":""}],"badges":[],"createdAt":"2021-05-06 05:41:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-498240/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-498240/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":9113287,"identity":"f75cbc6c-8cad-417d-a484-ea3714ef15f3","added_by":"auto","created_at":"2021-05-12 22:55:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":506224,"visible":true,"origin":"","legend":"The anticancer effect of honeysuckle on CT26 tumors in vivo. a Schematic diagram of assessing the anticancer effect of honeysuckle after the implantation of CT26 cells. b Gross view of the tumors in the honeysuckle-treated group and the control group. c The average sizes of tumors of the mice with different treatments. d Survive rates of the mice with different treatments. Data represent mean ± s.e.m., n = 5 per group. *P \u003c 0.05 was significant different between mock group and honeysuckle-treated group.","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-498240/v1/0c096d7664adca55b18780a3.png"},{"id":9113496,"identity":"a277f21d-83cf-4343-bc6a-e7be01a18015","added_by":"auto","created_at":"2021-05-12 22:58:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":121893,"visible":true,"origin":"","legend":"miR2911 is highly enriched in honeysuckle decotion and possesses a high stability. a The levels of miRNAs in honeysuckle decoction. b The miR2911 levels in honeysuckle decoction boiled for 0 h, 1 h, 2h or 3 h. c The miR2911 levels in honeysuckle decoction acidified by HCl to pH 2.0 and incubated for 0 h, 1 h, 2 h, or 3 h. Data represent mean ± s.e.m., n = 3 per group. *P \u003c 0.05 was significant different.","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-498240/v1/1afe69393f27c591067aea2e.png"},{"id":9113494,"identity":"b112dcb6-b865-496e-a35e-b86a4f3b3939","added_by":"auto","created_at":"2021-05-12 22:58:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":177481,"visible":true,"origin":"","legend":"Identification of TGF-β1 as a target of miR2911 in vitro. a Schematic description of the base pairing between miR2911 and TGF-β1 mRNA (mouse and human), and their binding energies. b Luciferase activity of HEK293T cells co-transfected with firefly luciferase reporters with miR2911 or NC miRNA treatment. WT: wild type firefly luciferase reporters with normal TGF-β1, Mutant: firefly luciferase reporters with mutant TGF-β1 3’-UTR. c The expression levels of TGF-β1 of CT26 cells in the mock group, the ncRNA-treated group and the miR2911-treated group. Data represent mean ± s.e.m., n = 3 per group. *P \u003c 0.05 was significant different between ncRNA-treated group and miR2911-treated group.","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-498240/v1/ae98e8ba2482ee2cdc312e17.png"},{"id":9113495,"identity":"7f78f5f4-0088-4b73-adef-d25a11943eb3","added_by":"auto","created_at":"2021-05-12 22:58:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":363331,"visible":true,"origin":"","legend":"SIDT1 helped miR2911 to transfer between species and realize the down-regulation of TGF-β1. a Diagram of miR2911 gavage feeding or intravenous injection treatment after the implantation of CT26 cells. b The miR2911 levels in blood of Sidt+/+ or Sidt-/- mice after gavage feeding or intravenous injection. c The miR2911, d TGF-β1 levels in CT26 tumor of Sidt1+/+ or Sidt1-/- mice after gavage feeding or intravenous injection. Data represent mean ± s.e.m., n = 5 per group. *P \u003c 0.05 was significant different.","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-498240/v1/761c4ff751d259144710f41b.png"},{"id":9113531,"identity":"49cde7be-723d-433c-bf99-0c24b1721496","added_by":"auto","created_at":"2021-05-12 23:01:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1578327,"visible":true,"origin":"","legend":"The anticancer effect of miR2911 in vivo. a Schematic diagram of assessing the anticancer effect of honeysuckle after the implantation of CT26 cells. b Gross view of the tumors in mock group and miR2911-treated group. c The tumor sizes of the mice with different treatments. d Representative photographs of the colon tumors with H\u0026E staining. e Survive rates of the mice after different treatments. Data represent mean ± s.e.m., n = 5 per group. *P \u003c 0.05 was significant different between ncRNA-treated group and miR2911-treated group.","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-498240/v1/c327fe7ab3cd0323fa3d4800.png"},{"id":9113558,"identity":"2514b0fa-e016-426f-8edb-34083c5c531b","added_by":"auto","created_at":"2021-05-12 23:04:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1997138,"visible":true,"origin":"","legend":"The role of T cells in the anti-tumor effect of miR2911. a, b Representative immunostaining images for CD4/CD8 immunostaining of the tumors in ncRNA-treated group and the miR2911-treated group, and the quantitative measurement of positive/negetive area percentage. c Gross view of the tumor-bearing nude mice treated ncRNA and miR2911, and the corresponding tumors. d The tumor sizes from the nude mice after different treatments. Data represent mean ± s.e.m., n = 5 per group. *P \u003c 0.05 was significant different between ncRNA-treated group and miR2911-treated group.","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-498240/v1/8dac432e176d0508d3a9cd8f.png"},{"id":13631219,"identity":"c7ebc889-1ae8-48ac-ab6e-e27bafdf4ca9","added_by":"auto","created_at":"2021-09-17 08:15:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":767227,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-498240/v1_covered.pdf"},{"id":9113559,"identity":"12e11c39-7343-4bdc-863e-98e9ce2681f2","added_by":"auto","created_at":"2021-05-12 23:04:25","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":763596,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-498240/v1_covered.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eHoneysuckle-Derived MicroRNA2911 Inhibits Tumor Growth by Targeting TGF-β1\u003c/p\u003e","fulltext":[{"header":"Full Text","content":"This preprint is available for \u003ca href='/article/rs-498240/latest.pdf' target='_blank'\u003edownload as a PDF\u003c/a\u003e."}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"honeysuckle, miR2911, colon cancer, TGF-β1","lastPublishedDoi":"10.21203/rs.3.rs-498240/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-498240/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Honeysuckle is a time‐honored herb with anticancer activity in traditional Chinese medicine. Recently, accumulating reports are suggesting that the microRNAs in this medicinal plant not only play a physiological role in its original system, but also can be transmitted to another species as potential therapeutic components. In the numerous bioactive investigations, the anti-tumor effects of these microRNAs in the magical herb are rarely studied, especially the special miR2911, a honeysuckle-encoded atypical microRNA, with high stability during the boiling process and unique biological activity to target TGF-β1 mRNA. \u003c/p\u003e\u003cp\u003eMethods: Luciferase assay was conducted to test the ability of miR2911 to target TGF-β1 mRNA. ELISA was performed to determine the expression level of TGF-β1 of mouse colorectal adenocarcinoma cells CT26 when treated with miR2911 and tumor tissue in Sidt1 +/+ and Sidt1 -/- mice. qRT-PCR was performed to examine the level of expression of miR2911. Tumor-bearing wild and nude mice were employed to evaluate the anti-tumor effect of honeysuckle and miR2911 in vivo . Tumor tissue necrosis was observed by H\u0026amp;E staining. Besides, the infiltration of T lymphocytes across solid tumors was tested by immunostaining staining. \u003c/p\u003e\u003cp\u003eResults: Our results showed that honeysuckle slowed the development of colon cancers down. Further research showed that miR2911 could bind strongly to TGF-β1 mRNA and down-regulated the expression of TGF-β1 and had a high stability under boiling and acid condition. Moreover, SIDT1 mediated dietary miR2911 inter-species absorption. And we found that miR2911 had a similar anticancer effect as honeysuckle. Mechanistically, miR2911 reversed the tumor-promoting effect of TGF-β1 by an increase of T lymphocytes infiltration, resulting in slowing the colon cancer process in immunocompetent mice. Consistent with this inference, the anti-tumor effect of miR2911 was revealed to be abolished in T cell immune deficiency mice. \u003c/p\u003e\u003cp\u003eConclusion: Taken together, honeysuckle-derived miR2911 showed an anti-tumor effect in colon cancers though targeting TGF-β1 mRNA. The down-regulation of TGF-β1 promoted T lymphocytes infiltration, and accordingly impeded the colon tumor development.\u003c/p\u003e","manuscriptTitle":"Honeysuckle-Derived MicroRNA2911 Inhibits Tumor Growth by Targeting TGF-β1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-05-12 22:55:08","doi":"10.21203/rs.3.rs-498240/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-05-19T05:43:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-05-10T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2021-05-08T03:49:00+00:00","index":0,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-05-08T01:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-05-08T00:00:00+00:00","index":1,"fulltext":""},{"type":"editorAssigned","content":"","date":"2021-05-07T00:00:00+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-05-07T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-05-06T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-05-06T23:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chinese Medicine","date":"2021-05-05T06:18:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4210030d-196d-45e2-8420-9ae2b55ebffc","owner":[],"postedDate":"May 12th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":4265901,"name":"Clinical Pharmacology"},{"id":4265902,"name":"Toxicology"}],"tags":[],"updatedAt":"2021-06-01T15:33:44+00:00","versionOfRecord":[],"versionCreatedAt":"2021-05-12 22:55:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-498240","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-498240","identity":"rs-498240","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}