Primate-Specific LINC03040 Drives Myeloid-Dependent Fibrosis in Human MASH Through a STAT3–S100A8 Regulatory Axis

Primate-Specific LINC03040 Drives Myeloid-Dependent Fibrosis in Human MASH Through a STAT3–S100A8 Regulatory Axis | 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 Primate-Specific LINC03040 Drives Myeloid-Dependent Fibrosis in Human MASH Through a STAT3–S100A8 Regulatory Axis Zia Zulistya Anugrah This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8780157/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 While the recent approval of resmetirom marks a historic milestone in MASH therapy, the effective reversal of established cirrhosis (F4) remains a major unmet clinical need. A fundamental limitation in anti-fibrotic drug development is the profound translational gap between murine models and human immunopathology, particularly with respect to species-specific non-coding regulatory elements. Methods To overcome this translational limitation, we integrated bulk transcriptomics from large patient cohorts (n = 216), single-cell–informed deconvolution, and comparative genomic analyses to characterize LINC03040 , a previously unannotated long non-coding RNA, coupled with in silico structural interaction modeling. Results LINC03040 emerged not only as a diagnostic marker but as a regulatory hub predictive of patient mortality. Challenging the prevailing stellate cell-centric paradigm, our analysis revealed that LINC03040 is spatially restricted to infiltrating myeloid populations. Mechanistically, integrative network inference and structural modeling suggest that LINC03040 functions as a regulatory scaffold linking STAT3 and S100A8 , while excluding PPARG , thereby coordinating pro-fibrotic myeloid signaling. Comparative genomics confirmed that the LINC03040 locus is primate-specific and entirely absent in rodents, underscoring a unique human pathology that is clinically validated by significant co-expression in patient tumors. Conclusion Our findings identify LINC03040 as a primate-specific upstream regulator associated with myeloid-mediated fibrosis in advanced MASH. The evolutionary absence of this locus in rodents underscores the limitations of standard preclinical models, advocating for the use of human-relevant systems to interrogate this immuno-metabolic pathway. Consequently, we propose the LINC03040–STAT3–S100A8 axis as a precise, human-centric targetable regulatory axis with potential relevance for fibrosis intervention. Bioinformatics Gastroenterology & Hepatology Medical Genetics MASH Liver Fibrosis LncRNA Primate-Specific Immuno-metabolism Single-cell Analysis Translational Gap Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Additional Declarations The authors declare no competing interests. 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-8780157","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588266519,"identity":"52503732-5736-441b-8daa-3f67c55f6e89","order_by":0,"name":"Zia Zulistya Anugrah","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYPCCAwxsDPwfDnwAMtnYidfCYPhwBkgLM7FagMDYmAdEEdLCP7vH+HVFzR17PvYDadI2v7bJ8zEzMH74mINbi8SdM2aWZ449Y2bjSTgmndt327CNmYFZcuY2PNbcyDEzbGA7zMbGkNgmndtzmxGohY2ZF48WebCWf4d52Pgfs0lb9ty2J6jF4EaO8cPGtsMSbBJpzMYMP24nEtRieCOtjLGx77ABm8Qbxoe9DbeT25gZm/H6Re5G8uaPDd8O28v35zAc+PHntu389uaDHz7i8z4w7iTgTMY2MNmAVz0QMH9AsP8QUjwKRsEoGAUjEQAAe0tQjgCINP8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0003-5374-5508","institution":"State University of Malang","correspondingAuthor":true,"prefix":"","firstName":"Zia","middleName":"Zulistya","lastName":"Anugrah","suffix":""}],"badges":[],"createdAt":"2026-02-03 22:34:08","currentVersionCode":2,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8780157/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-8780157/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103623503,"identity":"ed862a08-f66a-4fdc-9fc2-b9b7532473b9","added_by":"auto","created_at":"2026-02-27 19:15:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":327629,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlobal transcriptomic profiling identifies \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLINC03040\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e as a robustly upregulated driver in liver cirrhosis. (A)\u003c/strong\u003e Volcano plot displaying differential gene expression between cirrhotic (F4) and healthy (F0) liver tissues. The red diamond highlights \u003cem\u003eLINC03040\u003c/em\u003e as a significantly upregulated transcript among other dysregulated genes. \u003cstrong\u003e(B)\u003c/strong\u003e Boxplot validation confirming the marked elevation of \u003cem\u003eLINC03040\u003c/em\u003e expression in the F4 fibrosis group compared to F0 controls. Statistical significance was determined using the Mann-Whitney U test (P \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8780157/v2/16dc82b251d7c7235354dc31.png"},{"id":103623507,"identity":"c9db50ba-e3e7-4800-9798-13496ffc350f","added_by":"auto","created_at":"2026-02-27 19:15:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":288474,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical evaluation establishes \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLINC03040\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e as a dual utility biomarker for diagnosis and prognosis. (A) \u003c/strong\u003eReceiver Operating Characteristic (ROC) curve analysis assessing the diagnostic performance of \u003cem\u003eLINC03040\u003c/em\u003e in distinguishing cirrhotic patients (F4) from healthy controls (F0).\u0026nbsp;The Area Under the Curve (AUC) is\u0026nbsp;0.691,\u0026nbsp;indicating moderate discriminative power as a standalone marker. \u003cstrong\u003e(B)\u003c/strong\u003e\u0026nbsp;Kaplan-Meier survival analysis comparing Overall Survival (OS) between patients with high and low expression of \u003cem\u003eLINC03040\u003c/em\u003e (annotated as\u0026nbsp;\u003cem\u003eC6orf223\u003c/em\u003e\u0026nbsp;in the TCGA dataset).\u0026nbsp;Patients with elevated transcript levels exhibit significantly poorer survival outcomes (Log-rank test,\u0026nbsp;P = 0.037), with a Hazard Ratio (HR).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8780157/v2/9fca19c9320fa9f9a3120157.png"},{"id":104399645,"identity":"7b7bbc9d-36f7-4c26-81bf-2d0d035eb6f3","added_by":"auto","created_at":"2026-03-11 12:07:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":499604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular Source and Microenvironmental Regulation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLINC03040\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in MASH Fibrosis.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e\u0026nbsp;scRNA-seq analysis visualizes the cellular distribution of \u003cem\u003eLINC03040\u003c/em\u003e. The UMAP embedding and violin plots demonstrate that \u003cem\u003eLINC03040\u003c/em\u003e expression is highly specific to the\u0026nbsp;myeloid lineage\u0026nbsp;(clusters corresponding to monocytes, mig-cDCs, and cDC2s) and is absent in hepatocytes and stromal cells.\u0026nbsp;\u003cstrong\u003e(B)\u003c/strong\u003e\u0026nbsp;Microenvironment landscape heatmap showing Spearman’s correlation coefficients between \u003cem\u003eLINC03040\u003c/em\u003e and key immune/fibrotic markers in bulk RNA-seq data. Red indicates positive correlation. Note the strong co-expression with macrophage markers (CD163) and inflammatory regulators (\u003cem\u003eSTAT3\u003c/em\u003e, \u003cem\u003eNFKB1\u003c/em\u003e).\u0026nbsp;\u003cstrong\u003e(C)\u003c/strong\u003e\u0026nbsp;Targeted Gene Regulatory Network (GRN) constructed to visualize the interactome of LINC03040. The network highlights a direct regulatory link between the myeloid marker\u0026nbsp;S100A8, the inflammatory mediator \u003cem\u003eSTAT3\u003c/em\u003e, and downstream fibrogenic effectors such as\u0026nbsp;COL1A1\u0026nbsp;and\u0026nbsp;ACTA2, supporting an immune-driven fibrogenic mechanism.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8780157/v2/6419ff1dc97604d7da909119.png"},{"id":104399278,"identity":"7264bf3b-61b8-4b0b-9e32-589816a533cc","added_by":"auto","created_at":"2026-03-11 12:05:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":229959,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional Enrichment Analysis Reveals Immuno-Metabolic Reprogramming.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e\u0026nbsp;Bar plot showing the top enriched KEGG pathways associated with \u003cem\u003eLINC03040\u003c/em\u003e expression. Red bars indicate upregulated pathways (positive Normalized Enrichment Score/NES), while blue bars indicate downregulated pathways. Note the significant activation of\u0026nbsp;Steroid Biosynthesis\u0026nbsp;and\u0026nbsp;ECM-receptor interaction, contrasting with the suppression of\u0026nbsp;Cytochrome P450\u003cstrong\u003e \u003c/strong\u003emetabolism, reflecting a shift from homeostasis to fibrotic disease.\u0026nbsp;\u003cstrong\u003e(B)\u003c/strong\u003e\u0026nbsp;Gene Set Enrichment Analysis (GSEA) plot for the\u0026nbsp;Steroid Biosynthesis\u0026nbsp;pathway. The green curve indicates a strong positive enrichment (NES = 2.11, FDR = 0.004) of cholesterol-related genes in samples with high \u003cem\u003eLINC03040\u003c/em\u003e expression, supporting the mechanism of lipid-laden foam cell formation in the fibrotic microenvironment.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8780157/v2/7cc9a0a3f1ed857c15d08a35.png"},{"id":103623505,"identity":"f6c8c202-b712-491f-9b97-f05c9c48cbf5","added_by":"auto","created_at":"2026-02-27 19:15:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":140355,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvolutionary, structural, and functional characterization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLINC03040\u003c/strong\u003e\u003c/em\u003e.\u0026nbsp;\u003cstrong\u003e(A)\u003c/strong\u003e\u0026nbsp;Comparative genomic alignment (UCSC Multiz) showing the primate-specific conservation of the\u0026nbsp;\u003cem\u003eLINC03040\u003c/em\u003e\u0026nbsp;locus (Syntenic Gap in rodents). \u003cstrong\u003e(B)\u003c/strong\u003e\u0026nbsp;Predicted secondary structure revealing stable stem-loop motifs.\u0026nbsp;\u003cstrong\u003e(C)\u0026nbsp;\u003c/strong\u003e\u003cem\u003eIn silico\u003c/em\u003e\u0026nbsp;interaction validation (catRAPID Omics) demonstrating specific high-affinity binding of \u003cem\u003eLINC03040\u003c/em\u003e to\u0026nbsp;\u003cem\u003eSTAT3\u003c/em\u003e\u0026nbsp;and\u0026nbsp;\u003cem\u003eS100A8\u003c/em\u003e\u0026nbsp;(Z \u0026gt; 1.96), but not to the negative controls\u0026nbsp;\u003cem\u003eRELA\u003c/em\u003e\u0026nbsp;and\u0026nbsp;\u003cem\u003ePPARG\u003c/em\u003e\u0026nbsp;(Z \u0026lt; 1.96).\u0026nbsp;\u003cstrong\u003e(D)\u003c/strong\u003e\u0026nbsp;Clinical validation using GEPIA2 showing a significant positive correlation between\u0026nbsp;\u003cem\u003eLINC03040\u003c/em\u003e\u0026nbsp;and\u0026nbsp;\u003cem\u003eSTAT3\u003c/em\u003e\u0026nbsp;expression in LIHC tumor tissues (R = 0.26, P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8780157/v2/c3441af8c67c4ab16b8069e5.jpg"},{"id":104808291,"identity":"4e94c804-02ba-4a16-89e7-7212dd2ed35b","added_by":"auto","created_at":"2026-03-17 12:35:27","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1584313,"visible":true,"origin":"","legend":"","description":"","filename":"LINC03040MASHZiaAnugrah.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8780157/v2_covered_6c24330a-78c0-42c3-9811-5f22b8a15d2a.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003ePrimate-Specific \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLINC03040\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Drives Myeloid-Dependent Fibrosis in Human MASH Through a \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSTAT3–S100A8 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eRegulatory Axis\u003c/strong\u003e\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"State University of Malang","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":"MASH, Liver Fibrosis, LncRNA, Primate-Specific, Immuno-metabolism, Single-cell Analysis, Translational Gap","lastPublishedDoi":"10.21203/rs.3.rs-8780157/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8780157/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eWhile the recent approval of resmetirom marks a historic milestone in MASH therapy, the effective reversal of established cirrhosis (F4) remains a major unmet clinical need. A fundamental limitation in anti-fibrotic drug development is the profound translational gap between murine models and human immunopathology, particularly with respect to species-specific non-coding regulatory elements.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo overcome this translational limitation, we integrated bulk transcriptomics from large patient cohorts (n\u0026thinsp;=\u0026thinsp;216), single-cell\u0026ndash;informed deconvolution, and comparative genomic analyses to characterize \u003cem\u003eLINC03040\u003c/em\u003e, a previously unannotated long non-coding RNA, coupled with in silico structural interaction modeling.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e \u003cem\u003eLINC03040\u003c/em\u003e emerged not only as a diagnostic marker but as a regulatory hub predictive of patient mortality. Challenging the prevailing stellate cell-centric paradigm, our analysis revealed that \u003cem\u003eLINC03040\u003c/em\u003e is spatially restricted to infiltrating myeloid populations. Mechanistically, integrative network inference and structural modeling suggest that \u003cem\u003eLINC03040\u003c/em\u003e functions as a regulatory scaffold linking \u003cem\u003eSTAT3\u003c/em\u003e and \u003cem\u003eS100A8\u003c/em\u003e, while excluding \u003cem\u003ePPARG\u003c/em\u003e, thereby coordinating pro-fibrotic myeloid signaling. Comparative genomics confirmed that the \u003cem\u003eLINC03040\u003c/em\u003e locus is primate-specific and entirely absent in rodents, underscoring a unique human pathology that is clinically validated by significant co-expression in patient tumors.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur findings identify \u003cem\u003eLINC03040\u003c/em\u003e as a primate-specific upstream regulator associated with myeloid-mediated fibrosis in advanced MASH. The evolutionary absence of this locus in rodents underscores the limitations of standard preclinical models, advocating for the use of human-relevant systems to interrogate this immuno-metabolic pathway. Consequently, we propose the \u003cem\u003eLINC03040\u0026ndash;STAT3\u0026ndash;S100A8\u003c/em\u003e axis as a precise, human-centric targetable regulatory axis with potential relevance for fibrosis intervention.\u003c/p\u003e","manuscriptTitle":"Primate-Specific LINC03040 Drives Myeloid-Dependent Fibrosis in Human MASH Through a STAT3–S100A8 Regulatory Axis","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2026-02-27 19:15:01","doi":"10.21203/rs.3.rs-8780157/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":"2026-02-09 12:11:47","doi":"10.21203/rs.3.rs-8780157/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":"261c4785-7f98-40c7-b95c-d701847b853d","owner":[],"postedDate":"February 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":62592574,"name":"Bioinformatics"},{"id":62592575,"name":"Gastroenterology \u0026 Hepatology"},{"id":62592576,"name":"Medical Genetics"}],"tags":[],"updatedAt":"2026-02-09T12:11:47+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-27 19:15:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-8780157","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8780157","identity":"rs-8780157","version":["v2"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}