Literature-Identified Serum miRNA Signatures for Cognitive Decline: Integrated Analysis and Machine-Learning Diagnostics in Alzheimer’s Disease

Literature-Identified Serum miRNA Signatures for Cognitive Decline: Integrated Analysis and Machine-Learning Diagnostics in Alzheimer’s Disease | 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 Literature-Identified Serum miRNA Signatures for Cognitive Decline: Integrated Analysis and Machine-Learning Diagnostics in Alzheimer’s Disease Zhiyan Chen, Yadi Liu, Huan Wang, Ke Liu, Yutong Li, Xiaohua Hu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8620145/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Apr, 2026 Read the published version in Alzheimer's Research & Therapy → Version 1 posted 10 You are reading this latest preprint version Abstract Background Because identification of preclinical Alzheimer’s disease (AD) still relies on costly or invasive A/T/N biomarkers, we systematically integrated published case-control studies to identify differentially expressed serum microRNAs (miRNAs) associated with cognitive function in patients with AD, and validated their diagnostic performance in the large public cohort GSE120584. Methods We searched Chinese- and English-language databases for studies reporting serum miRNA expression differences in AD and their associations with cognitive scale scores. Validated target genes were retrieved from miRTarBase; protein–protein interaction (PPI) networks were constructed using STRING; functional modules were identified with the Cytoscape plug-in MCODE; and enrichment analyses were performed for GO, KEGG, Reactome, and Hallmark gene sets. Differential expression analysis in GSE120584 was conducted using limma with covariate adjustment (age, sex, and APOE4), and partial correlations between miRNA expression and age, sex, and APOE4 were calculated. Literature-derived miRNAs were matched to GSE120584, and unmatched miRNAs were supplemented by same-family candidates when necessary. Based on correlation-network analysis and nested cross-validation, optimal miRNA combinations were selected to construct diagnostic models with age or age + sex as baseline predictors. Model performance was evaluated using out-of-fold ROC and precision–recall (PR) curves, calibration curves, and decision curve analysis (DCA). Results Twenty-three publications including 2,580 patients with AD and 2,261 controls were included. Twenty-one differentially expressed serum miRNAs were identified, including miRNAs positively (n = 15) or negatively (n = 7) correlated with Mini-Mental State Examination (MMSE) scores. Targets of positively correlated miRNAs were enriched in PI3K/AKT/mTOR, Wnt, and TNF-α/NF-κB signaling pathways, whereas targets of negatively correlated miRNAs were mainly involved in cell cycle regulation, the G2/M checkpoint, and oxidative stress responses. After matching and expansion in GSE120584, 25 significantly differentially expressed miRNAs were identified. The minimal miRNA signatures with optimal diagnostic value were miR-211-5p alone (K1) and the three-miRNA panel miR-211-5p, miR-128-1-5p, and miR-128-3p (K3). When age and sex were added, the “K3 + age + sex” model showed the best performance (AUC = 0.838, AP = 0.934, Brier score = 0.162), yielding the highest sensitivity (0.563) and the best PPV (0.954) at specificity ≥ 0.90. Conclusion Using dual validation from published literature and a large cohort, we identified cognition-associated serum miRNAs in AD and established a combined diagnostic model integrating miRNA levels with clinical characteristics (age and sex). miR-211-5p and miR-128 family members appear promising as peripheral blood biomarkers, but require validation in independent cohorts. Alzheimer’s disease microRNAs(miRNA) MMSE differential expression analysis Full Text Additional Declarations No competing interests reported. Table 15 and 16 are not available with this version Supplementary Files Additionalfile1SearchStrategiesforChineseandEnglishDatabases.xlsx Additionalfile2NOSScaleAssessmentResultsforIncludedStudies.xlsx Additionalfile3SummaryofmiRNADifferentialExpressionandCorrelationwithMMSEScoresfromIncludedStudies..docx Additionalfile4TargetGenesoftheIncludedmiRNAs.xlsx Additionalfile5PartialCorrelationBetweenIncludedmiRNAsandClinicalBaselineCharacteristics.csv Additionalfile6PerformanceofmiRNAPanelsUnderDifferentBaselineConditions.xlsx Additionalfile7DiagnosticModelsBasedonmiRNAPanelsandTheirBaselinePerformance.csv Additionalfile8RolesofdifferentiallyexpressedpositivelycorrelatedmiRNAsincludedandexpandedsetinthepathologicalprocessofAD.docx Additionalfile9RolesofdifferentiallyexpressednegativelycorrelatedmiRNAsincludedandexpandedsetinthepathologicalprocessofAD.docx Cite Share Download PDF Status: Published Journal Publication published 20 Apr, 2026 Read the published version in Alzheimer's Research & Therapy → Version 1 posted Editorial decision: Revision requested 10 Mar, 2026 Reviews received at journal 29 Jan, 2026 Reviews received at journal 29 Jan, 2026 Reviewers agreed at journal 27 Jan, 2026 Reviewers agreed at journal 27 Jan, 2026 Reviewers agreed at journal 25 Jan, 2026 Reviewers invited by journal 22 Jan, 2026 Editor assigned by journal 19 Jan, 2026 Submission checks completed at journal 19 Jan, 2026 First submitted to journal 16 Jan, 2026 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-8620145","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":580768961,"identity":"228c7e53-8d8d-4cee-9a16-83840926f119","order_by":0,"name":"Zhiyan Chen","email":"","orcid":"","institution":"Beijing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhiyan","middleName":"","lastName":"Chen","suffix":""},{"id":580768962,"identity":"b3b83644-caa9-4057-929a-63a4328d1595","order_by":1,"name":"Yadi Liu","email":"","orcid":"","institution":"Xiyuan 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reported.\u003c/p\u003e\n\u003cp\u003eTable 15 and 16 are not available with this version\u003c/p\u003e","formattedTitle":"Literature-Identified Serum miRNA Signatures for Cognitive Decline: Integrated Analysis and Machine-Learning Diagnostics in Alzheimer’s Disease","fulltext":[],"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":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"alzheimers-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"azrt","sideBox":"Learn more about [Alzheimer's Research and Therapy](http://alzres.biomedcentral.com/)","snPcode":"13195","submissionUrl":"https://submission.nature.com/new-submission/13195/3","title":"Alzheimer's Research \u0026 Therapy","twitterHandle":"@AlzheimersRes","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer’s disease, microRNAs(miRNA), MMSE, differential expression analysis","lastPublishedDoi":"10.21203/rs.3.rs-8620145/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8620145/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBecause identification of preclinical Alzheimer\u0026rsquo;s disease (AD) still relies on costly or invasive A/T/N biomarkers, we systematically integrated published case-control studies to identify differentially expressed serum microRNAs (miRNAs) associated with cognitive function in patients with AD, and validated their diagnostic performance in the large public cohort GSE120584.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe searched Chinese- and English-language databases for studies reporting serum miRNA expression differences in AD and their associations with cognitive scale scores. Validated target genes were retrieved from miRTarBase; protein\u0026ndash;protein interaction (PPI) networks were constructed using STRING; functional modules were identified with the Cytoscape plug-in MCODE; and enrichment analyses were performed for GO, KEGG, Reactome, and Hallmark gene sets. Differential expression analysis in GSE120584 was conducted using limma with covariate adjustment (age, sex, and APOE4), and partial correlations between miRNA expression and age, sex, and APOE4 were calculated. Literature-derived miRNAs were matched to GSE120584, and unmatched miRNAs were supplemented by same-family candidates when necessary. Based on correlation-network analysis and nested cross-validation, optimal miRNA combinations were selected to construct diagnostic models with age or age\u0026thinsp;+\u0026thinsp;sex as baseline predictors. Model performance was evaluated using out-of-fold ROC and precision\u0026ndash;recall (PR) curves, calibration curves, and decision curve analysis (DCA).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTwenty-three publications including 2,580 patients with AD and 2,261 controls were included. Twenty-one differentially expressed serum miRNAs were identified, including miRNAs positively (n\u0026thinsp;=\u0026thinsp;15) or negatively (n\u0026thinsp;=\u0026thinsp;7) correlated with Mini-Mental State Examination (MMSE) scores. Targets of positively correlated miRNAs were enriched in PI3K/AKT/mTOR, Wnt, and TNF-α/NF-κB signaling pathways, whereas targets of negatively correlated miRNAs were mainly involved in cell cycle regulation, the G2/M checkpoint, and oxidative stress responses. After matching and expansion in GSE120584, 25 significantly differentially expressed miRNAs were identified. The minimal miRNA signatures with optimal diagnostic value were miR-211-5p alone (K1) and the three-miRNA panel miR-211-5p, miR-128-1-5p, and miR-128-3p (K3). When age and sex were added, the \u0026ldquo;K3\u0026thinsp;+\u0026thinsp;age\u0026thinsp;+\u0026thinsp;sex\u0026rdquo; model showed the best performance (AUC\u0026thinsp;=\u0026thinsp;0.838, AP\u0026thinsp;=\u0026thinsp;0.934, Brier score\u0026thinsp;=\u0026thinsp;0.162), yielding the highest sensitivity (0.563) and the best PPV (0.954) at specificity\u0026thinsp;\u0026ge;\u0026thinsp;0.90.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eUsing dual validation from published literature and a large cohort, we identified cognition-associated serum miRNAs in AD and established a combined diagnostic model integrating miRNA levels with clinical characteristics (age and sex). miR-211-5p and miR-128 family members appear promising as peripheral blood biomarkers, but require validation in independent cohorts.\u003c/p\u003e","manuscriptTitle":"Literature-Identified Serum miRNA Signatures for Cognitive Decline: Integrated Analysis and Machine-Learning Diagnostics in Alzheimer’s Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 05:46:53","doi":"10.21203/rs.3.rs-8620145/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-11T02:36:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-29T11:18:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-29T10:44:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"130552155355715291531417979461083007171","date":"2026-01-27T14:49:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167481096538736017155384859882471178720","date":"2026-01-27T11:01:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55173615599356969165646404798881960064","date":"2026-01-25T15:37:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-22T13:43:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-19T08:32:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-19T08:27:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Alzheimer's Research \u0026 Therapy","date":"2026-01-16T14:22:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"alzheimers-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"azrt","sideBox":"Learn more about [Alzheimer's Research and Therapy](http://alzres.biomedcentral.com/)","snPcode":"13195","submissionUrl":"https://submission.nature.com/new-submission/13195/3","title":"Alzheimer's Research \u0026 Therapy","twitterHandle":"@AlzheimersRes","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a4bd0d83-2581-4e24-b690-e3f5404a0fad","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:09:49+00:00","versionOfRecord":{"articleIdentity":"rs-8620145","link":"https://doi.org/10.1186/s13195-026-02048-x","journal":{"identity":"alzheimers-research-and-therapy","isVorOnly":false,"title":"Alzheimer's Research \u0026 Therapy"},"publishedOn":"2026-04-20 15:59:56","publishedOnDateReadable":"April 20th, 2026"},"versionCreatedAt":"2026-01-28 05:46:53","video":"","vorDoi":"10.1186/s13195-026-02048-x","vorDoiUrl":"https://doi.org/10.1186/s13195-026-02048-x","workflowStages":[]},"version":"v1","identity":"rs-8620145","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8620145","identity":"rs-8620145","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}