Kern, FabianKrammes, LenaDanz, KarinDiener, CarolineKehl, TimKüchler, OliverFehlmann, TobiasKahraman, MustafaRheinheimer, StefanieAparicio-Puerta, ErnestoWagner, SylviaLudwig, NicoleBackes, ChristinaLenhof, Hans-Petervon Briesen, HagenHart, MartinKeller, AndreasMeese, Eckart2023-02-092023-02-092020-11-13Kern F, Krammes L, Danz K, Diener C, Kehl T, Küchler O, et al. Validation of human microRNA target pathways enables evaluation of target prediction tools. Nucleic Acids Res. 2021 Jan 11;49(1):127-144.http://hdl.handle.net/10668/16774MicroRNAs are regulators of gene expression. A wide-spread, yet not validated, assumption is that the targetome of miRNAs is non-randomly distributed across the transcriptome and that targets share functional pathways. We developed a computational and experimental strategy termed high-throughput miRNA interaction reporter assay (HiTmIR) to facilitate the validation of target pathways. First, targets and target pathways are predicted and prioritized by computational means to increase the specificity and positive predictive value. Second, the novel webtool miRTaH facilitates guided designs of reporter assay constructs at scale. Third, automated and standardized reporter assays are performed. We evaluated HiTmIR using miR-34a-5p, for which TNF- and TGFB-signaling, and Parkinson's Disease (PD)-related categories were identified and repeated the pipeline for miR-7-5p. HiTmIR validated 58.9% of the target genes for miR-34a-5p and 46.7% for miR-7-5p. We confirmed the targeting by measuring the endogenous protein levels of targets in a neuronal cell model. The standardized positive and negative targets are collected in the new miRATBase database, representing a resource for training, or benchmarking new target predictors. Applied to 88 target predictors with different confidence scores, TargetScan 7.2 and miRanda outperformed other tools. Our experiments demonstrate the efficiency of HiTmIR and provide evidence for an orchestrated miRNA-gene targeting.enAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/Cell LineGenes, ReporterMesencephalonNeuronsSensitivity and SpecificityTransforming Growth Factor beta1-Methyl-4-phenylpyridinium3' Untranslated RegionsCell Line, TumorGene Expression RegulationHigh-Throughput Screening AssaysHumansMicroRNAsNeuroblastomaParkinson DiseasePredictive Value of TestsSignal TransductionTranscriptomeTumor Necrosis Factor-alpharesearch article33305319open access1-Metil-4-fenilpiridinioEnfermedad de ParkinsonEnsayos analíticos de alto rendimientoFactor de necrosis tumoral alfaLínea celular tumoralMicroARNsNeuroblastomaRegiones no traducidas 3'Regulación de la expresión génicaTranscriptomaTransducción de señalValor predictivo de las pruebas10.1093/nar/gkaa11611362-4962PMC7797041https://academic.oup.com/nar/article-pdf/49/1/127/35597117/gkaa1161.pdfhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7797041/pdf