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dc.contributor.authorWoods, Steven
dc.contributor.authorBarter, Matt J
dc.contributor.authorElliott, Hannah R
dc.contributor.authorMcGillivray, Catherine M
dc.contributor.authorBirch, Mark
dc.contributor.authorClark, Ian M
dc.contributor.authorYoung, David A
dc.date.accessioned2018-11-17T00:31:28Z
dc.date.available2018-11-17T00:31:28Z
dc.date.issued2019-04
dc.identifier.issn0945-053X
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/285368
dc.description.abstractThe Hedgehog (Hh) signalling pathway plays important roles during embryonic development and in adult tissue homeostasis, for example cartilage, where its deregulation can lead to osteoarthritis (OA). microRNAs (miRNAs) are important regulators of gene expression, and have been implicated in the regulation of signalling pathways, including Hh, thereby impacting upon development and disease. Our aim was to identify the function of miRNAs whose expression is altered in OA cartilage. Here we identified an increase in miR-324-5p expression in OA cartilage and hypothesised that, as in glioma, miR-324-5p would regulate Hh signalling. We determined that miR-324-5p regulates osteogenesis in human mesenchymal stem cells (MSCs) and in mouse C3H10T1/2 cells. Luciferase reporter assays demonstrated that miR-324-5p directly regulated established targets GLI1 and SMO in human but not in mouse, suggesting species-dependent mechanism of Hh pathway regulation. Stable Isotope Labelling with Amino acids in Cell culture (SILAC), mass spectrometry and whole genome transcriptome analysis identified Glypican 1 (Gpc1) as a novel miR-324-5p target in mouse, which was confirmed by real-time RT-PCR, immunoblotting and 3'UTR-luciferase reporters. Knockdown of Gpc1 reduced Hh pathway activity, and phenocopied the effect of miR-324-5p on osteogenesis, indicating that miR-324-5p regulates Hh signalling in mouse via direct targeting of Gpc1. Finally, we showed that human GPC1 is not a direct target of miR-324-5p. Importantly, as well as identifying novel regulation of Indian Hedgehog (Ihh) signalling, this study demonstrates how a miRNA can show conserved pathway regulation in two species but by distinct mechanisms and highlights important differences between human diseases and mouse models.
dc.format.mediumPrint-Electronic
dc.languageeng
dc.publisherElsevier BV
dc.rightsAttribution 4.0 International
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectCartilage
dc.subjectCell Line
dc.subjectMesenchymal Stem Cells
dc.subjectAnimals
dc.subjectHumans
dc.subjectMice
dc.subjectOsteoarthritis
dc.subjectDisease Models, Animal
dc.subjectLuciferases
dc.subjectMicroRNAs
dc.subjectRNA, Small Interfering
dc.subject3' Untranslated Regions
dc.subjectSignal Transduction
dc.subjectSpecies Specificity
dc.subjectGene Expression Regulation
dc.subjectGenes, Reporter
dc.subjectAdult
dc.subjectHedgehog Proteins
dc.subjectGlypicans
dc.subjectZinc Finger Protein GLI1
dc.subjectSmoothened Receptor
dc.titlemiR-324-5p is up regulated in end-stage osteoarthritis and regulates Indian Hedgehog signalling by differing mechanisms in human and mouse.
dc.typeArticle
prism.endingPage100
prism.publicationDate2019
prism.publicationNameMatrix Biol
prism.startingPage87
prism.volume77
dc.identifier.doi10.17863/CAM.32734
dcterms.dateAccepted2018-08-20
rioxxterms.versionofrecord10.1016/j.matbio.2018.08.009
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserved
rioxxterms.licenseref.startdate2019-04
dc.contributor.orcidElliott, Hannah R [0000-0002-1500-3533]
dc.identifier.eissn1569-1802
rioxxterms.typeJournal Article/Review
pubs.funder-project-idMedical Research Council (MC_PC_12009)
pubs.funder-project-idTCC (None)
cam.issuedOnline2018-09-05


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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International