Detecting new microRNAs in human osteoarthritic chondrocytes identifies miR-3085 as a human, chondrocyte-selective, microRNA

Crowe, Natalie, Swingler, Tracey, Le, Linh, Barter, Matt, Wheeler, Guy, Pais, Helio, Donell, Simon, Young, David, Dalmay, Tamas ORCID: https://orcid.org/0000-0003-1492-5429 and Clark, Ian (2016) Detecting new microRNAs in human osteoarthritic chondrocytes identifies miR-3085 as a human, chondrocyte-selective, microRNA. Osteoarthritis and Cartilage, 24 (3). pp. 534-543. ISSN 1063-4584

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Abstract

Objective: To use deep sequencing to identify novel microRNAs in human osteoarthritic cartilage which have a functional role in chondrocyte phenotype or function. Design: A small RNA library was prepared from human osteoarthritic primary chondrocytes using in-house adaptors and analysed by Illumina sequencing. Novel candidate microRNAs were validated by northern blot and qRT-PCR. Expression was measured in cartilage models. Targets of novel candidates were identified by microarray and computational analysis, validated using 3’-UTR-luciferase reporter plasmids. Protein levels were assessed by western blot and functional analysis by cell adhesion. Results: We identified 990 known microRNAs and 1621 potential novel microRNAs in human osteoarthritic chondrocytes, 60 of the latter were expressed in all samples assayed. MicroRNA-140-3p was the most highly expressed microRNA in osteoarthritic cartilage. Sixteen novel candidate microRNAs were analysed further, of which 6 remained after northern blot analysis. Three novel microRNAs were regulated across models of chondrogenesis, chondrocyte differentiation or cartilage injury. One sequence (novel #11), annotated in rodents as microRNA-3085-3p, was preferentially expressed in cartilage, dependent on chondrocyte differentiation and, in man, is located in an intron of the cartilage-expressed gene CRTAC-1. This microRNA was shown to target the ITGA5 gene directly (which encodes integrin alpha5) and inhibited adhesion to fibronectin (dependent on alpha5beta1 integrin). Conclusion: Deep sequencing has uncovered many potential microRNA candidates expressed in human cartilage. At least three of these show potential functional interest in cartilage homeostasis and osteoarthritis. Particularly, novel #11 (microRNA-3085-3p) which has been identified for the first time in man.

Item Type: Article
Additional Information: Licenced under CC BY NC ND: http://creativecommons.org/licenses/by-nc-nd/4.0/
Uncontrolled Keywords: osteoarthritis,cartilage,microrna,integrin
Faculty \ School: Faculty of Science > School of Biological Sciences
Faculty of Science > School of Computing Sciences
Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: Pure Connector
Date Deposited: 17 Feb 2016 17:00
Last Modified: 23 Sep 2022 02:07
URI: https://ueaeprints.uea.ac.uk/id/eprint/57155
DOI: 10.1016/j.joca.2015.10.002

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