microRNA-seq of cartilage reveals an over-abundance of miR-140-3p which contains functional isomiRs

Steven Woods, Sarah Charlton, Kathleen Cheung, Yao Hao, Jamie Soul, Louise N. Reynard, Natalie Crowe, Tracey E. Swingler, Andrew J. Skelton, Katarzyna A. Piróg, Colin G. Miles, Dimitra Tsompani, Robert M. Jackson, Tamas Dalmay, Ian M. Clark, Matt J. Barter, David A. Young

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miR-140 is selectively expressed in cartilage. Deletion of the entire Mir140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology; however, the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5' and 3' end, with >99% having one of two seed sequences (5' bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes, respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3'UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140- null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published data sets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140- 3p.1 is more functional than original consensus miR-140-3p seed containing isomiR.

Original languageEnglish
Pages (from-to)1575-1588
Number of pages14
Issue number11
Early online date13 Jul 2020
Publication statusPublished - Nov 2020

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