Pirog, K. A. | bioRxiv Science

bioRxiv2023

miR-324 mediates bone homeostasis through the regulation of osteoblast and osteoclast differentiation and activity

microRNAs (miRNAs) are non-coding RNAs which modulate the expression of other RNA molecules. One miRNA can target many transcripts, allowing each miRNA to play key roles in many biological pathways. miR-324 is a miRNA previously implicated in bone and cartilage maintenance, defects of which result in common age-related diseases, such as osteoporosis or osteoarthritis (OA). In global miR-324-null mice cartilage damage was increased in both surgically and ageing-induced OA, despite minimal changes to the cartilage transcriptome, with few predicted miR-324 targets dysregulated. However, micro-computed tomography and histology demonstrated that global miR- 324-null the mice had an increase in bone mineral density, trabecular thickness and cortical thickness, with many parameters increasing with age. The bone marrow of miR-324-null mice also had reduced lipid content while and in vivo TRAP staining revealed a decrease in osteoclasts, with histomorphometry demonstrating an increased rate of bone formation in miR-324-null mice. Ex vivo assays revealed that the high bone mass phenotype of the miR-324-null mice resulted from increased osteoblast activity and decreased osteoclastogenesis. RNA-seq and qRT-PCR followed by miR-324 target prediction and validation in osteoblasts, osteoclasts and bone marrow macrophages identified the osteoclast fusion regulator Pin1 as a miR-324 target in the osteoclast lineage and the master osteogenic regulator Runx2 as a target of miR-324-5p in osteoblasts, the in vitro overexpression of which recapitulated the increased osteogenesis and decreased adipogenesis phenotype observed in vivo. These data point to important roles of miR-324 in skeletal biology with altered bone homeostasis in miR-324-null mice potentially causal for the increased cartilage damage observed during OA and ageing. Elucidation of pathways regulated by miR-324 offer promise for the treatment of bone diseases such as osteoporosis.

cell biology↗

bioRxiv2020

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

MiR-140 is selectively expressed in cartilage. Deletion of the entire miR-140 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 datasets, 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.

molecular biology↗