miR-324 regulates both osteoblast and osteoclast differentiation and function to control bone homeostasis II

microRNAs are non-coding RNAs which modulate the expression of other RNA molecules. One microRNA can target many transcripts, allowing each microRNA to play key roles in many biological pathways. miR-324 is a microRNA previously implicated in bone and cartilage maintenance, defects of which result in common age-related diseases, such as osteoporosis or osteoarthritis (OA). Cartilage damage was increased in both surgically and ageing-induced OA however changes in the cartilage transcriptome were minimal, with few miR-324 predicted targets dysregulated. However, in vivo micro-computed tomography and histology demonstrated that global miR-324-null mice had an increase in bone mineral density, trabecular thickness and cortical thickness, with many parameters increasing with age. The bones of the miR-null mice also had decreased osteocytes numbers and lipid droplets. 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, bone marrow macrophages and osteocytes, revealed that the osteoclast fusion regulator Pin1 was a miR-324 target in the osteoclast lineage, Hoxa9 and Samd5 were osteocyte target genes and the master osteogenic regulator Runx2 was 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. Elucidation of pathways regulated by miR-324 offers promise for the treatment of bone diseases such as osteoporosis. Overall design: Rib cages were dissected from individual mice (four per genotype; wild-type and miR-324-null). Tissue was digested with collagenase as described previously (PMID: 32660984) to release the cells (costal chondrocytes). RNA was extracted from the cells using trizol/mirVana miRNA Isolation Kit (Thermo Fisher Scientific), following the manufacturer's protocol to isolate total RNA.