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

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. Medial knee cartilage caps were dissected from individual mice (five per genotype; wild-type and miR-324-null). Tissue was washed three times with sterile DPBS, placed in cryogenic vials and immediately frozen in liquid nitrogen. For grinding, the tissue was placed in an autoclaved chamber with a ball (Retsch, Verder Scientific UK Ltd, UK) and 250µl QIAzol lysis reagent (QIAGEN, Manchester, UK). The chambers were transferred to Retsch MM200 mixer mill and tissue ground at vibration frequency of 25 Hz for 90 seconds. To this was added an additional 250µl QIAzol lysis reagent and the mixture transferred to an RNase free tube and incubated at room temperature for 5 minutes. 100µl of chloroform was added and the sample vortexed for 15 s, incubated at room temperature for 10 minutes then centrifuged at 12,500 x g at 4oC for 5 minutes. The upper RNA containing aqueous phase was transferred into a new Eppendorf and RNA and miRNA were purified using a mirVana™ miR Isolation Kit (Ambion, ThermoFisher Scientific, Loughborough, UK) followed by DNAse treatment (DNA-free™ DNA Removal Kit, Invitrogen, ThermoFisher Scientific) following the manufacturer's protocol.