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. Overall design: Osteoblasts and haematopoietic stromal cells were isolated from leg bone chips and marrow respectively, dissected from the long bones of 20-week-old mice. The epiphyses were removed to expose the marrow, which was flushed from the bones by centrifugation and cultured directly with a-MEM, supplemented with 100 ng/mL M-CSF, for 3 days to obtain M-CSF-dependent bone marrow macrophages (BMMs). The remaining bone was cut into small chips of approximately 2 mm3 and collagenase digested for 1 hour. Subsequently, the bone chips were washed with PBS and cultured in supplemented a-MEM and cultured for approximately 6 weeks until semi confluent layers of osteoblasts were present. Osteoblasts were subsequently plated in osteogenic growth media (complete a-MEM supplemented with 2 mM ß-glycerophosphate (BGP) and 50 µg/mL L-ascorbic acid (both Sigma-Aldrich)) for 18 days, before RNA was extracted. BMMs were plated in complete a-MEM, supplemented either with 30 ng/mL M-CSF and 50 ng/mL RANK-L to stimulate osteoclastogenesis (samples labelled osteoclasts) or 30 ng/mL M-CSF only (samples labelled BMMs) and stimulated for 5 days, before RNA was extracted. Total RNA was extracted from BMMs, osteoclasts and osteoblasts using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific), following the manufacturer's protocols, before all samples were purified using the DNA-free™ DNA Removal Kit (ThermoFisher Scientific) to remove any DNA contamination.