The Role And Mechanism Of Bone Marrow Mesenchymal Stem Cell-derived Exosomal Mir-24-3p In Regulating Osteogenesis

BackgroundOsteoporosis is a disease that results in increased bone fragility and fracture risk due to reduced bone mass and degradation of bone microarchitecture.The pathogenesis is due to an imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption,resulting in a negative balance of bone formation at the systemic tissue level.Since the effective prevention and treatment of osteoporosis still faces great problems,it is of great significance to strengthen the research on the pathogenesis of osteoporosis and explore new therapies.Exosomes are extracellular vesicles secreted by cells that mediate intercellular communication.Studies have found that bone marrow mesenchymal stem cell(BMSC)-derived exosomes(BMSC-Exos)improve osteoporosis by promoting proliferation and differentiation of osteoblast.In addition,the potential of osteoporotic bone marrow BMSC-derived exosomes to promote osteoblast differentiation is weaker than that of non-osteoporotic,but the specific mechanism is still unclear.Some research has indicated that bone marrow mesenchyml stem cell(BMSC)-derived exosomes(BMSC-Exos)can improve osteoporosis by promoting osteoblast proliferation and differentiation.Some studies have reported that osteoporotic BMSC-derived exosomes have a lower potential to promote osteoblast differentiation in vitro compared to non-osteoporotic BMSC-derived exosomes,but the specific mechanism is still unclear.The microRNAs(miRNAs)carried by exosomes are considered to be key regulators of osteoporosis and are expected to be new targets for osteoporosis treatment.MicroRNAs(miRNAs)carried by exosomes may be key regulators and are expected to become new targets for osteoporosis treatment.ObjectiveTo clarify the differences in the effects of BMSC-derived exosomes from ovariectomized(OVX)-induced postmenopausal osteoporotic rat(OVX-Exos)and sham-operated(Sham)non-osteoporotic rat(Sham-Exos)in osteogenesis.Identification of differentially expressed miRNA between OVX-Exos and Sham-Exos.To explore the regulatory role and targets of BMSC-derived exosomal miRNA in osteogenesis and provide a theoretical basis for the development of drugs for the prevention and treatment of osteoporosis.Methods1.BMSC was isolated from the bone marrow of OVX and Sham rats,and exosomes were isolated and purified from the supernatant of BMSC using differential ultracentrifugation.2.OVX-Exos and Sham-Exos were co-cultured with osteoblasts,respectively,to verify their effects on osteoblastic bone formation in vitro.OVX-Exos and Sham-Exos were infused into OVX rats through the tail vein,respectively,to verify their effects on bone losss.3.The miRNA expression profiles of OVX-Exos and Sham-Exos were detected by second-generation sequencing.4.Validation of the regulatory effects of BMSC-derived exosomal miRNA on bone formation and bone loss in OVX rats by gain-and loss-of function experiments.5.The target genes of candidate miRNAs were predicted and their targeting relationships were verified with dual luciferase reporter assays.Results1.The potential of OVX-Exos to promote osteoblastic bone formation in vitro was weaker than that of Sham-Exos,as evidenced by the lower ALP activity,number of mineralized nodules,and expression levels of osteogenic markers in osteoblasts co-cultured with OVX-Exos compared to Sham-Exos.The potential of OVX-Exos to prevent bone loss in OVX rats in vivo was weaker than that of Sham-Exos.2.Next-generation sequencing identified 20 miRNAs with significant differences between OVX-Exos and Sham-Exos,among which miR-24-3p had the largest differential expression fold.The expression of miR-24-3p showed a decreasing trend during osteoblast mineralization.Overexpression of miR-24-3p inhibited osteoblastic bone formation,while inhibition of miR-24-3p expression promoted bone formation.3.The expression of fibroblast growth factor receptor 3(FGFR3)was negatively correlated with miR-24-3p.Dual luciferase reporter assay confirmed that FGFR3 is a target gene of miR-24-3p and is involved in the regulation of bone formation by miR-24-3p.Conclusion1.OVX-Exos were weaker than Sham-Exos in promoting osteoblastic bone formation in vitro and preventing bone loss in OVX rats in vivo.2.There were many differentially expressed miRNAs including miR-24-3p between OVX-Exos and Sham-Exos.3.BMSC-derived exosomal miR-24-3p negatively regulated osteoblastic bone formation by targeting FGFR3,and inhibiting the expression of miR-24-3p in vivo prevented bone loss in OVX rats.