MicroRNA-9-5p promotes osteoporosis development through inhibiting osteogenesis and promoting adipogenesis via targeting Wnt3a

H.-G. Zhang, X.-B. Wang, H. Zhao, C.-N. Zhou

Department of Traumatology, Yantaishan Hospital, Yantai, China. fanba05328829@163.com

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• Orthopedics

OBJECTIVE: To explore the role of microRNA-9-5p in regulating osteoporosis (OS) development and its underlying mechanism.

PATIENTS AND METHODS: MicroRNA-9-5p expression in peripheral blood of 30 OS patients and 30 healthy subjects was examined by quantitative Real-Time-Polymerase Chain Reaction (qRT-PCR). During the processes of osteogenesis and adipogenesis, mRNA levels of microRNA-9-5p, osteogenesis-related genes, and adipogenesis-related genes in marrow stromal stem cells (MSCs) were detected by qRT-PCR as well. After overexpression or knockdown of microRNA-9-5p, the regulatory effects of microRNA-9-5p on osteogenesis-related genes and adipogenesis-related genes in MSCs were accessed by detecting their mRNA and protein levels. Alizarin red staining and oil red staining were performed to determine the osteogenic and adipogenic capacities of MSCs after microRNA-9-5p overexpression, respectively. The dual-luciferase reporter gene assay was conducted to verify the binding condition of microRNA-9-5p and Wnt3a. Finally, rescue experiments were performed to confirm whether microRNA-9-5p could regulate OS development via targeting Wnt3a.

RESULTS: Higher expression of microRNA-9-5p was found in OS patients than that of healthy controls. MicroRNA-9-5p expression was downregulated with the prolongation of osteogenic induction, whereas it was upregulated during the process of adipogenic differentiation. Overexpression of microRNA-9-5p downregulated mRNA levels of osteogenesis-related genes (ALP, RUNX2, and OPN), whereas upregulated adipogenesis-related genes (PPARγ, Adipsin, and C/EBPα) in MSCs. The number of calcified nodules became fewer after microRNA-9-5p overexpression in MSCs. MSCs that overexpressed microRNA-9-5p showed more lipid droplets than that of controls. Subsequently, the dual-luciferase reporter gene assay verified that Wnt3a is the target gene of microRNA-9-5p. Both mRNA and protein levels of Wnt3a were negatively regulated by microRNA-9-5p. Rescue experiments indicated that the regulatory effects of microRNA-9-5p on osteogenesis and adipogenesis of MSCs were reversed by Wnt3a overexpression.

CONCLUSIONS: MicroRNA-9-5p is lowly expressed in the peripheral blood of OS patients. MicroRNA-9-5p promotes the occurrence and progression of OS through inhibiting osteogenesis and promoting adipogenesis via targeting Wnt3a.

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