MicroRNA-579-3p promotes the progression of osteoporosis by inhibiting osteogenic differentiation of mesenchymal stem cells through regulating Sirt1

Abstract

Objective: To explore whether microRNA-579-3P was involved in the development of osteoporosis, and to investigate the possible molecular mechanisms.

Patients and methods: The messenger RNA (mRNA) expression levels of microRNA-579-3P, alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2) and bone sialoprotein (BSP) in serum samples of osteoporosis patients and normal controls were detected by quantitative Real-time polymerase chain reaction (qRT-PCR), respectively. Meanwhile, the expressions of the above genes during osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) were examined as well. To investigate the effect of microRNA-579-3P on osteogenesis, microRNA-579-3P was overexpressed and knocked down in hMSCs. Subsequently, the mRNA and protein expression levels of osteogenesis-related genes, such as ALP, RUNX2 and BSP, were detected by qRT-PCR and Western blot, respectively. In addition, ALP activity and mineralization forming ability were evaluated by ALP staining and alizarin red staining. Bioinformatics predicted that Sirt1 was the target gene of microRNA-579-3P. Subsequent luciferase reporter gene assay was performed to verify the binding relationship of microRNA-579-3P to Sirt1. Meanwhile, qRT-PCR and Western blot were used to detect the changes in the mRNA and protein expression levels of Sirt1, respectively. After overexpression of microRNA-579-3P and Sirt1, qRT-PCR, Western blot, ALP staining and alizarin red staining assays were performed to detect the osteogenic differentiation of hMSCs.

Results: The expression of microRNA-579-3P in serum of patients with osteoporosis was significantly higher than that of normal controls. Meanwhile, the expression of microRNA-579-3P decreased gradually during osteogenic differentiation of hMSCs. Overexpression of microRNA-579-3P significantly reduced the expressions of osteogenic related genes, including ALP, RUNX2 and BSP. Besides, ALP activity and mineralized nodule formation ability decreased obviously as well. Luciferase reporter gene assay showed that microRNA-579-3P could bind to Sirt1. After overexpression of microRNA-579-3P, the mRNA and protein expression levels of Sirt1 were significantly reduced, which were reversed after silence of microRNA-579-3P. Simultaneous overexpression of microRNA-579-3P and Sirt1 could reverse the inhibition of osteogenic differentiation of hMSCs caused by overexpression of microRNA-579-3P alone.

Conclusions: MicroRNA-579-3P could inhibit osteogenic differentiation of hMSCs by regulating Sirt1, thereby promoting the development of osteoporosis.