The miR-224-5p/SIRT3/AMPK/mTOR axis is involved in the melatonin-mediated inhibition of glucocorticoid-induced osteoporosis by activating autophagy.

Abstract

Melatonin has been shown to exert an inhibitory effect on osteoporosis. This study investigates the function of the miR-224-5p/SIRT3/AMPK/mTOR axis in melatonin-mediated effects against osteoporosis. Human bone marrow mesenchymal stem cells (hBMSCs) were treated with glucocorticoid dexamethasone to induce an in vitro osteoporosis model. After melatonin treatment, miR-224-5p and SIRT3 levels were measured by RT‒PCR. Transmission electron microscopy and immunofluorescence were conducted for evaluating autophagy. Western blotting was carried out to determine the expression of osteogenesis-related proteins (Runx2, OSX, OPN, and OCN), SIRT3-AMPK-mTOR axis, and autophagy-related markers (LC3 and p62). Alizarin red staining was used to measure matrix mineralization. The data showed that melatonin inhibited dexamethasone-induced osteoporosis in vitro, and enhanced autophagic levels (as indicated by increased LC3 puncta, LC3II/I ratio, and autophagic vacuoles). In terms of the mechanisms, melatonin decreased miR-224-5p expression and increased SIRT3. SRIT3 was shown to be a direct target of miR-224-5p. miR-224-5p upregulation or SIRT3 downregulation reversed the effects of melatonin on osteoporosis and suppressed autophagy. Additionally, miR-224-5p inhibited SIRT3 expression and AMPK pathway activation. In summary, we discovered that melatonin suppressed glucocorticoid-induced osteoporosis and autophagy inhibition via the miR-224-5p/SIRT3/AMPK/mTOR axis.