Melatonin promotes osteogenic differentiation of BMSCs by regulating circZFAND1/miR-433-3p/DKK1 pathway through melatonin receptor-dependent m6A modification of circZFAND1.

Abstract

An effective approach for treating osteoporosis (OP) is to focus on restoring the osteogenic ability of bone marrow-derived mesenchymal stem cells (BMSCs). Melatonin (MEL) has been found to possess the capability to enhance osteogenesis. Nevertheless, the precise mechanisms through which MEL regulates the differentiation of BMSCs and the development of OP are still not well understood. In the present study, circZFAND1 characterization was validated using Sanger sequencing, RNase R treatment, and RNA fluorescence in situ hybridization assay. To investigate the relationship between circZFAND1, DKK1 and miR-433-3p, we performed a series of experiments including the luciferase reporter assay, RNA immunoprecipitation assay, and rescue experiments. In addition, the MEL-mediated m6A modification of circZFAND1 was evaluated using dot-blot assay and Methylated RNA immunoprecipitation (Me-RIP) assay. Finally, the osteogenic differentiation of BMSCs was detected through function assays, including immunofluorescence staining, 5-ethynyl-20-deoxyuridine (EdU) assay, western blotting, and staining with alkaline phosphatase and alizarin red. The results showed that MEL promoted osteogenic differentiation of BMSCs by regulating circZFAND1/miR-433-3p/DKK1 pathway through melatonin receptor 2 (MT2) -dependent m6A modification of circZFAND1. These findings offer novel biomarkers for managing OP and may lay the theoretical foundation for applying MEL for OP management in the clinical setting.