Melatonin Promotes Bone Marrow Mesenchymal Stem Cell Osteogenic Differentiation and Prevents Osteoporosis Development Through Modulating circ_0003865 that Sponges miR-3653-3p
Abstract Background: To investigate circRNAs in Melatonin (MEL)-regulated bone marrow mesenchymal stem cell (BMSC) differentiation and osteoporosis.Methods: BMSC osteogenic differentiation was measured by qRT-PCR, western blot (WB), Alizarin Red and alkaline phosphatase (ALP) staining. Differential circRNA and mRNA profiles of BMSCs treated by MEL were characterized by deep sequencing, followed by validation using RT-PCR, Sanger sequencing, and qRT-PCR. Silencing and overexpression of circ_0003865 were conducted for functional investigations. The sponged microRNAs and targeted mRNAs were predicted by bioinformatics and validated by qRT-PCR, RNA pull-down, and dual-luciferase reporter assay. The function of miR-3653-3p and circ_0003865/miR-3653-3p/growth arrest-specific gene 1 (GAS1) cascade were validated for the osteogenic differentiation of BMSCs by qRT-PCR, WB, Alizarin Red, and ALP staining. The effects of circ_0003865 on osteoporosis (OP) development was tested in murine osteoporosis model.Results: MEL promoted osteogenic differentiation of BMSCs. RNA sequencing revealed significant alterations in circRNA and mRNA profiles associated with multiple biological processes and signaling pathways. Circ_0003865 expression in BMSCs was significantly decreased by MEL treatment. Silencing of circ_0003865 promoted osteogenic differentiation of BMSCs. Overexpression of circ_0003865 abrogated the promotion of BMSCs osteogenic differentiation induced by MEL. Furthermore, circ_0003865 sponged miR-3653-3p to promote GAS1 expression in BMSCs. BMSC osteogenic differentiation was enhanced by miR-3653-3p overexpression. By contrast, miR-3653-3p silencing mitigated the promoted BMSC osteogenic differentiation caused by circ_0003865 silencing. Finally, circ_0003865 silencing repressed OP development in mouse model.Conclusion: MEL promotes BMSC osteogenic differentiation and inhibits osteoporosis pathogenesis by suppressing the expression of circ_0003865, which regulates GAS1 gene expression via sponging miR-3653-3p.