Abstract
Bone marrow-derived mesenchymal stem cells (BM-MSCs), the common progenitor cells of adipocytes and osteoblasts, have been recognized as the key mediator during bone formation. Herein, our study aim to investigate molecular mechanisms underlying circular RNA (circRNA) AFF4 (circ_AFF4)-regulated BM-MSCs osteogenesis. BM-MSCs were characterized by FACS, ARS, and ALP staining. Expression patterns of circ_AFF4, miR-135a-5p, FNDC5/Irisin, SMAD1/5, and osteogenesis markers, including ALP, BMP4, RUNX2, Spp1, and Colla1 were detected by qRT-PCR, western blot, or immunofluorescence staining, respectively. Interactions between circ_AFF4 and miR-135a-5p, FNDC5, and miR-135a-5p were analyzed using web tools including TargetScan, miRanda, and miRDB, and further confirmed by luciferase reporter assay and RNA pull-down. Complex formation between Irisin and Integrin αV was verified by Co-immunoprecipitation. To further verify the functional role of circ_AFF4 in vivo during bone formation, we conducted animal experiments harboring circ_AFF4 knockdown, and born samples were evaluated by immunohistochemistry, hematoxylin and eosin, and Masson staining. Circ_AFF4 was upregulated upon osteogenic differentiation induction in BM-MSCs, and miR-135a-5p expression declined as differentiation proceeds. Circ_AFF4 knockdown significantly inhibited osteogenesis potential in BM-MSCs. Circ_AFF4 stimulated FNDC5/Irisin expression through complementary binding to its downstream target molecule miR-135a-5p. Irisin formed an intermolecular complex with Integrin αV and activated the SMAD1/5 pathway during osteogenic differentiation. Our work revealed that circ_AFF4, acting as a sponge of miR-135a-5p, triggers the promotion of FNDC5/Irisin via activating the SMAD1/5 pathway to induce osteogenic differentiation in BM-MSCs. These findings gained a deeper insight into the circRNA-miRNA regulatory system in the bone marrow microenvironment and may improve our understanding of bone formation-related diseases at physiological and pathological levels.
Highlights
Bone formation, termed as osteogenesis or ossification, is a well-orchestrated dynamic process that is executed by osteoblasts[1]
Bone marrow-derived mesenchymal stem cells (BM-MSCs) treated with osteogenic medium (OM) displayed positive staining with both Alizarin red S (ARS) and Alkaline phosphatase (ALP) and demonstrated efficient osteogenic differentiation induction in comparison to those grown in a normal medium (NM)
Our quantitative real-time polymerase chain reaction results showed that these osteogenesis-related genes were greatly upregulated in OM-treated BM-MSCs as compared to those grown in NM (Fig. 1C), further confirming the osteoinduction effect in OMtreated BM-MSCs
Summary
Bone formation, termed as osteogenesis or ossification, is a well-orchestrated dynamic process that is executed by osteoblasts[1]. Liu et al Cell Death and Disease (2021)12:631 increases fat deposits in bone marrow may promote osteoclast proliferation and contribute to subsequent bone loss. Risk factors including aging, nutrition, corticosteroid medication usage in high doses, and an inactive lifestyle are recognized as main contributors to these bonerelated disorders, underlying molecular mechanisms for the BM-MSCs lineage commitment during the progression of the variety of bone diseases are highly diverse and not well clarified so far. It was reported to stimulate osteoblast differentiation and increase cortical bone strength by activating mitogenactivated protein kinase signaling pathway[10,11]. Despite its considerable roles in bone metabolism, detailed analysis is crucial to explain how Irisin exactly regulated osteogenic differentiation through recruitment of upstream and downstream signaling pathways
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