Circular RNA AFF4 modulates osteogenic differentiation in BM-MSCs by activating SMAD1/5 pathway through miR-135a-5p/FNDC5/Irisin axis

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.

IRF4 suppresses osteogenic differentiation of BM-MSCs by transcriptionally activating miR-636/DOCK9 axis

“…To induce osteogenic differentiation, BM-MSCs were divided into Control and OIM groups, and BM-MSCs in OIM groups were treated by a Human Mesenchymal Stem Cell (hMSC) Osteogenic Differentiation Medium Bullet Kit (Lonza, Switzerland) as previously reported. 21 …”

Impact of Exercise and Aging on Mitochondrial Homeostasis in Skeletal Muscle: Roles of ROS and Epigenetics

“…MiR-135a-5P inhibits the expression of FNDC5 [196]. Acting as a sponge of miR-135a-5p, circRNA ATF4 could upregulate the expression of FNDC5/irisin [17]. Exercise induced neurogenesis in the mouse hippocampus and the proliferation of neural precursor cells of the dentate gyrus of aged mice by downregulating miR-135a-5p [197].…”

“…Skeletal muscle can secrete a variety of myokines, which promote crosstalk between skeletal muscle and other organs. Recent evidence suggests that the expression of myokines is also regulated by epigenetic modifications [16][17][18][19][20][21].…”

The Emerging Role of Non-Coding RNAs in Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells

“…During NOTCH/RBPJ signaling pathway, the Notch intracellular domain translocates to the nucleus and binds with RBPJ and co-activators, forming a complex that induces the transcription of downstream gene DAB1 ( Luo et al, 2019 ). CircRNA AFF4 activates the expression of fibronectin type III domain-containing protein 5 (FNDC5)/Irisin through Smad1/5 pathway via sponging miR-135a-5 p, which induces the osteogenic differentiation of BMSCs in vitro and ectopic bone formation in vivo ( Liu et al, 2021 ). Moreover, circ_AFF4 was reported to promote osteoblastic proliferation by acting as a miR-7223-5p sponge ( Mi et al, 2019 ).…”