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

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.

A Chemotherapy-Driven Increase in Mcl-1 Mediates the Effect of miR-375 on Cisplatin Resistance in Osteosarcoma Cells.

BACKGROUND: Osteosarcoma (OS) is one of the most difficult cancers to treat due to its resistance to chemotherapy. The essential role played by Mcl-1 in promoting chemoresistance has been observed in a variety of cancers, including OS, while the underlying mechanism remains unclear. METHODS: We investigated the expression of Mcl-1 in 42 paired OS specimens obtained before and after adjuvant chemotherapy, and its correlation with clinicopathological characteristics. Loss and gain of function studies were performed to analyze the effects of Mcl-1 modulations on the chemosensitivity, and the mechanism involved in the deregulation of Mcl-1 in OS cells. RESULTS: In OS specimens, the expression of Mcl-1 was significantly upregulated after chemotherapy, and high Mcl-1 expression was associated with poorer overall survival and an increased recurrence rate. Furthermore, we demonstrated that chemotherapy-driven increased Mcl-1 decreased chemosensitivity by promoting tumour proliferation and inhibiting DNA damage. Moreover, Mcl-1 was found to be a direct target of miR-375 in OS cells. The knockdown of Mcl-1 phenocopied miR-375 downregulation, and the overexpression of miR-375 rescued the effects of cisplatin-induced DNA damage mediated by Mcl-1. CONCLUSION: Our data indicated that chemotherapy-driven increase in the expression of Mcl-1 plays a critical role in chemoresistance, and the intervention of the miR-375/Mcl-1 axis may offer a novel strategy to enhance chemosensitivity in OS treatment.

In vivo ossification of a scaffold combining ß-tricalcium phosphate and platelet-rich plasma.

Tricalcium phosphate (TCP) and platelet-rich plasma (PRP) are commonly used in bone tissue engineering. The aim of the present study was to investigate a composite that combined TCP with PRP and assess its effectiveness in the treatment of bone defects. Cavity-shaped bone defects were established on the tibiae of 27 beagle dogs, and were repaired by pure ß-TCP with bone marrow stromal cells (BMSCs), ß-TCP/PRP with BMSCs and autogenic ilium. The samples were harvested at 4, 8 and 12 weeks, and bone regeneration was evaluated using X-ray radiography, immunocytochemical staining of osteocalcin (OCN), hematoxylin and eosin staining and reverse transcription-polymerase chain reaction analyses. Biomechanical tests of the scaffolds were performed at the 12th week after scaffold implantation. When using pure ß-TCP as a scaffold, the scaffold-bone interface was clear and no material adsorption and bone healing was observed. Substantial bone regeneration was observed when the tibial defects were restored using ß-TCP/PRP and autogenic ilium. Furthermore, the mRNA expression levels of OCN, alkaline phosphatase and collagen type I α1 were significantly higher in the animals with ß-TCP/PRP scaffolds at 8 and 12 weeks following implantation compared with those in the animals with the pure ß-TCP scaffolds. The maximum load and compressive strength of the ß-TCP/PRP scaffolds were similar to those of the autogenic ilium; however, they were significantly higher than those of the pure ß-TCP scaffold. Thus, the ß-TCP/PRP composite may be used as a potential scaffold to carry in vitro cultured BMSCs to treat bone defects.