ABSTRACT
Although the interaction between tumor cells and tumor-associated macrophages (TAMs) has been widely studied; however, the mechanism of osteosarcoma cells in regulating the polarization of TAMs remains unclear. Exosomes from SAOS-2 cells were isolated and validated by electron microscopy and Western blot. Transfection of indicated plasmids was applied to modify the expressions of miR-181a-5p and RAR-related orphan receptor alpha (RORA). Flow cytometric analysis was carried out to analyze M1/M2 macrophage polarization. Quantitative real-time PCR was performed to determine the levels of miR-181a-5p and RORA. Protein levels of CD63, CD81, RORA, CD163, CD206, IL-10, CXCL10, and IL-1ß were evaluated by Western blot. The direct interaction of miR-181a-5p and RORA was validated by dual-luciferase activity assay. The expression of miR-181a-5p was upregulated in osteosarcoma tissues and presented in SAOS-2-derived exosomes. SAOS-2-derived exosomes promoted the polarization of M2 macrophages by transferring miR-181a-5p. In addition, RORA was downregulated in osteosarcoma tissues and showed a negative correlation with miR-181a-5p. RORA was found to be the downstream target of miR-181a-5p in SAOS-2 cells. Inhibition of RORA reversed the effects of miR-181a-5p knockdown on the polarization of M2 macrophages. The results showed that exosomal miR-181a-5p derived from osteosarcoma cells induced polarization of M2 macrophages via targeting RORA.
Subject(s)
Bone Neoplasms , Exosomes , MicroRNAs , Osteosarcoma , Humans , Exosomes/genetics , Macrophages , Osteosarcoma/genetics , MicroRNAs/genetics , Nuclear Receptor Subfamily 1, Group F, Member 1ABSTRACT
Fracture healing is a complicated process affected by many factors, such as inflammatory responses and angiogenesis. Omentin-1 is an adipokine with anti-inflammatory properties, but whether omentin-1 affects the fracture healing process is still unknown. Here, by using global omentin-1 knockout (omentin-1-/-) mice, we demonstrated that omentin-1 deficiency resulted in delayed fracture healing in mice, accompanied by increased inflammation and osteoclast formation, and decreased production of platelet-derived growth factor-BB (PDGF-BB) and osteogenesis-promoting vessels that are strongly positive for CD31 and Endomucin (CD31hiEmcnhi) in the fracture area. In vitro, omentin-1 treatment suppressed the ability of the tumor necrosis factor-α (TNF-α)-activated macrophages to stimulate multi-nuclear osteoclast formation, resulting in a significant increase in the generation of mono-nuclear preosteoclasts and PDGF-BB, a pro-angiogenic protein that is abundantly secreted by preosteoclasts. PDGF-BB significantly augmented endothelial cell proliferation, tube formation and migration, whereas direct treatment with omentin-1 did not induce obvious effects on angiogenesis activities of endothelial cells. Our study suggests a positive role of omentin-1 in fracture healing, which may be associated with the inhibition of inflammation and stimulation of preosteoclast PDGF-BB-mediated promotion of CD31hiEmcnhi vessel formation.
Subject(s)
Cytokines/genetics , Femoral Fractures/genetics , Fracture Healing , GPI-Linked Proteins/genetics , Lectins/genetics , Sialoglycoproteins/metabolism , Animals , Cell Movement , Disease Models, Animal , Female , Femoral Fractures/etiology , Femoral Fractures/immunology , Gene Knockout Techniques , Mice , Osteoclasts/metabolism , Osteogenesis , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , RAW 264.7 Cells , X-Ray MicrotomographyABSTRACT
BACKGROUND: Ras-associated domain family protein1 isoform A (RASSF1A) was significantly absent in clinical samples and many osteosarcoma (OS) cell lines. Overexpression of RASSF1A could suppress OS metastasis, which may be mediated by tumor-associated macrophages polarized M2 (M2-TAMs). However, the relationship between IL-1ß secreted by M2-TAMs and RASSF1A remains unknown. METHODS: The expression levels of M2-TAMs markers CD68 and CD204 were measured by flow cytometry, and arginase-1 (Arg-1) and interleukin-1ß (IL-1ß) secreted by M2-TAMs were examined by real-time quantitative PCR (RT-qPCR). MTT assay was employed to determine the proliferation of OS cells, while scratch wound healing assay and Transwell assay were used to evaluate their migration and invasion, respectively. The level of miR-181α-5p was measured by RT-qPCR, while the levels of RASSF1A, GSK-3ß, p-GSK-3ß, ß-catenin, MMP-2 and MMP-9 were evaluated by Western blot. The direct binding of miR-181α-5p and RASSF1A was identified using dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. RESULTS: The levels of CD68, CD204, Arg-1 and IL-1ß were elevated in M2-TAMs compared with control group. Overexpression of RASSF1A and knockdown of miR-181α-5p could both suppress invasion and migration of OS cells through Wnt pathway. IL-1ß secreted by M2-TAMs facilitated the OS metastasis via RASSF1A/Wnt pathway, which could be targeted by miR-181α-5p and affected by nuclear factor-kappa B (NF-κB). CONCLUSIONS: IL-1ß secreted by M2-TAMs contributed to OS metastasis, which could be suppressed by knockdown of miR-181α-5p or overexpression of RASSF1A through NF-κB/miR-181α-5p/RASSF1A/Wnt pathway. These findings can guide new target discovery for drug development in OS treatment.
