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Updated: Aug 19, 2025
Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation
Published on: April 3, 2017
Exosomal miR-181a-5p derived from SAOS-2 cells promotes macrophages M2 polarization by targeting RORA
Zheng-Guang Wang1, Ming-Si Deng2, Ji-Qing Su3
1Department of Spinal Surgery, The Third Xiangya Hospital of Central South University, Changsha, People's Republic of China.
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-1β 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.
Insights
Osteosarcoma cells release exosomes containing miR-181a-5p, which promote M2 macrophage polarization by targeting RORA. This interaction drives tumor progression in osteosarcoma.
Area of Science:
- Oncology
- Cell Biology
- Immunology
Background:
- Tumor-associated macrophages (TAMs) play a crucial role in cancer progression.
- The specific mechanisms by which osteosarcoma cells influence TAM polarization are not fully understood.
- Understanding these interactions is vital for developing targeted therapies.
Purpose of the Study:
- To elucidate the role of osteosarcoma-derived exosomes in regulating TAM polarization.
- To investigate the involvement of miR-181a-5p and RORA in this process.
- To determine the therapeutic potential of targeting this pathway.
Main Methods:
- Isolation and characterization of exosomes from SAOS-2 osteosarcoma cells.
- Analysis of macrophage polarization (M1/M2) using flow cytometry.
- Quantitative real-time PCR and Western blot to assess miR-181a-5p and RORA expression.
- Dual-luciferase reporter assay to confirm direct interaction between miR-181a-5p and RORA.
Main Results:
- Osteosarcoma tissues and SAOS-2 exosomes showed elevated miR-181a-5p levels.
- SAOS-2 exosomes promoted M2 macrophage polarization via exosomal miR-181a-5p transfer.
- RORA expression was downregulated in osteosarcoma and negatively correlated with miR-181a-5p.
- RORA was identified as a direct downstream target of miR-181a-5p.
- Inhibition of RORA mimicked the effects of miR-181a-5p knockdown on macrophage polarization.
Conclusions:
- Exosomal miR-181a-5p from osteosarcoma cells induces M2 macrophage polarization by targeting RORA.
- This mechanism contributes to the pro-tumorigenic microenvironment in osteosarcoma.
- Targeting the exosomal miR-181a-5p/RORA axis may offer a novel therapeutic strategy for osteosarcoma.
