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Updated: Oct 4, 2025
Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models
Published on: October 28, 2021
IL-1β secreted by macrophage M2 promotes metastasis of osteosarcoma via NF-κB/miR-181α-5p/RASSF1A/Wnt pathway
Zhi-Peng Han1, Dong-Biao Liu1, Liu-Qing Wu1
1Department of Orthopedics, The Third Xiangya Hospital of Central South University, Changsha 410013, China.
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-1β 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-1β (IL-1β) 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-3β, p-GSK-3β, β-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-1β 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-1β 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-1β 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.
Insights
Interleukin-1β (IL-1β) from M2 tumor-associated macrophages promotes osteosarcoma (OS) metastasis. Targeting the NF-κB/miR-181α-5p/RASSF1A/Wnt pathway with RASSF1A or miR-181α-5p can suppress OS spread.
Area of Science:
- Oncology
- Molecular Biology
- Immunology
Background:
- Ras-associated domain family protein1 isoform A (RASSF1A) is often absent in osteosarcoma (OS) and can suppress metastasis.
- Tumor-associated macrophages (M2-TAMs) may mediate RASSF1A's effect on OS metastasis.
- The role of IL-1β secreted by M2-TAMs in relation to RASSF1A in OS metastasis is unclear.
Purpose of the Study:
- To investigate the relationship between IL-1β secreted by M2-TAMs and RASSF1A in osteosarcoma (OS) metastasis.
- To elucidate the underlying molecular mechanisms involving miR-181α-5p, NF-κB, and the Wnt pathway.
Main Methods:
- Flow cytometry to quantify M2-TAM markers (CD68, CD204).
- RT-qPCR to measure IL-1β, Arg-1, and miR-181α-5p levels.
- MTT, scratch wound healing, and Transwell assays to assess OS cell proliferation, migration, and invasion.
- Western blot to evaluate RASSF1A, GSK-3β, p-GSK-3β, β-catenin, MMP-2, and MMP-9.
- Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays to confirm direct binding of miR-181α-5p and RASSF1A.
Main Results:
- M2-TAMs exhibited elevated CD68, CD204, Arg-1, and IL-1β levels.
- Overexpression of RASSF1A and knockdown of miR-181α-5p suppressed OS cell invasion and migration via the Wnt pathway.
- IL-1β from M2-TAMs promoted OS metastasis through the RASSF1A/Wnt pathway, modulated by miR-181α-5p and NF-κB.
Conclusions:
- IL-1β secreted by M2-TAMs drives OS metastasis.
- Suppression of OS metastasis can be achieved by inhibiting miR-181α-5p or overexpressing RASSF1A.
- The NF-κB/miR-181α-5p/RASSF1A/Wnt pathway is a key mechanism in OS metastasis and a potential therapeutic target.
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