IL-1β serves as a biomarker for poor prognosis in osteosarcoma and drives tumor cell proliferation through the IL-1R1/NF-κB signaling axis

To investigate the expression profile of interleukin-1β (IL-1β) in osteosarcoma tissues, explore its correlation with clinical prognosis, and to elucidate the underlying molecular mechanisms of IL-1β regulating the proliferation of osteosarcoma cells.

Four osteosarcoma patients admitted to Department of Orthopedics of our hospital from December 2020 to January 2024 were enrolled, and their tumor and adjacent tissues were collected to detect differential mRNA and protein expression of IL-1β by immunohistochemistry, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and Western blotting. Additionally, 15 paraffin-embedded osteosarcoma specimens collected between March 2018 and August 2021 were immunohistochemically stained and scored using ImageJ, and then divided into high IL-1β expression group (3+, 2+) and low expression group (1+, 0). Kaplan-Meier analysis was used to determine the association between the scoring results and patient survival rates. In in vitro experiments, osteosarcoma cell line U2OS was subjected and treated with 0 to 100 ng/mL recombinant IL-1β protein. EdU incorporation assay, flow cytometry, CCK-8 assay and colony formation assay were performed to screen the optimal proliferation-stimulating concentration. Transcriptome sequencing was carried out on the U2OS cells with 40 ng/mL IL-1β treatment and untreated cells to analyze significantly up-regulated signals involved in regulating U2OS proliferation. The obtained key regulatory molecule, nuclear factor kappa B (NF-κB) was then verified by RT-qPCR, Western blotting, and immunofluorescence staining. After IL-1 receptor type 1 (IL-1R1) was knocked down by lentiviral transfection, or the cells were treated with NF-κB inhibitor embelin, the changes in cell proliferation were detected with EdU incorporation assay, flow cytometry, CCK-8 assay, and colony formation assay. In in vivo experiments, 12 female nude mice (5 weeks old) were randomly divided into control group, IL-1R1 knockdown group, and IL-1R1 inhibitor (anakinra) injection group, with 4 animals in each group. An orthotopic osteosarcoma model was established by intratibial injection, and tumor growth was compared across groups.

Immunohistochemical staining and survival analysis demonstrated that osteosarcoma patients with high IL-1β expression level had poorer prognosis (P<0.05). In vitro experiments showed that IL-1β treatment significantly increased the EdU incorporation rate (P<0.05), enhanced G2/M cell cycle arrest (P<0.05), and improved the colony formation ability (P<0.05) in U2OS cells, indicating that IL-1β promotes osteosarcoma cell proliferation. Transcriptome data analysis revealed 4165 differentially expressed genes(1688 up-regulated and 2477 down-regulated) between IL-1β-treated and untreated U2OS cells. IL-1β treatment obviously promoted cell cycle and up-regulated the proteins related to NF-κB signaling pathway in U2OS cells. RT-qPCR confirmed the mRNA levels of cell cycle-related molecules were enhanced by IL-1β treatment, which was further confirmed by Western blotting at protein level. IL-1R1 knockdown resulted in notably decreased proliferation ability in U2OS cells compared with the control cells (P<0.05). Both RT-qPCR and Western blotting confirmed that IL-1R1 knockdown reduced the expression levels of cell cycle-related proteins and NF-κB. Addition of NF-κB inhibitor, embelin, significantly inhibited the IL-1β-induced EdU incorporation rate (P<0.05), G2/M phase arrest (P<0.05), and colony formation ability (P<0.05) in U2OS cells, and suppressed the expression of cell cycle-related proteins. In vivo experimental results showed that the tumor weight in the IL-1R1 knockdown group (0.75±0.10 g) and anakinra injection group (0.93±0.06 g) was statistically lower than that in the control group(1.46±0.09 g) (both P<0.05).

Intratumoral IL-1β level is positively correlated with poor prognosis in osteosarcoma patients. IL-1β promotes the proliferation ability of osteosarcoma cells through the IL-1R1/NF-κB signaling axis.