Abstract: Objective To investigate the role and underlying mechanisms of neutrophil extracellular traps ( NETs ) in the proliferation, migration, and invasion of osteosarcomacells. Methods A total of 20 osteosarcoma patients and 20 healthy subjects were enrolled. Levels ofPAD4 and H3cit in serum were measured by ELISA. Neutrophils were isolated from peripheral blood of both groups to compare NETs formation. Co-culturing experiments were conducted by incubating NETs with osteosarcoma cells (MG63), with the addition of DNA degrading enzyme DNaseⅠ . The groups included Control, NETs, and NETs + DNase Ⅰ groups. CCDC25 protein expression level was detected using Western blotting, cell proliferation was assessed using CCK-8 assay, and cell migration and invasion were evaluated using Transwell assay. MG63 cells were transfected with si-NC and si-CCDC25, then co-cultured with NETs, cells were then divided into 2 groups: si-NC and siCCDC25 groups. CCDC25 protein expression, cell proliferation, and invasion were assessed as described above. Results Compared to healthy subjects, osteosarcoma patients showed elevated levels of PAD4 and H3cit in serum ( P < 0. 05 ), indicating enhanced NETs formation capacity. Compared to the Control group, the NETs group exhibited elevated CCDC25 protein expressionlevel, increased cell proliferation and cell migration and invasion (P < 0. 05). The NETs + DNaseⅠ group showed reduced CCDC25 protein expression level, decreased cell proliferation, cell migration and invasion compared to the NETs group (P < 0. 05). The si-CCDC25 group, when compared to the si-NC group, demonstrated decreased CCDC25 protein expression level, cell proliferation, cell migration and invasion (P<0.05). Conclusion  NETs activate osteosarcoma cell CCDC25 expression through released DNA, thereby promoting tumor cell proliferation, and cell migration and invasion.

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