Abstract: Objective To investigate the promoting effect of Palladin on the stemness characteristics of glioma and explore potential novel therapeutic targets for glioma treatment. Methods Tumor stem cells were enriched from glioma U251 cells by sphere culture, and the expression level of Palladin was compared between these stem cells and parental cells using Western blot. Stable U251 cell lines with Palladin overexpression and knockdown were established. Sphere formation, Transwell invasion and drug resistance assays were performed to evaluate the self-renewal capacity, metastatic potential and drug resistance of these cells. Additionally, colony formation and CCK-8 assays were used to analyze proliferative capacity across all cell groups. The molecular mechanism of Palldin was investigated using Western blot and rescue experiments. Results Sphere-forming cells exhibited statistically significant upregulation of Palladin expression compared to parental cells. Sphere formation assays, Transwell invasion assays, and drug resistance assays demonstrated that Palladin overexpressing U251 cells exhibited significantly enhanced self-renewal capacity, invasive potential, and drug resistance compared to control cells, whereas Palladin-knockdown U251 cells showed markedly reduced self-renewal, invasion, and drug resistance capabilities. Colony formation and CCK-8 assays demonstrated that over-expression of Palladin enhanced proliferation of U251 cells, while knockdown of Palladin significantly reduced their proliferation. Western blot and rescue experiments further revealed that Palladin exerts its functional effected through activation of the mTOR signaling pathway. Conclusions Palladin promotes glioma stemness characteristics through activation of the mTOR pathway, highlighting its potential value as a therapeutic target.

Key words: glioma, Palladin, stem feature, mTOR