Hsa-miR-25-3p Inhibition Sensitizes Patient-Derived Glioblastoma Cells to Temozolomide via β-catenin Downregulation

Abstract MicroRNAs (miRNAs) play pivotal roles in glioblastoma (GBM) progression and therapy resistance. Among them, miR-25-3p has emerged as a key oncogenic miRNA that promotes tumor growth, invasiveness, and resistance to temozolomide (TMZ). In this study, we profiled miRNA expression in primary GBM specimens ( n = 50) stratified by MGMT methylation and TP53 mutation status and assessed the functional impact of miR-25-3p inhibition in seven patient-derived GBM cell lines. Quantitative PCR analysis revealed upregulation of miR-135b in MGMT -methylated tumors and miR-10b in TP53 -mutant cases. Both miR-25-3p and miR-10b were significantly elevated in 3D spheroid cultures compared to 2D monolayers. Notably, both miRNAs were secreted via tumor-derived extracellular vesicles, implicating a role in cell-cell communication. Inhibition of miR-25-3p in GBM cell lines consistently suppressed β-catenin and re-induced FBXW7 expression across all cases, correlating with inhibitor uptake. In four of seven cell lines, miR-25-3p inhibition enhanced TMZ sensitivity and reduced invasiveness, although the anti-invasive effect was not further potentiated by the addition of TMZ. In addition, RNA-Seq and methylome analyses revealed genetic and epigenetic reprograming toward a less aggressive, less invasive phenotype with reduced stemness potential. These findings highlight the interplay between tumor microenvironment and molecular heterogeneity in shaping miRNA dynamics in GBM. Collectively, our results identify miR-25-3p as a promising dual-action therapeutic target to mitigating both invasion and chemoresistance in GBM, warranting further translational investigation. Graphical Abstract miRNA-mediated regulation of invasion, protein synthesis, and drug sensitivity in glioblastoma. Schematic overview of miRNA-mediated regulation of glioblastoma cell behavior. miRNAs modulate mRNA targets and protein synthesis, influencing β-catenin signaling, invasion capacity, and temozolomide sensitivity through FBXW7-associated pathways, ultimately affecting apoptosis.