USP4-Mediated Deubiquitination of PTBP1 Promotes Glioma Progression and Radioresistance by Enhancing PHB2-Dependent Mitophagy.

Mitophagy plays a key role in glioma radioresistance, but its upstream regulatory mechanism is not fully understood. This study aims to investigate the function and potential molecular mechanism of polypyrimidine tract-binding protein 1 (PTBP1) in mitophagy and radioresistance of glioma. The GSE16011 dataset was used to screen the differentially expressed genes, combined with weighted gene co-expression network analysis and machine learning to screen the key genes related to mitophagy in glioma. The expression levels of PTBP1, ubiquitin-specific protease 4 (USP4) and prohibitin 2 (PHB2) were detected by qRT-PCR and western blot. After transfection of overexpression vectors and shRNAs, EdU, flow cytometry, JC-1 staining, MitoSOX staining, and clonogenic survival assays were used to evaluate cell proliferation, apoptosis, mitochondrial membrane potential, ROS level, and radiosensitivity under 8 Gy irradiation. The interaction between PTBP1 and PHB2 mRNA was verified by RIP, dual-luciferase reporter assay and actinomycin D assay. Co-IP, ubiquitination assay and cycloheximide experiment were used to verify the deubiquitination regulation of USP4 on PTBP1. The effect of USP4/PTBP1 axis on the radioresistance of glioma in vivo was verified by constructing a subcutaneous xenograft tumor model combined with 10 Gy irradiation. Bioinformatics analysis identified PTBP1 as a key gene related to mitophagy in glioma, which was highly expressed in glioma tissues and cells. PTBP1 overexpression promoted glioma cell proliferation, inhibited apoptosis, enhanced mitophagy, maintained mitochondrial membrane potential, reduced mitochondrial ROS level, and promoted radioresistance under the irradiation conditions. These effects were reversed by mitophagy inhibitor BafA1. Mechanistically, PTBP1 directly bound to the 3'UTR of PHB2 to enhance its mRNA stability and expression. Overexpression of PHB2 reversed the inhibitory effect of PTBP1 knockdown on mitophagy and radioresistance. Moreover, USP4 inhibited the degradation of PTBP1 by deubiquitination to stabilize its protein level. USP4 knockdown inhibited mitophagy and radioresistance, while PTBP1 overexpression reversed these effects. In vivo experiments further confirmed that USP4 overexpression promoted glioma radioresistance and tumor growth, while PTBP1 knockdown abolished these effects. USP4 stabilizes PTBP1 protein to enhance PHB2 mRNA stability, thereby promoting mitophagy and driving glioma radioresistance. Targeting the USP4/PTBP1/PHB2 axis may represent a potential strategy to sensitize glioma cells to radiation, although this requires further validation with appropriate 0 Gy controls and direct mitophagic flux assays.