Mechanism Of PTRF-mediated Lipid Remodeling In Glioblastoma

Objective:Metabolism remodeling is a hallmark of glioblastoma（GBM）that regulates tumor proliferation and the immune microenvironment.Previous studies have reported that increased polymerase 1 and transcript release factor（PTRF）levels are associated with a worse prognosis in glioma patients.This study intends to explore the biological role and potential molecular mechanisms of PTRF in GBM metabolism through metabolomics methods and molecular biology experiments,indicating new therapeutic targets and methods for GBM.Methods:The relationship between PTRF and lipid metabolism in GBM and the effect of PTRF on lipid metabolism were detected by nontargeted metabolomics profiling and subsequent lipidomics analysis.Western blotting,quantitative real-time PCR,immunofluorescence,and protein immunoprecipitation were conducted to explore the key metabolic enzymes and the molecular mechanism of PTRF in lipid metabolism.Flow cytometry,cell mitochondrial stress test,plate cloning experiment,and CCK8proliferation experiment were detected to explore the biological role and phenotype of PTRF in GBM.A sequence of experiments in vivo（both xenograft tumor and intracranial tumor mouse models）combined with bioluminescence imaging,survival curve,and immunohistochemical staining were used to detect the tumor-specific impacts of PTRF in GBM.Further,key metabolic enzyme inhibitors and mitochondrial oxidative respiration inhibitors combined with animal experiments were detected to explore whether the effect of PTRF in GBM is reversed.Results:In this study,we found that PTRF remodeled phospholipid metabolism to promote tumor proliferation and suppress immune responses in GBM by stabilizing cPLA2.We initially established multiple primary GBM cell lines from GBM patients and then used a lentivirus vector to overexpress PTRF.Non-targeted metabolomics profiling and a subsequent focused lipidomics analysis demonstrated that PTRF remodeled the phospholipid composition and content of GBM cells by substantially increasing lysophosphatidylcholine levels.We then found that the PTRF-triggered lipid remodeling is mediated by the phospholipid-metabolizing enzyme cytoplasmic phospholipase A2（cPLA2）,and showed that PTRF decreased the extent of ubiquitination of cPLA2,thereby promoting its enzymatic activity via increased protein stability in GBM cells.A sequence of experiments in vitro revealed that the PTRF-cPLA2 lipid remodeling pathway apparently enhanced both the quantity and the rate of endocytosis in GBM cells,and regulated mitochondrial respiration to increase the level of energy metabolism and promote the proliferation of GBM cells.Animal experiments in vivo（xenograft tumors and intracranial tumor mouse models）showed that PTRF promoted the proliferation of GBM and decreased numbers of CD8⁺tumor-infiltrating lymphocytes（TILs）through ATP-adenosine metabolism.Finally,we used cPLA2 inhibitors（AACOCF₃）and mitochondrial oxidative respiration inhibitors（metformin）in animal experiments and found that inhibiting cPLA2 activity and energy metabolism can reversed the proliferation of GBM and the reduction of CD8⁺TILs induced by PTRF.Conclusions:The PTRF-cPLA2 lipid remodeling pathway promotes tumor proliferation and suppresses immune responses in GBM.In addition,our findings highlight the supportive role of phospholipid metabolism in GBM,providing new therapeutic targets and immunotherapy strategies for further investigation.