The Study Of ARPCUB To Promote Mesenchymal Phenotype Maintenance And Radioresistance In Glioma Stem Cells

Background:Glioblastoma(GBM)is the most malignant and prevalent primary central nervous system tumour.The presence of glioma stem cells(GSCs)in GBM and its proneuralto-mesenchymal transition(PMT)during progression leads to radioresistance in GBM.Actin-related protein-2/3 complex subunit 1B(ARPC1B)is one of the molecules that constitute the actin-related protein-2/3 complexes(ARPs),is associated with malignant progression in a variety of cancers,whereas the impact of ARPC1B on the phenotype and radioresistance of GSCs is unclear.Therefore,exploring the role and mechanisms of ARPC1B in the phenotype and radioresistance of GSCs could help provide a theoretical basis for clinical solutions to the GBM radioresistance challenge.Methods:1.Potential ARPs regulating PMT were mined by multiple analyses based on TCGA and CGGA databases combined with single-cell sequencing data,and in vitro knockdown and overexpression of ARPC1B was examined by Western blotting for effects on phenotypic markers in GSCs.2.Tumour stem cell sphere formation assay and ELDA assay were applied to assess the effect of ARPC1B on the self-renewal ability of GSCs,and the role of ARPC1B in the radioresistance of GSCs was examined by apoptosis assay,comet assay,y-H2AX IF staining and cell cycle assay.3.To validate the role of ARPC1B on malignant progression and radioresistance in GSCs by in vivo experiments.4.Proteins interacting with ARPC1B were detected by co-IP and mass spectrometry and validated with in vivo and in vitro experiments.MG132,CHX treatment and co-IP experiments were used to verify whether ARPC1B affects downstream proteins through a ubiquitination mechanism.5.Design deletion mutant plasmids to identify binding sites for ARPC1B to interact with IFI16 and HuR.6.Find the key ubiquitinating enzymes involved in the mechanism of ARPC1B interaction with IFI16 and HuR ubiquitination and validate them by rescue experiments.7.To postulate the downstream pathways affected by ARPC1B-regulated IFI16 and HuR and validate them by multiple rescue experiments.8.Screen for sensitive drugs associated with ARPC1B expression in GBM cells by Spearman correlation algorithm.The drug efficacy was validated by in vivo and in vitro experiments.Results:1.Based on TCGA and CGGA databases combined with single-cell sequencing data,multiple analyses showed that ARPC1B was significantly associated with the MES phenotype in ARPs and was associated with poor prognosis of GBM.ARPC1B was highly expressed in MES GSCs and lowly expressed in PN GSCs in in vitro experiments.Knockdown of ARPC1B in GSCs suppressed the expression of MES markers,while overexpression of ARPC1B suppressed the expression of PN markers.2.Knockdown of ARPC1B resulted in decreased self-renewal ability of GSCs,whereas overexpression enhanced self-renewal ability of GSCs.It was demonstrated in vitro by apoptosis assay,comet assay,y-H2AX IF staining and cell cycle assay that knockdown of ARPC1B attenuated the radioresistance of MES GSCs,while overexpression of ARPC1B enhanced the radioresistance of PN GSCs.3.In vivo experiments demonstrated that knockdown of ARPC1B inhibited tumor aggressiveness and radioresistance,whereas overexpression of ARPC1B did the opposite.Knockdown of ARPC1B significantly prolonged the overall survival of mice in both IR and non-IR groups,while overexpression of ARPC1B shortened the overall survival of GSC8-11 mice in both IR and non-IR groups.4.Co-IP experiments demonstrated that ARPC1B interacted with IF116 and HuR,and in vitro and in vivo validation demonstrated that IFI16 and HuR expression was altered upon intervention with ARPC1B.MG132,CHX treatment and co-IP experiments demonstrated that ARPC1B could regulate IFI16 and HuR expression by affecting protein ubiquitination modifications.5.ARPC1B was shown to interact with the Pyrin domain of IFI16 and the RRM2 domain of HuR by constructing a deletion mutant plasmid in combination with co-IP experiments.6.ARPC1B was shown to bind competitively with TRIM21 to IFI16 and HuR by rescue experiments,thereby inhibiting TRIM21-mediated ubiquitination degradation.co-IP experiments validated that knockdown of ARPC1B significantly promoted TRIM21/IFI16 and TRIM21/HuR interactions,while overexpression of ARPC1B inhibited TRIM21/IFI16 and TRIM21/HuR interactions.7.ARPC1B was demonstrated by multiple rescue assays in vitro to promote PMT and radioresistance in GSCs through the IFI16/NF-κB pathway and HuR/STAT3 pathway,respectively.8.High expression of ARPC1B in GBM cells responded to high sensitivity to the ATR inhibitor AZD6738,and in vitro and in vivo experiments demonstrated that AZD673 8 combined with radiotherapy could effectively inhibit the growth of MES GSCs.Conclusion:1.ARPC1B was significantly associated with the MES phenotype and associated with poor prognosis of GBM,and it promoted PMT and radioresistance in GSCs in vitro and in vivo.2.ARPC1B competitively binds IFI16 and HuR with TRIM21,thereby inhibiting TRIM21-mediated ubiquitination degradation,which in turn promotes PMT and radioresistance in GSCs via the IFI16/NF-κB pathway and HuR/STAT3 pathway,respectively.3.AZD6738 is a potential radiosensitizer and has good therapeutic efficacy in GBM when combined with radiotherapy.