The Effect Of CHAF1B On The Proliferation And Self-renewal Of Glioma Stem Cells

Background:Glioblastoma multiforme(GBM)is the most malignant and common type of primary astrocytoma,accounting for over 60%of adult brain tumours,with a peak incidence between the ages of 50 and 60.Due to its complex heterogeneity,plasticity and the emergence of drug resistance after treatment,GBM remains a fatal disease with a very poor prognosis after the use of combination therapies,including surgical resection within safety limits,adjuvant radiotherapy combined with temozolomide(TMZ).Typically,the median survival time for GBM patients is only 15 months.Glioma stem cells(GSCs)are a group of tumour cells that have the ability to self-renew,proliferate indefinitely and differentiate in multiple lineages.Many studies in recent years have suggested that GSCs may be involved in tumour initiation,development,invasion and migration,as well as resistance to chemotherapeutic agents,leading to tumour recurrence.Given the key role of GSCs in the pathogenesis of GBM,better characterisation of the molecular and phenotypic features of GSCs will contribute to the understanding of the underlying biological functions of GBM and the development of new diagnostic approaches and,in particular,more effective therapeutic strategies.Chromatin Assembly Factor 1B(CHAF1B)is the p60 subunit of the chromatin assembly factor 1 complex(CAF-1)and plays an important role in DNA replication and chromatin assembly in proliferating tissues.Several studies have shown that CAF1 controls specific chromatin reorganization in the S phase and promotes cell cycle progression,which in turn affects cell proliferation and apoptosis.In recent years,CHAF1B has been found to be involved in the genesis and development of various malignancies.However,the expression and function of CHAF1B in GBM is still unclear.Therefore,we explored the specific mechanism of action of CHAF1B affecting the proliferation of glioma stem cells through GSCs cells CSC2078 and TS576 cell lines to provide a theoretical basis for the clinical application of CHAF1B in GBM.Objective:After inhibiting the expression of CHAF1B,the effect of inhibiting CHAF1B on the proliferation ability of glioma stem cells was observed through in vivo and ex vivo experiments,thus exploring the function and mechanism of CHAF1B in GBM.Methods:1.Expression of CHAF1B query through TCGA database.2.The proliferative activity of CHAF1B on CSC2078 and TS576 glioma stem cells was detected by CCK8 method,growth curve and Soft AGAR clone formation experiment.3.The self-renewal ability to detect the self-renewal capacity of CHAF1B acting on GSCs.4.Flow cytometry for cell cycle detection.5.Flow cytometry to detect changes in apoptosis following the action of CHAF1B.6.Western-blot assay to detect changes in related proteins and apoptosis-related proteins after the action of CHAF1B.7.Establishment of a mouse subcutaneous glioma transplantation model to observe the effect of CHAF1B on the proliferation of brain glioma stem cells.Results:1.CHAF1B may be a potential therapeutic target for GBM We found significantly higher expression levels of CHAF1B in human GBM tissues than in normal tissues in the TCGA database.The Western-blot assay revealed that siCHAF1B could down-regulate the protein expression level of CHAF1B in glioma stem cells.2.The effect of siCHAF1B on the proliferation of glioma stem cells by in vitro experimentsThe results of CCK8 showed that the expression of siCHAF1B significantly inhibited the cellular activity of GSCs.Growth curve assays showed that inhibition of CHAF1B expression suppressed the proliferative capacity of GSCs.Soft AGAR clone formation experiment demonstrated that inhibition of CHAF1B expression decreased the clonogenic ability of GSCs.The results of The self-renewal ability assay showed that the number of neurospheres was significantly reduced after silencing CHAF1B compared to the control group(P<0.01),indicating that inhibition of CHAF1B activity inhibited the self-renewal ability of GSCs.Flow cytometry results showed that silencing of CHAF1B significantly reduced the proportion of G1 phase and increased the proportion of S phase in the cell cycle,suggesting that,silencing of CHAF1B could block the cell cycle.Apoptosis was detected by flow cytometry and Western blotting,and the results showed that inhibition of CHAF1B expression in GSCs was confirmed to increase the rate of apoptosis.3.The effect of siCHAF1B on the proliferation of glioma stem cells by in vivo experimentsThe establishment of a mouse subcutaneous glioma transplantation model revealed by measuring the size of the tumors and the weight of the mice that the tumors grew more slowly after CHAF1B silencing compared to the control group,and the tumor volume and weight growth trends were reduced.Conclusion:1.Down-regulation of CHAF1B expression can significantly inhibit the cell proliferation and self-renewal ability of GSCs.2.Reduction/silencing of CHAF1B expression slows cell growth,leads to cell cycle arrest and accelerates apoptosis in GSCs.3.CHAF1B may provide a new target for the treatment of GBM.