ANAPC11 Drives The Malignant Phenotype Of Glioblastoma Cells

Objective:Glioblastoma(GBM)is the most deadly malignant tumor in the central nervous system.Its proliferation rate is fast,and it is easy to relapse early after surgical resection.Therefore,inhibition of excessive proliferation of GBM cells driven by various tumor-related factors and exploration of new therapeutic targets have always been the focus of neurosurgery.This study intends to find out the molecules that play a key role in the malignant progression of GBM through the single-cell sequencing analysis of the pathological tissues of GBM patients,and explore the mechanism of action.Methods:Firstly,the samples of brain GBM tumor tissue center were sequenced by single cell sequencing,and the sequencing data and four public single cell database data were analyzed by infer Copy number variation(CNV)to screen the key molecules that affect the CNV accumulation of GBM.Then,a variety of strategies were used to verify the expression,localization and function of important molecules in GBM:Quantitative polymerase chain reaction(Q-PCR)was used to detect mRNA expression in tumor cells or tissues;Immunohistochemistry was used to detect protein expression in tissues;Lentivirus transfection strategy was adopted to over-express target genes;Western blot was used to detect proteins in tissues or cells.Finally,small interfering RNA(siRNA)technique was used to knock down the corresponding gene expression;Transcriptome sequencing technique was used to verify the transcriptome changes after siRNA knockout;Cell Counting Kit-8(CCK8)was used to detect the cell viability of GBM cells;Laser confocal technique was used to detect the localization and expression of different proteins in GBM cells;Plate cloning technique was used to evaluate the ability of cell proliferation.The proliferation ability of cells in vivo was evaluated by subcutaneous tumor-bearing test and in situ tumor formation test in nude mice.Results:In this study,we analyzed the CNV of GBM cells at the single cell level,and divided GBM cells into different groups according to their CNV status.The differential expression of anaphase-promoting complex subunit11(ANAPC11)among different CNV groups,and its expression is largely independent of CNV.The results of tissue mass transcriptional group sequencing,single cell transcriptional group sequencing,quantitative polymerase chain reaction and Western blotting analysis showed that ANAPC11 in glioma tissues was up-regulated at mRNA and protein levels compared with normal brain tissue,and its high expression was significantly correlated with poor prognosis.In GBM,transcripts(transcript variants 2 to 11)encoding ANAPC11 protein subtype 2 account for the largest proportion of all transcripts in ANAPC11,and these transcripts are necessary for ANAPC11 to regulate GBM cell proliferation and neuronal differentiation.Knocking down ANAPC11 can make GBM cells break away from cell cycle,inhibit the proliferation of GBM cells and promote their differentiation into neurons.Finally,the experiment of intracranial tumorigenesis and subcutaneous tumorigenesis showed that the decrease of ANAPC11 expression significantly reduced the tumor-bearing volume,and the size of intracranial tumor-bearing tumor was reduced by 14.5 times.The median survival time of nude mice was prolonged.Conclusion:To sum up,this study found that ANAPC11 is a key regulatory factor in the differentiation of GBM cells into neurons,which provides a new target for the development of differentiation induction therapy for GBM.