The Role And Mechanism Of FOXM1/CEP55 Axis In The Development Of Glioma

BackgroundGlioma is the most common malignant brain tumor,accounting for more than 60%of the primary intracranial tumors.Because of its aggressive nature and unclear borders,it is difficult to remove it completely by operation alone.Although considerable progress has been made in the diagnosis and treatment of gliomas,the mortality rate of gliomas remains high,and the 5-year survival rate of patients is still very low.At present,the pathophysiological mechanism of gliomas is not fully understood,and many researches on alternative therapies for gliomas are still in progress.Therefore,further elucidation of the molecular mechanism of glioma occurrence and development will help early accurate diagnosis of gliomas,timely intervention and development of effective treatment strategies.Centrosome is the main microtubule tissue center of human cells.It is mainly involved in cell polarity and normal separation of chromatin in the process of cytokinesis.It can participate in the formation of spindle and ensure the accurate distribution of genetic information of daughter cells.Therefore,the structure,function and quantity of centrosomes are strictly controlled within the cells.Centrosome structural,functional,or quantitative abnormalities can cause chromosome mis-segregation,which can lead to the occurrence of aneuploidy,chromosome instability,and failure of cytokinesis.These can cause genomic instability and thus mediate tumorigenesis.Recent studies have found that the abnormal number of centrosomes is closely related to the occurrence and development of various malignant tumors.Centrosome protein 55(CEP55)is a recently discovered highly coiled helix protein in the centrosome-related protein family,which can participate in the regulation of cell division and play an important role in the progress of the cell cycle.When its expression is abnormal,it can lead to the dysfunction of cytokinesis,the appearance of multipolar spindle and aneuploid cells,and then the instability of chromosome state,which in turn can cause chromosomal instability,leading to tumorigenesis and development.A large number of studies have found that CEP55 is up-regulated in a variety of human tumor cells and tissues,and its expression level is closely related to the clinicopathological characteristics and prognosis of patients.The universality and widespread expression of CEP55 in tumor cells and tissues shows that it plays an important biological function in tumorigenesis and development.As for its expression level,prognostic value and biological function in the development of malignant phenotype of glioma cells,it is still not completely clear.Revealing the possible mechanism of CEP55 in the origin and development of glioma to provide important reference value for further research on the potential treatment and accurate diagnosis of glioma.Eukaryotic gene expression is regulated by many aspects,among which the regulation of gene transcription plays an important role.During transcription,transcription factors can bind to specific binding sites to start the transcription and expression of specific genes.At present,more than 2000 transcription factors have been found in the human genome.These transcription factors regulate the expression of many human genes,forming an extremely complex gene transcription regulation network,which is of great significance for the normal physiological activities of the body.At present,there are more and more researches on transcription factors in regulating the expression of tumor-related genes.The imbalance of some transcription factors may lead normal cells to obtain tumor related characteristics.Therefore,the study of differential expression of transcription factors in tumors and related regulatory genes provides important evidence for clarifying the mechanism of tumor development and the discovery of possible therapeutic targets.FOXM1 belongs to the forkhead box family and is a transcription factor involved in regulating normal cell proliferation.In addition,a large number of studies have found that FoxM1 is up-regulated in a variety of tumor tissues and cells,regulate the expression of different oncogenes,play different biological functions in the process of tumor development,and can be used as a potential tumor marker and treatment target.In glioma,FoxMl expression is up-regulated,and it can promote the proliferation,invasion and migration of glioma cells,but its specific mechanism is not fully understood.In this study,we examined the relationship between CEP55 expression in glioma tissues,explored its relationship with clinical pathological features,and guided the prognosis of patients.Then we analyzed the correlation between the expression of FOXM1 and CEP55 in gliomas,and discussed the possible mechanism of them in the development of gliomas.This study will be divided into the following three sections for elaboration.Part Ⅰ Expression of CEP55 in human glioma tissue and its prognostic significanceObjective1.To explore the expression of CEP55 mRNA and protein in glioma tissues;2.To analyze the relationship between the expression level of CEP55 protein and the clinicopathological characteristics of glioma patients,and to guide the prognosis of patients.Materials and Methods1.qRT-PCR and western blot were used to detect the CEP55 mRNA and protein expression in glioma tissues and normal brain tissue samples;2.The expression of CEP55 protein in glioma tissue microarrays was detected by immunohistochemistry,and the patients were divided into groups according to the immunohistochemical scores;3.χ2 test was used to statistically analyze the relationship between the expression level of CEP55 protein and the clinicopathology of glioma patients,and the Log-rank test was used to examine the effect of CEP55 expression level on the overall survival and progression-free survival of glioma patients.Cox regression analysis was used to determine the relationship between clinicopathological characteristics,the CEP55 protein expression and clinical prognosis.Results1.CEP55 was up-regulated in glioma tissuesBy qRT-PCR and western blot detection of 53 human gliomas and 5 normal human brain tissues,the expression of CEP55 mRNA and protein in glioma tissues was significantly up-regulated compared with normal brain tissues.Moreover,with the increase of tumor grade,the expression level also increased,the difference was statistically significant(P<0.05).2.The relationship between CEP55 expression level and clinicopathological characteristics and patient prognosisWe detected the expression of CEP55 in glioma tissue microarrays by immunohistochemistry and grouped them according to the staining results.The analysis of clinical pathological data showed that the high expression of CEP55 was closely related to pathological grade(P<0.0001)and recurrence(P<0.0001),but there was no significant correlation with other clinicopathological features such as age,gender and position(P>0.05).Log-rank test results showed that the overall survival and progression-free survival of patients with high expression of CEP55 were reduced(P<0.0001).Cox regression analysis found that the expression level of CEP55 protein was an independent prognostic factor affecting the overall survival(P=0.001)and progression-free survival(P=0.011)of glioma patients.Conclusions1.Compared with normal glioma tissues,the expression of CEP55 is significantly overexpressed in glioma tissues;2.The expression level of CEP55 protein is significantly related to the pathological grade of glioma;3.Upregulation of CEP55 expression is associated with poor prognosis in glioma patients.Part Ⅱ Study on the Biological Functions of CEP55 in GliomaObjective1.To investigate the role of CEP55 in glioma cell proliferation,invasion and migration,apoptosis and autophagy.Materials and Methods1.qRT-PCR and western blot were used to detect the expression of CEP55 mRNA and protein in four glioma cell lines(U87,U251,SW1783,and HS683).The cells with higher expression level of CEP55 were selected for interference experiment and the cells with lower expression level were used for over expression experiment;2.Chemically synthesized CEP55 shRNA and overexpression lentivirus were transfected into glioma cell lines separately,and selected stable transfected cells for subsequent functional experiments;3.The effects of CEP55 on the proliferation of glioma cells were detected by CCK-8 and EdU cell staining methods.Transwell assays were performed to detect the effects of CEP55 on the invasion and migration of glioma cells.The expression of epithelial-mesenchymal transition markers(E-cadherin,N-cadherin,Vimentin,and Snail)were detected by western blot;4.Annexin V-FITC/PI double staining was used to determine the effect of CEP55 on glioma cell apoptosis.Western blot was used to detect the expression of Bcl-2,Bax,cleaved-caspase-9,cleaved-parp 1 and other apoptosis related proteins;5.Transmission electron microscopy was used to detect the formation of autophagosomes.Western blot was used to detect changes in the expression of autophagy-related proteins.And the formation of autophagolysosomes and autophagosomes were detected by mRFP-GFP-LC3 adenovirus.Results1.Expression and transfection efficiency of CEP55 in human glioma cell linesThe results of qRT-PCR and western blot showed that CET55 was expressed in U87,U251,SW1783,and HS683 glioma cell lines,and its mRNA and protein expression levels were higher in U87 and U251,while lower in SW1783 and HS683.Compared with control and Scramble groups,the mRNA and protein expression of CEP55 were significantly decreased in U87 and U251.Similarly,compared with control and Vector groups,the mRNA and protein expression of CEP55 were significantly increased in HS683 and SW1783.2.CEP55 can promote the proliferation of glioma cellsThe results of CCK-8 assays showed that the proliferation ability and cell viability of U87 and U251 transfected with sh-CEP55 were reduced compared with the Scramble group,the difference was statistically significant(P<0.05).Conversely,after up-regulating the expression of CEP55,the proliferation ability and cell viability were significantly higher than the Vector group(P<0.05).EdU cell staining showed that after silenced CEP55 expression,the proportion of EdU positive cells was significantly lower than that of Sramble group.While,compared to the Vector group,the proportion of EdU-positive cells was significantly increased.3.CEP55 can promote epithelial-mesenchymal transition and promote invasion and migration in glioma cellsWestern blot results showed that the expression of N-cadherin,Vimentin,and Snail decreased,and the expression of E-cadherin increased after CEP55 down-regulated.In contrast,the expression of E-cadherin decreased,and the expression of N-cadherin,Vimentin,and Snail proteins increased after CEP55 overexpressed.Transwell assay results showed that the number of cells in the chamber was significantly lower in the sh-CEP55 group than that in the Scramble group(P<0.01),while compared with Vector group,the number of cells in the chamber was significantly increased(P<0.01).4.Down-regulation of CEP55 can promote glioma cell apoptosisAnnexin V-FITC/PI double staining results showed that the proportion of apoptotic cells in sh-CEP55 group was significantly higher than that in Scramble group.Western blot results showed that cleaved-caspase 9,cleaved-parp 1 and Bax protein were increased in sh-CEP55 group than those in Scramble group,while Bcl-2 protein was decreased compared with Scramble group.5.Down-regulation of CEP55 promotes autophagy in glioma cellsTransmission electron microscopy showed that a large number of autophagosomes were formed in sh-CEP55 group compared to the Scramble group.Western blot results showed that silencing CEP55 expression significantly increased LC3 and Beclin 1 protein expression,and decreased P62 protein expression.The mRFP-GFP-LC3 double-labeled adenovirus detection results showed that compared with Scramble group,the number of autophagosomes and autolysosomes in sh-cep55 group increased significantly.Conclusions1.CEP55 can promote the proliferation,migration and invasion of glioma cells;2.Down-regulation of CEP55 can induce glioma cell apoptosis;3.Down-regulation of CEP55 can induce autophagy in glioma cells.Part Ⅲ FOXM1 regulates CEP55 expression and promotes glioma cell proliferation,invasion and migrationObjective1.To explore the relationship between FOXM1 and CEP55 expression in gliomas;2.To explore the mechanism of FOXM1 regulating CEP55 expression and promoting the proliferation,invasion and migration ability of glioma cells.Materials and Methods1.Analyze the expression of FOXM1 and CEP55 genes in glioma tissues and normal brain tissues in TCGA and GTEx databases by bioinformatics technology,and perform correlation analysis.Then analyze the correlation between the expression level and the overall survival of glioma patients;2.qRT-PCR was used to detect the expression of FOXM1 mRNA in glioma tissues,and the relationship between FoxMl mRNA and CEP55 mRNA was analyzed;3.Chemically synthesized CEP55 shRNA and overexpression lentivirus were transfected into glioma cell lines separately,and selected stable transfected cells for subsequent functional experiments;4.qRT-PCR and western blot were used to detect changes in CEP55 mRNA and protein expression in glioma cells after FOXM1 interference or overexpression;5.Double luciferase assay was detected to verify whether CEP55 is the target gene of transcription factor FOXM1;6.CCK-8,clonogenic assay,EdU cell staining,transwell assay,and western blot were used to detect the effects of FOXM1 regulating CEP55 expression on glioma cell proliferation,invasion,and migration.7.Orthotopic xenograft model further confirmed the positive effect of FOXM1/CEP55 on glioma progress.Results1.FOXM1 and CEP55 are overexpressed in glioma tissues,and their expressions are positively correlatedBioinformatics analysis showed that the expression levels of FOXM1 and CEP55 mRNA in glioma patients were significantly up-regulated compared with normal brain tissue,and the expression levels in GBM were higher than in LGG patients,and correlation analysis showed a positive correlation between FOXM1 and CEP55 expression levels(r=0.78,P<0.0001).qRT-PCR results showed that the expression of FOXM1 mRNA in glioma tissues was significantly up-regulated compared with normal brain tissue.Moreover,as the tumor level increased,the expression level also increased.Statistical analysis of the expression levels showed that there was a significant correlation between FOXM1 and CEP55 expression levels in glioma tissues(r=0.82,P<0.0001).2.High expression of FOXM1 and CEP55 predicts poor prognosis in glioma patientsKaplan-Meier survival curve shows that in LGG patients,the expression levels of CEP55 and FOXM1 are significantly related to the total survival time of patients(P=0.0012,P=0.0035).Although there was no significant difference in the total survival time of GBM(P=0.069,P=0.18),the patients with lower expression level of CEP55 and FoxM1 had longer total survival time than the patients with higher expression level.For all glioma patients,we found that patients with higher levels of these two proteins had lower survival rates(P<0.0001,P<0.0001).3.CEP55 is a downstream target gene of transcription factor FOXM1,and FOXM1 can promote its transcription and expressionqRT-PCR and western blot showed that down-regulating FOXM1 significantly decreased CEP55 mRNA and protein expression levels.In contrast,up-regulating FOXM1 significantly increased CEP55 mRNA and protein expression levels.The luciferase reporter assay showed that in FOXM1-overexpressing cells,the full-length promoter construct showed increased luciferase activity,indicating that FOXM1 directly activates CEP55 gene transcription.Accordingly,mutation of the binding site abolished the luciferase activity induced by FOXM1.4.Up-regulation of CEP55 can rescues the prornotive effects of FOXM1 on the proliferation,migration and invasion of glioma cellsWe transfected CEP55 overexpression and negative control lentivirus in sh-FOXM1 glioma cell lines.CCK-8,clonogenic assay,and EdU cell staining tests showed that CEP55 could abrogate the sh-FOXM1-mediated inhibition of glioma cell proliferation.Similarly,Transwell and western blot results showed that CEP55 could partially restore the FOXM1 down-regulation induced effect on glioma cell migration and invasion.5.FOXM1/CEP55 axis modulates tumor growth in orthotopic xenograft modelTo confirm whether the FoxMl/CEP55 axis regulated the tumorigenicity of glioma cells in vivo,we intracranially injected nude mice with U87 cells stably expressed with sh-FOXM1+Vector or sh-FOXM1+CEP55.We observed a significant decrease in tumor formation,increased the biologic status and survival of mice in the tumor-bearing mice when FOXM1 was knockdown,but this effect was abrogated by CEP55 overexpression.These data indicated that CEP55 was a critical target for FOXM1 promoting the tumorigenic potential of glioma cells,and FoxMl/CEP55 axis promoted tumor progress in vivo.Conclusions1.The expression of FOXM1 and CEP55 is upregulated and the expression of them is positively correlated in gliomas.Also,the expression level of them were related to the poor prognosis of patients;2.CEP55 is a downstream target gene of FOXM1,and FOXM1 can regulate the expression of CEP55 by binding to the CEP55 promoter region;3.The effect of FOXM1 on the proliferation,migration and invasion of glioma cells partly depends on CEP55.