The Mechanism Of CEP55 On The Sensitivity Of Glioblastoma To Temozolomide

Objective:Glioblastoma（GBM）is one of the most aggressive and refractory malignant tumors,accounting for 52%of primary brain tumors.Its five year fatality rate ranks the third in the whole body malignant tumors,second only to pancreatic cancer and lung cancer.It is a serious threat to human health.Therefore,it is of great significance to study its pathogenesis and treatment strategy.Current treatment options include surgical resection,radiotherapy,systemic therapy（temozolomidom-based chemotherapy,targeted therapy）,and supportive therapy.However,even with the multimodal combination treatment strategy,the mean survival was only 14.6 months.The inefficiency and drug resistance of Temozolomide（TMZ）is an important cause of glioblastoma recurrence and death.Therefore,it is imperative to further explore the occurrence,development and molecular mechanism of drug resistance of glioblastoma,and to develop new precision treatment methods and strategies on this basis.Centrosomal protein 55（CEP55）is a key regulator of centrosomal related proteins and cytokinesis,and is involved in the regulation of cell cycle.Previous studies of our project team have found that abnormal expression of CEP55 is common in astrocytomas,and its expression level increases with tumor grade,and patients with high expression of CEP55 have a longer survival period.This topic proposed on the basis of existing research,by bioinformatics prediction and clinical samples found CEP55 patients with glioblastoma TMZ sensitivity,preliminarily by in vivo and in vitro experiments in glioblastoma CEP55 by combining affect the possiblemechanism of TMZ sensitivity,YB-1 for clarifying the molecular mechanism of glioblastoma resistance molecular intervention strategy research foundation,optimization of treatment.Methods:1.The expression of CEP55 in Glioma in The Cancer Genome Atlas（TCGA）database was analyzed using GEPIA website,and verified in The CGGA（Chinese Glioma Genome Atlas）database.The relationship between CEP55 expression and clinicopathological characteristics and prognosis was analyzed online.2.A total of 254 glioma paraffin tissue specimens（including 81 WHO2grade astrocytomas;97 cases of WHO3 grade astrocytoma;76 cases of WHO4grade glioma）,the expression level of CEP55 in the samples was detected by immunohistochemical staining and the relationship between CEP55expression and clinicopathological characteristics was analyzed.GBM patients were divided into TMZ-sensitive group and insensitive group based on whether they had MGMT promoter methylation,and the relationship between CEP55 and TMZ sensitivity was analyzed.Meanwhile,12 fresh GBM surgical specimens were collected and Western Blot was used to detect the expression of CEP55 in tumor tissues and paratumoral tissues.3.GBM cell lines U87 and U251 were cultured in vitro,CEP55 was silenced by si RNA technology,GBM cells were treated with TMZ at different concentrations,and cell proliferation was detected by CCK-8.Flow cytometry to detect the cell cycle change,q PCR and Western Blot key factor CDC2,observe the cell cycle Cdc25c,P27,Cyclin D1,cyclin B expression changes.4.The co-expression of CEP55 and YB-1 was detected by immuno histochemical staining and immunofluorescence agent immunoprecipitatio n.Results:1 Relationship between high expression of CEP55 and clinicopathological features in glioblastoma1.1 CEPP55 is highly expressed in glioblastoma1.1.1 The m RNA level of CEP55 in glioblastoma was significantly higher than that in non-tumor brain tissue by TCGA and CGGA database analysis.The expression level of CEP55 in glioblastoma was significantly higher than that in low-grade glioma.The expression level of glioblastoma was the highest among different histological types of gliomas observed in CGGA database.1.1.2 Twelve pairs of fresh GBM surgical specimens（tumor tissues and adjacent tissues）were collected.Western Blot was used to detect the expression of CEP55 protein,and the expression level of CEP55 in tumor tissues was significantly higher than that in adjacent tissues.1.2 Clinicopathological significance of high expression of CEP551.2.1 Relationship between CEP55 height and clinicopathological features Online analysis of the CGGA database showed that CEP55 was highly expressed in IDH1 wild-type,1p/19q co-deletion,radio-chemotherapy-resi stant and relapsed glioma tissue samples.1.2.2 Relationship between the expression level of CEP55 and prognosis of gliomaKaplan-Meier survival curves of both TCGA and CGGA databases indicated that patients with high expression of CEP55 had a shorter survival.Univariate and multivariate analyses of online Cox proportional risk regression model in CGGA database indicated that CEP55 was an independent prognostic factor.Univariate and multivariate Cox regression analysis of 254 glioma patients showed that the expression level of CEP55 protein,WHO grade and age were independent prognostic factors.2 CEP55 affects TMZ sensitivity of glioblastoma2.1 MGMT promoter methylation as a marker of TMZ sensitivity was divided into drug resistant group（MGMTU）and sensitive group（MGMTM）.Immunohistochemical method was used to detect the expression level of CEP55 in glioma,and the results showed that the expression level of CEP55in TMZ insensitive group was significantly higher than that in TMZ sensitive group.2.2 GSEA（Gene Set Enrichment Analysis）Gene Enrichment Analysis was used to predict the interaction between CEP55 and Cell Cycle and G₂/M checkpoint,and the results showed positive correlation.2.3 GBM cell CEP55 was silenced by si RNA technology,and the proliferation of GBM cells was inhibited by CCK-8 assay.Flow cytometry results indicated that the silenced CEP55 cells were blocked in the G₂/M phase.Next,q PCR and WB were used to detect the key molecules in the G₂/M phase,and the results showed that cyclin B was decreased.3 CEP55 may affect TMZ sensitivity by binding with YB-13.1 Bioinformatics of TCGA database predicted that CEP55 might be positively correlated with YB-1,TP53,MELK and YB-1,among which YB-1was the most correlated with correlation coefficient of 0.593.3.2 The expression of CEP55 and YB-1 in glioblastoma was detected by immunofluorescence method,and the number of cells co-expressing CEP55and YB-1 in the same area was correlated.3.3 CEP55 and YB-1 were knocked down in U87 and U251 cells,respectively,and the expression levels of YB-1 and CEP55 were detected by WB and q PCR.3.4 Co immunoprecipitation（CO-IP）method was used to explore the relationship between CEP55 and YB-1,and to determine the interaction between CEP55 and YB-1 in glioblastoma.3.5 CEP55 and YB-1 staining were performed on paraffin tissue of 34 cases of glioblastoma by immunohistochemical method,and correlation analysis was conducted.The results showed that there was a positive correlation between the two（r=0.255）.Conclusion:1 The high expression of CEP55 in glioblastoma is associated with susceptibility to temozolomide2.CEP55 affects the proliferation of GBM cells,and silencing CEP55mediates G₂/M arrest,which may be involved in the regulation of TMZ sensitivity.3.CEP55 may affect the drug sensitivity of TMZ through its interaction with YB-1...