| Background:Glioma is a common malignant tumor in the brain.It originates from the central nervous system,which accounts for about 45% of the tumors in the central nervous system and 80% of the malignant tumors in the primary central nervous system.Glioma is characteristic as infiltrating growth,high invasion and unclear boundary.So the operation is difficult to excise completely but easy to postoperative recurrence.Although surgery primarily integrated scheme of combined radiotherapy and chemotherapy has become the treatment guidelines,but for high grade glioma(WHO class Ⅲ,Ⅳ)patients,under the new technology of image guided and neural electrophysiological monitoring,the result of resection is not very ideal,improve prognosis was not significant.The average survival period after surgery is only 12 ~ 15 months,and the 5-year survival rate is less than 10%.Clinical imaging showed that recurrent lesions,often appeared within 2cm around the tumor,were still abundant in blood supply.At present,the pathogenesis of glioma is not yet clear,and the study on the invasion function of glioma is still not perfect.Therefore,it is very important to further explore the development of glioma and the related molecular biological mechanism.In addition,new biomarkers or therapeutic targets are explored,and they are important to diagnose glioma and predict the risk of recurrence and improve the prognosis of patients.One of the members of the Polycomb Group family proteins,the Chromobox Protein Homolog7(CBX7),plays an important role in the development of multiple tumors.In the current study,CBX7 plays a role in the tumor suppressor genes in thyroid cancer,breast cancer and pancreatic cancer.Epithelial mesenchymal transition(EMT)process,represents an important part of the tumor invasion.Cubic epithelium removes the adhesion of the surface,and morphologically transforms into spindle fiber cell morphology,which is conducive to the movement of cells,so it is also beneficial to the metastasis of tumor cells.Although there is no large amount of epithelial tissue in the intracranial brain tissue,there is a process similar to EMT,called EMT-like process.The Wnt/β-catenin signaling pathway is an important pathway in the study of tumor invasion and metastasis,and ZEB1 is the downstream regulator.DKK1 is the inhibitory protein of this signaling pathway,by which the EMT-like process can be adjusted.At present,the relationship between CBX7 and glioma is still unclear,especially in the invasion process of glioma.Objective:To investigate the expression of CBX7 in human glioma,and the effect of CBX7 on the invasion of glioma cells.Methods:First,The expression of CBX7 was analyzed by The Chinese Glioma Genome Atlas,CGGA,The Cancer Genome Atlas(TCGA)and GSE16011 database.The expression of CBX7 in normal astrocytes and Glioma cells(U118,A172,Ln229,T98G)were detected by Western blot.Human specimens(5 normal brain tissues and 27 glioma specimens)were used to detect the differences in the expression via immunohistochemistry and Western blot.Then,lentivirus Lv-CBX7 was transfected into glioma cell lines Ln229 and T98 G.Through Transwell experiment,wound healing,and the 3D invasion experiment,the effect of CBX7 on the migration and invasion function of glioma cells was detected.Western blot technology was used to detect the changes of Wnt/ beta-catenin signaling pathway,EMT-like process related proteins(Vimentin,E-cadherin and N-cadherin)and MMP2 and MMP9.Immunofluorescence technique was used to observe the location distribution of beta-catenin protein.Chromatin Immunoprecipitation(Ch IP)and biluciferase reporter gene were used to verify the binding sites between CBX7 and DKK1 promoter region.Through the use of Small interfering RNA(Small interfering RNA,si RNA),the effect of CBX7 was interfered by si-DKK1,which was detected by western blot.In addition,the influence of DKK1 on the invasion function of glioma was observed by Transwell experiment,wound healing and 3D invasion experiment.Intracranial tumor test was performed to analyze the survival of the nude mice and the size of the tumor.The results of the HE section were directly observed to indicate the impact of CBX7 on the invasion ability of glioma.Immunohistochemical technique was used to detect the expression of invasion related proteins after lentivirus Lv-CBX7 treatment.Result:The database analysis of CGGA,TCGA and GSE16011 showed that the m RNA expression of CBX7 in glioma tissues was significantly lower than that of normal brain tissue,and the expression was lower in glioma tissues with high malignancy.Western blot and immunohistochemical results verify that CBX7 has low expression in glioma.Lentivirus Lv-CBX7 enhances the expression of CBX7 in glioma cells,and Transwell experiment,wound healing,and 3D invasion experiment confirmed that up-regulated CBX7 can inhibit the migration and invasion of glioma cells.The expression of Vimentin and N-cadherin as well as MMP2 and MMP9 were detected by Western blot technology.The expression of MMP2 and MMP9 was down-regulated after CBX7,but E-cadherin was up-regulated.Immunofluorescence observation of beta-catenin protein was transferred from nucleus to cytoplasm after up-regulation of CBX7.Using Chromatin Immunoprecipitation(Ch IP)and biluciferase reporter gene validated CBX7 can bind to the promoter region of DKK1 and promote the transcription of DKK1.The expression of DKK1 was down-regulated by si-DKK1,which was detected via Western blot.Si-DKK1 which could reactivate the Wnt/ beta-catenin signaling pathway,promote the expression of Vimentin,N-cadherin,MMP2 and MMP9 but inhibit E-cadherin and effect of CBX7.Through Transwell experiment,wound healing,and the 3D invasion experiment,the reduction of DKK1 can partially reverse the effect of CBX7 on the invasion function of glioma.In the intracranial tumor test,the survival period of the nude mice with LV-CBX7 tumor cells was significantly better than that of the normal tumor group.The results of HE section were directly observed after the increase of CBX7,and there was a significant decrease in the peripheral metastases of glioma.Immunohistochemical technique was used to prove that the increased expression of DKK1 in the intracranial tumors after the increase of CBX7 could decrease the expression of Vimentin and N-cadherin and MMP2 and MMP9.Conclusion:CBX7 reduces the activity of Wnt/ beta-catenin signaling pathway by promoting transcription of DKK1,which can effectively inhibit the invasion of glioma cells.Therefore,the further study on the mechanism of CBX7 inhibiting the invasion of glioma cells will provide a new direction for the clinical diagnosis and treatment of glioma,which is of great clinical significance. |
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