The Study Of E3 Ubiquitin Ligase Cullin-7 Promoting Malignant Progression Of Glioma Via NF-κB Activation

Objective:The highest incidence rate of intracranial malignant tumour is glioma.Even after surgery combined with chemoradiotherapy,the prognosis of patients with high-grade glioma(WHOⅢ-Ⅳ)is still very poor.Cullin-7(CUL7)is a E3 ubiquitin ligase in Cullin family,which participates in the cell biological behavior regulation.This study aims to analyze the CUL7 clinical significance in glioma,explore its molecular mechanism of promoting the malignant progression of glioma,and provide a new theoretical basis for molecular targeted therapy of glioma.Methods:1.Genome sequencing data of glioma patients were collected from Chinese Glioma Genome Atlas(CGGA)as well as the Cancer Genome Atlas(TCGA)databases.The protein expression of 38 glioma patients treated in Qilu hospital was analyzed by immunohistochemical.CUL7 expression levels in patients with different WHO grades were compared.2.The clinical information and molecular genetic characteristics such as gender,KPS scores,age,IDH1 mutation,1p/19q codeletion,TERT expression,ATRX mutation,MGMT promoter methylation as well as survival times of the patients were collected.The relationship between clinical information and CUL7 expression was analyzed used χ2 test,and survival was analyzed by Kaplan Meier and COX proportional hazard regression models.3.Gene Set Enrichment Analysis(GSEA)was used to calculate mechanism of CUL7 promoting tumorigenesis.4.CUL7 was knocked down by siRNA or lentivirus in U87MG and U251 glioma cell lines,and CUL7 was overexpressed by GV141-CUL7 plasmid.5.5-ethyl-2’-deoxyuridine(EdU),flow cytometry and Transwell assays were used to identify the ability of cell proliferation,invasion as well as migration in vitro.6.The intracranial xenogenic tumor model was constructed in nude mice.The effects of CUL7 knockdown on glioma tumorigenesis and malignant progression were evaluated by small animal imaging and survival analysis.7.Co-immunoprecipitation(co-IP)and Western blot were used to detect the effect of the interaction between CUL7 and MST1 on NF-κB activation.8.The CUL7’s direct inhibitory molecule,miR-3940-5p was predicted by Targetscan website,which was estimated by double luciferase reporter gene analysis.9.EdU,flow cytometry and Transwell assays were used to verify the biological role of miR-3940-5p in glioma.Results:1.In CGGA as well as TCGA databases,CUL7 expression in patients with high-grade glioma was increased.In 38 patients treated in Qilu hospital,the expression of CUL7 was also increased with the increase of WHO grade.2.Among 617 patients in TCGA database,CUL7 expression was correlated with age,KPS score,MGMT promoter methylation,IDH1 mutation,1p/19q codeletion,TERT expression as well as ATRX mutation,but not gender.Survival analysis showed that patients with high CUL7 expression had poor prognosis.Univariate COX proportional hazard regression model found that high CUL7 expression was high risk factor,while multivariate COX proportional hazard regression model obtained the same results.3.GSEA discovered that CUL7 was intimately correlated to the progression of glioma.4.In vitro,CUL7 knockdown inhibited migration,invasion as well as proliferation;overexpression of CUL7 promoted migration,invasion as well as proliferation of glioma cell lines.5.GSEA showed that CUL7 was positively correlated with NF-κB pathway.Western blot verified that CUL7 promoted NF-κB activation in glioma cells.6.Through the co-IP experiment,we discovered that CUL7 bound to MST1 resulting in ubiquitin-mediated degradation of MST,which promoted NF-κB activation.7.After negative control and CUL7 knockdown U87MG cells were implanted into nude mice,it was found that the tumor progression was inhibited and the survival time of animals was prolonged after CUL7 knockdown.8.MiR-3940-5p could directly bind to 3 ’UTR region and reduced CUL7 expression.9.MiR-3940-5p inhibited the migration,invasion as well as proliferation of glioma cells.Conclusion:1.High CUL7 expression was positively correlated with high WHO grade as well as poor prognosis.2.CUL7 promoted ubiquitin-mediated MST1 degradation as well as NF-κB activation,which promoted the progression of glioma.3.CUL7 played an important role in promoting malignant progression of glioma and might be an important target for the treatment of glioma.