| 1.BackgroundGlioma is the most common primary central nervous system tumor in clinic.The5-year survival rate of glioma patients is only 5%.Glioma is a highly malignant,invasive growth of tumor,which is not clearly demarcated from the surrounding normal brain tissue and easy to relapse,so the clinical efficacy is not ideal.At present,the widely applicated strategy for treatment of glioma is to resect the tumor in the greatest extent by surgery,combined with radiation and temozolomide-based chemotherapy,which has improved the quality and prolonged the life of glioma patients in recent years.However,the prognosis of high-grade glioma patients has not been significantly improved,and drug resistance to chemotherapeutics has emerged.Therefore,there is an urgent need to find novel targets for glioma.Thyroid Hormone Receptor Interacting Protein 13(TRIP13),an AAA+ATP enzyme,was first found to be associated with mitotic checkpoints in mice.Recent studies have shown that TRIP13 is low expressed in most normal tissues and highly expressed in many malignant tumors,such as colon cancer,breast cancer,non-small cell lung cancer and head and neck cancer,promoting the migration and proliferation of colon cancer cells and enhancing the proliferation and invasion of head and neck cancer as well as non-small cell lung cancer.It has been reported that TRIP13 is involved in the regulation of mitosis and repair of DNA damage,and plays an important role in the regulation of cancer cell cycle.However,the function and mechanisms of TRIP13 in glioma cells remain elusive.By analyzing the gene expression profile of glioma patients in TCGA(The Cancer Genome Atlas)public database,it was found that the survival rate of glioma patients was significantly reduced with high expression of TRIP13,suggesting that TRIP13 is a potential oncogene in glioma.Many studies have shown that epidermal growth factor receptor(EGFR)is specifically amplified in most glioma patients,and EGFR amplification and mutation are the critical causes of death in glioma patients.Our preliminary studies have found that TRIP13 interacts with EGFR,suggesting that TRIP13 may regulate glioma progression through EGFR pathway.The purpose of this study is to elucidate the function and mechanisms of TRIP13 in glioma progression,and to provide new insights for targeted therapy of glioma.2.Methods(1)The correlation was compared between TRIP13 expression and survival rate of glioma patients from the data bank GSE4271?U132A.Immunohistochemical analysis was performed on 80 glioma sections of different pathological grades to detect TRIP13.The expression levels of TRIP13 in established glioma cell lines was determined by Western blot assay.Laser confocal microscopy was used to examine the localization of TRIP13 in different tumor cells.(2)The cell lines of stable knockdown or overexpression TRIP13 were established using lentiviral infection.Transwell invasion test and chicken embryo invasion model were used to detect the effects of TRIP13 on the invasive ability of glioma cells.The effect of TRIP13 on the proliferation of glioma cells was analyzed by clonogenic assay.The effect of TRIP13 on the sphere formation ability of glioma cells was analyzed by culturing glioma cells under 3D conditions.Western blotting was used to detect the effect of TRIP13 on the expression of stemness-related marker proteins in glioma cells.(3)The signal pathways regulated by TRIP13 in glioma cells were detected by human Phospho-Kinase Array kit.The effect of TRIP13 on the expression of EGFR under EGF stimulation was detected by Western blot assay.Real-time PCR was used to analyze the EGFR expression regulated by TRIP13.(4)The interaction between TRIP13 and EGFR,the effect of TRIP13 on the ubiquitination level of endogenous EGFR and the phosphorylation of TRIP13 regulated by EGFR under EGF stimulation were detected by immunocoprecipitation assay.The co-localization of TRIP13 and EGFR in glioma cells was determined by laser confocal microscopy.(5)The expression of chemokine CXCL6 in culture medium was detected by a ELISA kit.(6)The IC50value of osimertinib in glioma cells was detected by CCK8 assay.The inhibitory effect of osimertinib on glioma cells was dissected by clone formation assay and Transwell assay.Western blotting was used to investigate the effect of TRIP13 on the expression of paraptosis protein.3.Results(1)TRIP13 is highly expressed in glioma tissuesTRIP13 was highly expressed in high-grade gliomas(grade III,IV)and low expressed in low-grade gliomas(grade I,II).Analysis of GSE4271?U132A database showed that the expression of TRIP13 was negatively correlated with the survival rate of glioma.TRIP13 is localized in the nucleus of most tumor cells,such as non-small cell lung cancer,colon cancer,breast cancer,but mainly in the cytoplasm of glioma cells.(2)TRIP13 promotes glioma cell proliferation,invasion and sphere formationKnockdown TRIP13 inhibited the ability of glioma cells to form clony and to invade.Overexpression TRIP13 promotes the formation of glioma spheres and increases the expression of stem cell marker proteins Nestin and SOX2.TRIP13 promoting glioma cell proliferation,invasion,and formation of spheres is not related to the ATPase activity of TRIP13.(3)TRIP13 promotes EGFR pathway activationOverexpression of TRIP13 resulted in an increase in the expression levels of EGFR,p-AKT(S473)and p-ERK1/2 in glioma cells cultured in 3D or stimulated by EGF.Furthermore,TRIP13 promotes the activation of EGFR pathway,which is not related to the ATPase activity of TRIP13.(4)TRIP13 interacts with EGFR and EGFRVIIIFlag-TRIP13,GFP-EGFR and myc-EGFRVIII were overexpressed in 293T cells.The results of immunoprecipitation showed that TRIP13 interacted with EGFR and EGFRVIII.The interaction was not related to the ATPase activity of TRIP13,but positively associated with the kinase activity of EGFR.In addition,endogenous EGFR in U87MG and LN-229 cells also interacted with TRIP13,and the interaction was enhanced upon EGF stimulation.(5)EGFR and EGFRVIII phosphorylate TRIP13 at Tyr56Activated EGFR or EGFRVIII phosphorylated TRIP13 at Tyr56.Overexpression of TRIP13-Y56F mutant could not promote glioma cell invasion and microsphere formation.(6)TRIP13 promotes the secretion of chemokine CXCL6The transcriptome sequencing results showed that knockdown TRIP13significantly decreased the chemokine pathway,and CXCL6 decrease was the most significant.The expression of CXCL6 increased in the supernatant of TRIP13-overexpressing cells.In addition,TRIP13 regulated CXCL6 expression through EGFR.Furthermore,CXCL6 promoted the invasion of glioma cells,but had no effect on the proliferation of glioma cells.(7)Knockdown of TRIP13 enhances the sensitivity of glioma cells to osimertinibOsimertinib inhibited the proliferation,invasion and sphere formation of glioma cells.Osimertinib inhibited the activation of EGFR pathway,promoted the expression of protein CHOP,and attenuated the expression of anti-apoptotic protein Bcl-2.Overexpression of TRIP13 reduced the death of glioma cells induced by Osimertinib and downregulated the expression of CHOP protein.4.Conclusions(1)High expression of TRIP13 reduces the survival rate of glioma patients(2)TRIP13 promotes glioma cell proliferation,invasion and sphere formation(3)TRIP13 promotes EGFR pathway activation upon EGF stimulation(4)EGFR and EGFRVIII phosphorylate TRIP13 at Tyr56(5)Knockdown of TRIP13 inhibits the secretion of chemokine CXCL6(6)Knockdown of TRIP13 enhances the sensitivity of glioma cells to osimertinib... |
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