TIPE1 Activates MAPK/ERK Pathway To Promote The Proliferation And Metastasis Of Glioma Cells

Background:Glioma is a kind of primary brain tumor with high incidence and mortality.The incidence increases with age and malignancy degree,among which glioma accounts for about 75.3%of primary and other central nervous system malignancies and 80%of malignancies.The median survival time of glioma patients is about 12～15 months.The complicated pathogenesis of glioma results in high heterogeneity of tumor tissues and high degree of vascularization,leading to the poor prognosis.However,the current clinical treatment still have limitations.Therefore,it is an urgent medical problem to find the novel potential targets to improve the therapeutic efficacy.Tumor necrosis factor induced-protein8-likel(TIPE1),belonging to the TIPE family,is widely expressed in various tissues and cells,mainly locates in the cytoplasm,and affects the activation of related signaling pathways through interacting with a variety of proteins.Furthermore,it regulates a variety of biological processes(such as cell apoptosis,proliferation,migration,etc.)and participates in the occurrence and development of tumors,non-alcoholic fatty liver,diabetic nephropathy and other diseases.Our previous studies have found that TIPE1 activates the PIP3/Akt/TGF-β axis and promotes the selective activation of macrophages,leading to the progression of liver cancer and melanoma.These results suggest that TIPE1 might be involved in tumor progression through multiple mechanisms.Studies have shown that TIPE1 is highly expressed in brain tissue,but the role of TIPE1 in the development of glioma has not been reported.Objectives:1.To clarify the expression of TIPE1 in glioma.2.To clarify the effects of TIPE1 on the proliferation and metastasis of glioma cells.3.To explore the molecular mechanisms of TIPE 1 involved in the development of glioma.Methods and results:1.TIPE1 expression is up-regulated in gliomaIn order to preliminarily analyze the possible role of TIPE1 in the progression of glioma,we first detected that the expression of TIPE1 in human glioma tissues and found that TIPE1 expression in glioma tissues was significantly higher than that in adjacent tissues.We further analyzed the clinical data set of glioma patients through TCGA,GEPIA,UALCAN and other database.Results showed that TIPE1 expression correlated with the grade of glioma.Moreover,the median survival time of patients with high TIPE1 expression in glioma tissues had shorter survival time than those with low TIPE1 expression.These results suggest that TIPE1 expression correlates with pathological degree and prognosis of glioma,indicating the potential involvement of TIPE1 in glioma development.2.TIPE1 promotes the growth,migration and invasion of glioma cellsIn order to further clarify the role of TIPE1 in the development of glioma,we constructed glioma cell lines U251 and U87 stably infected with TIPE1 shRNA lentivirus expression,and found that interference with TIPE1 significantly inhibited the proliferation of glioma cells through cell growth curve,clonal formation,and Edu staining.On the contrary,overexpression of TIPE1 promoted the proliferation of glioma cells.Scratch healing and Transwell experiments showed that TIPE1 knockdown significantly inhibited,migration and invasion ability of U251 and U87 cells,while overexpression of TIPE1 showed a promoting effect.3.TIPE1 activates MAPK/ERK pathway to promote glioma cell growth,migration and invasionTo further explore the mechanisms by which TIPE1 promotes glioma progression,we utilized transcriptomic sequencing(RNAseq)to analyze the differentially expressed genes in control and TIPE1 knockdown U87 cells.Kyoto Encylopaediaof Genesand Genomes(KEGG)pathway analysis showed a significant enrichment in MAPK signal pathway.Western blot further confirmed that interference of TIPE1 significantly down-regulated the phosphorylation levels of p38,JNK,ERK in U87 and U251 cells,while overexpression of TIPE1 had a promoting effect.Analysis of clinical samples showed that TIPE1 expression in glioma tissues positively correlated with the phosphorylation levels of p38,JNK and ERK.More importantly,Edu staining showed that although JNK and p38 phosphorylation inhibitors did not affect the effect of TIPE1 on the proliferation of glioma cells,ERK phosphorylation inhibitor eliminated the inhibition of TIPE1 interference on the proliferation of glioma cells.These results suggest that TIPE1 promotes the development of glioma through activating ERK pathway.4.TIPE1 upregulates VEGF expression through ERK pathway to facilitate glioma cell proliferation and metastasisFurther analysis of the above RNAseq data showed that the expression of VEGFA gene in MAPK signaling pathway related genes was significantly down-regulated in TIPE1 knockdown U87 cells.RT-qPCR and ELISA confirmed that interference with TIPE1 expression in U87 and U251 cells significantly inhibited VEGFA expression,while overexpression of TIPE1 elevated its level.Moreover,ERK phosphorylation inhibitors abrogate the regulation of TIPE1 on VEGFA expression.In order to further investigate whether TIPE1 affects the growth and metastasis of glioma cells by VEGFA,control or TIPE1 overexpressed U87 cells were transfected with small interference RNA for VEGFA.Edu staining showed that interference with VEGFA expression eliminated the promotion effect of TIPE1 overexpression on the proliferation,invasion and migration of glioma cells.These results suggest that TIPE1 promotes the proliferation,invasion and migration of glioma cells by regulating ERK/VEGF axis.In addition,to evaluate whether TIPE1 influences the effect of glioma cells on angiogenesis,HUVEC cells were treated with conditioned medium for control or TIPE1 knockdown glioma cells.Tube formation assay showed that compared to control groups,HUVEC cells treated with conditioned medium from TIPE1 knockdown glioma cells had the reduced number of branches and tubule length,while overexpression of TIPE1 has the opposite effect.In addition,GEPIA database analysis showed a significant positive correlation between TIPE1 and VEGFA expression in glioma tissues.Immunohistochemical staining showed that patients with high TIPE1 expression in glioma tissues had high level of CD31 expression.These results preliminarily suggest that TIPE1 promotes glioma cell proliferation,invasion and migration,as well as angiogenesis,by activating ERK signaling pathway to up-regulate VEGFA expression.Conclusion:1.TIPE1 is up-regulated in glioma and promotes tumor progression.2.TIPE1 enhances the proliferation,invasion and migration of glioma cells through MAPK/ERK signaling pathway.3.TIPE1 activates MAPK/ERK signaling pathway to up-regulate VEGFA expression and to promote glioma neovascularization and tumor progression.Innovation and significance:1.This study identifies the role of TIRE1 in promoting the development of glioma cells,and the high expression of TIPE1 might be one of the potential prognostic factors in glioma patients.2.This study has proved that TIPE1 activates ERK and up-regulates VEGF expression in glioma cells,thus promoting glioma growth,metastasis and neovascularization,providing a new theoretical basis for elucidating the pathogenesis of glioma.