To Study The Regulation Mechanism Of EGFRvⅢ And EGFRvⅢ Secretion Profiles In Human Glioma

Objectives:1.Establishing the mixed cultivating model of U87 EGFRvⅢ and wt U87,in order to confirm the growth promoting effect and its molecular mechanism of EGFRvⅢ in different proportions of the model.Furthermore,to validate the driving role of EGFRvⅢ for Glioma heterogeneity in vitro and in vivo,and giving us original ideas for understanding the development of glioma.2.Interfere the synthesis and assembly process of secreted proteins in endoplasmic reticulum of EGFRvⅢ cells through the specific binding of F25 P preproinsulin and Spase,to inhibit the growth support role of EGFRvⅢ for surrounding cells,For the targeted treatment of EGFRvⅢ positive glioma provides new ideas.3.To elucidate the molecular mechanism of SPP in glioma progression by interfering the SPP in endoplasmic reticulum and analyzing the EGFRvⅢ secreted expression profiles.Methods:1.Detecting the expression levels of EGFRvⅢ,EGFR,Ki-67,STAT3,NF-KB,EZH2,PKM2 by immunohistochemical method at different sites of clinical specimens.2.U87 EGFRvⅢ and wt U87 were mixed together,MTT and cell clone formation assay were used to detect the proliferation of different mixed proportion.3.Establishing nude mice glioma intracranial model for investigating the effect of different ratio of EGFRvⅢ on tumor growth.4.Treating U87 EGFRvⅢ cells using the F25 P preproinsulin Lentivirus,Collected the supernatant fluid to analyz the effects of supernatant on the phenotype and function of U87,LN229 and U251 in glioma cells.5.Detecting the F25 P preproinsulin antitumor effect after the treatment of DOX in vivo.6.Treating U87 EGFRvⅢ cells using the Lenti-si-HM13,analyze the expression of secreted protein and its influence on the phenotype and function of U87,LN229 and U251 cells.7.Detect the tumor inhibition effects of Lenti-si-HM13 in the intracranial GBM model.Results:1.The detected protein expression of EGFRvⅢ,EGFR,Ki-67,STAT3,NF-KB,EZH2,and PKM2 at different sites in the same GBM specimen showed significant heterogeneity.2.The proliferation rate of U87 cells mixed with different ratio of U87 EGFRvⅢ was significantly different,and the proliferation rate reached the plateau stage when EGFRvⅢ ratio was 30%.3.EGFRvⅢ played a driving role in promoting the growth of glioma in vivo experiments.4.The supernatant collected from U87 EGFRvⅢ treated with F25 P preproinsulin had significant effects on the phenotypic and functional changes of U87,LN229 and U251.5.Inducing expression of F25 P preproinsulin can inhibit the growth of glioma in vivo.6.Secretion profiles of U87 EGFRvⅢ treated with Lenti-si-HM13 change obviously,among which,the most significant change is TGF-β1.7.The in vivo experiments showed that Lenti-si-HM13 could significantly decrease the content of TGF-β1 and TGF-α and inhibit tumor growth in mice.Conclusions:1.There is obvious heterogeneity in GBM,and EGFRvⅢ has a driving effect on GBM growth.2.F25 P preproinsulin can specifically bind to Spase,Effecting the secreted proteins of EGFRvⅢ cells.3.The expression of F25 P preproinsulin could inhibit the growth and metastasis of glioma in vivo and in vitro studies.4.Lenti-si-HM13 can influence the synthesis and assembly of secreted proteins of EGFRvⅢ glioma cells in Endoplasmic Reticulum,then resulting the changes of expression profiles.5.Knocking down the relative expression of HM13 using Lentivirus in EGFRvⅢ suppresses the glioma tumor progression in vivo.