To Study The Mechanism Of EGFR/EGFRvⅢ Downregulates AJAP1 Expression Level In Glioma

Objectives: Though analysising the TCGA,CGGA and REMBRANT data,we confirmed a negative correlation between EGFR and AJAP1 expression in four GBM subtypes(classical,mesenchymal,neural,and proneural),especially in the classical subtype which frequently shares EGFR amplification and mutation.Combining with stablishing lentivirus containing EGFRvⅢ(a persistent activing EGFR subtype)and using small molecule inhibitors,we verify and prove that GBM can silence expression of AJAP1 to enhance invasion though EGFR pathway,and MK2206 can block EGFRvⅢ downstream activating.MK2206 may be a promising therapeutic for EGFR amplication ang EGFRvⅢ-positive GBMs.Methods:1.Though bioinformatic analysising the public database(TCGA,CGGA and Ranmbrant)about cope number,messaging RNA and protein to confirm the relationship between EGFR and AJAP1.We establish GBM cell lines with EGFRv Ⅲ lentivirus in which EGFR downstream pathway are permanently activated in vitro and found that activating downstream of EGFR leads to AJAP1 downregulation at the mRNA level.2.Establishing U87 and LN229 which stably express EGFRvⅢ though transfecting EGFRv Ⅲ lentivirus.Using real-time PCR to test mRNA level of AJAP1;Using western blot to test proterin level of EGFR,EGFRvⅢ,p-EGFR,AKT,p-AKT and AJAP1.Using immunofluorescent staining to detect rebuilding of glioma cell skeleton casused by activting EGFR pathway.Using transwell experiment to detect effect on glioma cell migration ability arise from EGFR pathway activation.3.To block EGFRvⅢ-PI3K/AKT-EZH2 signaling pathway on different nodes,we choice three small molecule inhibitors: LY294002(PI3K inhibitors),MK2206(p-AKT inhibitors),DZNEP(histone methylation inhibitors),then observe AJAP1 mRNA and protein expression changes and cytoskeleton and morphological changes after segmental block EGFR/EGFRvⅢ downstream.4.Using bioinformatics analysis forecast binding sites of H3K27Me3 in AJAP1 promoter region;Designing targeted AJAP1 promoter region Chip-PCR primer to identify difference of bingding quantity between control group,EGFR pathway persistent activation group and treatment group.5.Building EGFR/EGFRvⅢ glioma intracranial animal model,to explore the intracranial function of p-AKT small molecule inhibitor MK2206.Identifing expression quantity of AJAP1 and local invasion of EGFR/EGFRv Ⅲ glioma after blocking EGFR/EGFRv Ⅲ-PI3K-AKT-EZH2 in vivo.Results:1.Bioinformatic analysis showed that EGFR and AJAP1 have no significant correlation in the gene copy number,but in the mRNA level exists significant negative correlation;In TCGA,CGGA and Ranmbrandt three databases,especially in classcial subtypes which accompanys with enrichment of EGFR,AJAP1 and EGFR get an obvious negative correlation.These results prompt that there is a transcription regulation between EGFR and AJAP1.2.After transfected with EGFRvⅢ virus,expression level of p-EGFR and p-AKT raised obviously in glioma cells,and AJAP1 expression level decreased obviously;Confocal microscope imaging showed AJAP1 expression is reduced in glioma cells expressing EGFRvⅢ,and F actin staining showed increased filiform pseudopodia and tabular pseudopodia reducing;Transwell experiments showed expression of EGFRvⅢ significantly enhance glioma’s capability of cell migration.3.After expressing AJAP1,there will be reduced filiform pseudopodia and increased tabular pseudopodia in glioma cell lines.4.In EGFRv Ⅲ positive glioma cell lines,using PI3 K inhibitor(LY294002),p-AKT inhibitor(MK2206)and histone methylation inhibitors(DZNEP)along a concentration gradient,increase AJAP1 protein expression quantity;Confocal microscopy imaging showed that AJAP1 expression level is upregulated in EGFRvⅢ group after dealing with the p-AKT inhibitor,and fewer filiform pseudopodia and more tabular pseudopodia.5.Immunofluorescence staining showed raised H3K27Me3 expression quantity after expressing EGFRv Ⅲ in nucleus,but after treating with p-AKT inhibitor H3K27Me3 expression downregulated;In nucleoprotein western blot testing we get the same result.6.Chip-PCR experiment shows that binding quantity of H3K27Me3 in AJAP1 promoter region after expressing EGFRv Ⅲ is increased,and it is declined when combined with treatment of p-AKT inhibitor MK2206.7.Combined with 87-Vector/U87-EGFRvⅢ mixed glioma intracranial model,we confirmed that p-AKT inhibitor MK2206 can successfully block EGFR/EGFRv Ⅲ downstream AKT phosphorylation and raise AJAP1 expression level,HE staining showed tumor border is more clearly in treatment group compared to control group.Conclusions:1.EGFR/EGFRv Ⅲ signaling pathways activated can change the glioma cell skeletal structure,and increase the glioma migration ability.2.Inproved expression level of p-AKT and EZH2 can silence AJAP1 in vitro.3.Our results show that blocking EGFR/EGFRvⅢ-AKT-EZH2 can upregulate AJAP1 expression in vivo and in vitro.4.EGFR/EGFRv Ⅲ-AKT-EZH2 signaling pathway inproves combination of H3K27Me3 in AJAP1 promoter region,which can silence AJAP1 expression.5.Small molecule inhibitor MK2206 has therapy effect for abnormal activation of EGFR/EGFRvⅢ signal pathway in glioma.