Hypoxia Induced GIT2 Mediates HIF-1? Facilitates Tumor Invasion,migration And Proliferation By Regulating EMT Network In GBM

Research background:Glioblastomas(GBM)are most aggressive primary brain tumors characterized by features of high invasion,migration abilities.Despite advanced treatments of surgery,radiation and chemotherapy,the median survival of the patients is extremely poor with the most malignant glioma is still less than 16 months.In some cases,tumor cells undergo a process of epithelial-to-mesenchymal transition(EMT)to gain invasive capacity and disseminate which causes more difficult to eradicate the tumor,easier to recurrent and poorer prognosis.Git2 is a member of the GIT protein family characterized by an N-terminal GTPase-activating protein domain and exhibit efficient GAP activities toward Arfl and Arf6,function as GAPs in membrane trafficking and actin remodeling.GIT2 also interacts with p21-activated kinase(PAK)-interacting exchange factor(PIX)and paxillin,which serves an essential role in focal adhesion and cell motility.Recent studies found GIT2 regulates directional lung cancer cell migration and loss of GIT2 induces EMT by miRNAs in bladder carcinoma cell lines,however,the role of GIT2 in GBM remain undiscovered.here,we identify Git2 is highly expressed in GBM and serves as a EMT inducer that regulates EMT network and facilitates a mesenchymal shift of GBM.Hypoxia is a common feature of GBM owing to aberrant vascular function and rapid cell division and is a well-known inducer of the epithelial to mesenchymal transition(EMT)program in epithelial cancers like pancreatic ductal adenocarcinoma,hepatocellular carcinoma,ovarian carcinoma and lung cancer.Previous studies demonstrate that tumor hypoxia and up-regulation of hypoxia-inducible factors(HIFs)is linked with EMT-mediated increasing of metastasis and therapy resistance.Our work show that GIT2 expression is enhanced in hypoxia by HIF-1? transcriptionally through HIF-1 a binding to GIT2 promtor which leads to more invasive and migratory functions,facilitating a mesenchymal shift and regulates several EMT downstream targets.We also find GIT2 can stabilize three essential EMT transcription factors named SNAIL1?TWIST1and ZEB1 post-transcriptionally by deubiquitylation of their protein levels thus regulating the whole EMT network.Our findings reveal a brand new mechanism of GIT2 induced in hypoxia which facilitates tumor invasion,migration and proliferation by regulating EMT network in GBMMethods:1.qRT-PCR and Western blot were used to test the GIT2 mRNA and protein level in 5 GBM cells lines,as well as its expression in tumor tissues with paired paired peritumoral tissues in 8 patients.2.CCK8 assay,Colony Formation assay,Transwell invasion assay and wound healing assay were used to explore the function of GIT2 in U87 and A172 GBM cell lines with GIT2 siRNA.3.qRT-PCR and immunofluorescence assay were used to detect the expression of GIT2 in U87 cell under hypoxia or treated with CoC12.Western blot was used to test the expression of GIT2 and HIF-lain 4 GBM cell lines and in 2 primary GBM cells.the expression of EMT gene were also tested by qPCR and Western blot.4.qRT-PCR and Western blot were used to find the Targeted regulatory relationship between HIF-la and GIT2 with shHIF-la plasmid.CCK8 assay,Colony Formation assay,Transwell invasion assay and wound healing assay were used to verify the function of GIT2 in GBM cells under hypoxia with shGIT2 plasmid.5.luciferase reporter assay were used to prove whether the GIT2 was a transcriptional target of HIF-1?,and to explore binding site in GIT promoter,results were further verified through ChIP assay.6.qRT-PCR,Western blot assay,immunofluorescence assay were used to explore the targeted regulatory relationship between GIT2 and EMT genes.7.Western blot was used to test the stability of EMT transcription factors through knockdown of GIT2 in GBM cells of overexpression of GIT2 in 293T cells.The polyubiquitination and proteasomal degradation levels of SNAIL1,TWIST1and ZEB1 were tested by co-IP.8.correlation or the expression of GIT2,HIF-1?,SNAIL1,TWIST1 and ZEB1 in GBMs were explored by Database analysis.Kaplan-Meier curves of overall survival was analyzed with high or low GIT2 expression between patients with GBM.Results:1.GIT2 is markedly highly expressed in GBM cell lines and GBM tissues.2.knockdown of GIT2 remarkably decreased the invasion,migration and proliferation ability in GBM cells.3.GIT2 is highly expressed under hypoxia condition in GBM cells,and is transcriptionally regulated by HIF-1?,GIT2 depletion under hypoxia decreased the Hypoxia-induced invasion and migration ability in GBM cells.4.HIF-1 a regulates GIT2 transcriptionally and binds to the GIT2 promoter under hypoxia.5.the protein level of SNAIL1,TWIST1 and ZEBlwere decreased after knockdown of GIT2 gene,other EMT genes were reduced both transcriptionally and post-transcriptionally.6.knockdown of GIT2 in GBM cells increased SNAIL1,TWIST1 and ZEB1 ubiquitination and proteasomal degradation while over-expression of GIT2 in 293T cells decreased their ubiquitination level.7.GIT2 overexpression correlates with HIF-1?,SNAIL1,TWIST1 and ZEBland predicts poor prognosis in GBM patients.Conclusions:1 GIT2 is highly expressed in GBM cells and was transcriptionally regulated by HIF-1a through binding to GIT2 promoter under hypoxia.2 GIT2 inhibits SNAIL1,TWIST1,ZEB1 ubiquitination and stabilize the expression of these EMT transcription genes,thus enhance the ability of invasion,migration and proliferation of GBM cells under hypoxia.