GLI2 Promotes Cell Proliferation And Migration Through Transcriptional Activation Of ARHGEF16 In Human Glioma Cells

Background:Neurogliocytoma,generally called glioma,is derived from neurogliocyte and is the most common malignant tumors in central nervous system,of which 54% are glioblastoma.Glioblastomas present strong capabilities of proliferation,angiogenesis and invasion,leading to serious injury in adjacent normal tissues,and just one third of the patients with glioblastoma survive for over a year,with the 5-year survival rate being only 5%.The early diagnosis of gliomas and the 5-year survival rate of the patients have not improved significantly due to our deficient understanding of the underlying mechanisms of glioma initiation and progression.Therefore,exploration of these mechanisms along with identification of novel biomarker and drug target in gliomas is of great importance for the diagnosis and therapy of gliomas.Hedgehog（Hh）signalling pathway is very important in regulating animal embryogenesis,and its main components include Hh ligand,membrane receptor Patched（Ptch）,transmembrane protein Smoothened（Smo）,negative regulator Sufu and the zinc-finger transcription factors GLIs（GLI1,GLI2 and GLI3）.In germ cells,disregulated Hh signalling pathway leads to development disorders,while in somatic cells the hyperactive signalling pathway is often associated with ocurrence of a number of humna cancers.For instance,Hh signalling pathway is aberrantly activated to different extent among basal cell carcinoma,medulloblastoma,lung cancer,breast cancer and glioma,though the underlying mechnisms by which Hh signalling pathway promotes the initiation and progression of multiple human cancers need further illumination.The functions of Hh signalling pathway in cancers are mediated mainly through the target genes of GLI transcription factors.In this study,in order to identify novel target genes of GLIs and reveal their functions and molecular mechanisms in glioma,we compared the gene expression profiles between control glioma cells and GLI2A-overexpressing glioma cells to screen differentially expressed genes for sunsequent studies,with the hope to make some improvemnets in understanding mechanisms of glioma initiation and progression.Methods and results:1.Exploration of genes regulated by GLI2 in glioma cells Methods:1.The core components of Hh signalling pathway,such as PTCH11,Smo,Sufu,GLI1 and GLI2 were examined through western blottingting and real-time quantitative PCR（Q-PCR）in H4,U87 and U118 glioma cells to confirm the existence of the Hh signalling pathway in these cell lines.2.Microarray analysis between the GLI2A-overexpressing stable cell lines（U87 GLI2A）and the control U87 cells（U87 Control）was performed to discover the differentially expressed genes.3.Some of the most significantly upregulated genes in U87 GLI2 A cells was selected and confirmed via Q-PCR,and finally ARHGEF16 was identified as the candidate gene positively regulated by GLI2.4.A online software（http://www.genomatix.de/）was utilised to analyze the putative GLI binding site（GBS）within ARHGEF16 promoter,and the DNA fragments containing GBS were cloned to construct the pGL3 basicARHGEF16-Luci reporters.5.pGL3 basic-ARHGEF16-Luci constructs and pRL-TK were co-transfected into U87 cells that overexpress GLI2 A or the corresponding control cells,and 48 hours later the luciferse activity was assayed to examine the activating effect of GLI2 on different pGL3 basic-ARHGEF16-Luci constructs.6.Chromatin-immunoprecipitation was performed to detect the binding of GLI2 onto the promoter of ARHGEF16.Results:1.PTCH11,Smo,Sufu,GLI1 and GLI2 are all expressed among the H4,U87 and U118 glioma cells,though the expression level of each component showed variation in different cell types.This indicated the existence of Hh signalling in these cells.2.The microarray analysis revealed that compared with control cells,U87 GLI2 A cells presented 814 down-regulated genes and 1121 up-regulated genes,and ARHGEF16,one of the most significantly up-regulated genes,was selected for furhter study.In H4,U87 and U118 cells overexpressing GLI2 A,the levels of both mRNA and protein of ATHGEF16 were significantly up-regulated,and in contrast suppression of GLI2 via RNA interference or GANT61 in H4 cell（an inhibitor of GLI1 and GLI2）resulted in reduced expression level of ARHGEF16.3.Within the promoter and adjacent regions（-2500 +2500,the first 5‘ base of ARHGEF16 mRNA [NM₀14448.3] was designated as +1）of ARHGEF16,9 putative GBSes（GBS1-9）were discovered,and the DNA region containing these GBSes were cloned and inserted into pGL3 basic vector to construct a total of three pGL3 basic-ARHGEF16-Luci reporters,named Frag-I,-II,and-III.4.In dual-luciferase assay,it was shown that Frag-I,and Frag –III were significantly activated by GLI2 A.However,once GBS2 or GBS9 was mutated,Frag-I and Frag-III ARHGEF16-Luci reporters could no longer be activated by GLI2,indicating the crucial roles of GBS2 or GBS9 in activating ARHGEF16 transcription by GLI2.5.Compared with Normal rabbit IgG,GLI2 antibody enriched the ARHGEF16 promoter regions containing GBS2 or GBS9,while the negative DNA region was not enriched by GLI2 antibody.2.Study of the functions of ARHGEF16 in glioma cells Methods:1.Since ARHGEF16 expression level is much more higher in H4 than in U87 and in U118,lentiviruses are used to construct U87 and U118 stable cell lines that overxpress ARHGEF16 while H4 used for ARHGEF16 knockdown.2.Transwell assay and soft-agar colony formation assay were performed to compare the capabilities of migration and proliferation between ARHGEF16-overexpressing U87 and U118 cells along with the control cells.Plate clone formation assay was used to detect proliferation capacity of H4 sh-Control and H4 sh-ARHGEF16 cells.3.ARHGEF16 was knocked down in U87 GLI2 A cell.After the knockdown efficiency was confirmed via Q-PCR and western blottingting,transwell migration assay and soft agar colony formation assay were performed to detect migration and proliferation capacities of Control+sh-Control,GLI2A+sh-Control 和 GLI2A+shARHGEF16 U87 cells.4.U87 Control and U87 GLI2 A cells were injected subcutaneously in nude mice to form tumor xenorgrafts.A month after seeding of the cells in the nude mice,the formed tumors were removed and weighed after the mice were killed.Moreover the protein levels of GLI2,ARHGEF16,Ki67（proliferation marker）and MMP9（maker of cell invasion）in the two groups of tumors were analyzed via immunohistochemistry.Results:1.Compared with corresponding control cells,more of U87 and U118 cells overexpressing ARHGEF16 migrated to the other side of the Transwell chamber membrane,indicating that ARHGEF16 promoted the migration of U87 and U118 glioma cells.In soft agar colony formation assay,U87 and U118 cells overexpressing ARHGEF16 formed more and larger colonies compared with the corresponding control cells,indicating ARHGEF16 promoted proliferation of U87 and U118 glioma cells.Conversely,ARHGEF16 knockdown in H4 cell inhibited glioma cell proliferation as assessed via plate clone formation assay.2.Transwell migration assay and soft agar colony formation assay showed that GLI2A+sh-Control U87 cell presented stronger migratory and proliferative capacities than Control+sh-Control U87 cell.However,ARHGEF16 knockdown in GLI2A+sh-ARHGEF16 U87 significantly inhibited both cell migration and proliferation as compared with GLI2A+sh-Control U87 cell,indicating a promoting effect of GLI2/ARHGEF16 signaling on glioma cell migration and proliferation.3.Compared with U87 Control cells,U87 GLI2 A glioma cells proliferated faster and formed larger tumors.Furthermore GLI2,ARHGEF16,Ki67（proliferation marker）and MMP9（marker of cell invasion）presented higer protein levels in tumors of U87 GLI2 A group than in tumors of U87 Control group.3.Exploration of the mechanisms by which ARHGEF16 promotes migration and proliferation of glioma cells Methods:1.The fusion protein BD-ARHGEF16 formed by human ARHGEF16 protein and the DNA binding domain（BD）of yeast transcription factor GAL4 was used as the bait protein to screen ARHGEF16-interacting proteins through yeast two-hybrid.2.Immunoprecipitation assay and GST pull-down assay were used to confirm the interaction of ARHGEF16 and the candidate protein identified through yeas two-hybrid.3.CKAP5 was knocked down in U87 ARHGEF16 cell.After the knockdown efficiency was confirmed,transwell migration assay and soft agar colony formation assay were performed to detect migration and proliferation capacities of Control+sh-Control,ARHGEF16+sh-Control 和 ARHGEF16+sh-CKAP5 U87 cells.Results:1.On the most stringently selective medium agar plate SD/-Ade/-His/-Leu/-Trp/ X-α-Gal/AbA,one of the strongly positive colonies was identified to express a central fragment of humna CKAP5 protein.2.In immunoprecipitation assay and GST pull-down assay,it was shown that CKAP5 could be specifically enriched through ARHGEF16,indicating that ARHGEF16 could interact with CKAP5.3.Transwell migration assay and soft agar colony formation assay showed that ARHGEF16+sh-Control U87 cell presented stronger migratory and proliferative capacities than Control+sh-Control U87 cell.However,CKAP5 knockdown in ARHGEF16+sh-CKAP5 U87 cell significantly inhibited both cell migration and proliferation as compared with ARHGEF16+sh-Control U87 cell,indicating that ARHGEF16 promoted glioma cell migration and proliferation in a CKAP5-dependent manner.Conclusions:1.GLI2 directly activates ARHGEF16 transcriptipn.2.GLI2/ARHGEF16 signaling promotes migration and proliferation of glioma cells.3.ARHGEF16 coordinates with CKAP5 to promote glioma cell migration and proliferation induced by GLI2.