Study On The Molecular Mechanism Of Small Gtpase RhoJ In The Progression And Invasion Of Glioblastoma

[Objective]Glioblastoma Multiforme(GBM)is the most common fatal primary intracranial malignant tumor in the central nervous system(CNS).Despite standard clinical treatment regimens,including surgical treatment accompanied by adjuvant therapies such as chemoradiotherapy,median survival of GBM patients is still less than 15 months.Many molecularly targeted drugs have been developed over the years,but none of these drugs have improved overall survival in patients with primary GBM.Therefore,finding new targets and other comprehensive treatment methods is of great importance for the treatment of GBM patients.RhoJ,a member of the small GTPase family,is a homologue of CDC42.RhoJ is considered to be a key regulator of cell morphology,actin cytoskeleton,cell movement and cell cycle,and plays an important role in angiogenesis,metastasis and progression of various tumors,drug resistance and other aspects.However,the expression,its application in diagnosis,treatment and prognosis of RhoJ in glioblastoma have not been reported.We analyzed RhoJ expression in GBM through TCGA?CGGA and HPA dataset,discussed the role of RhoJ in GBM proliferation,metastasis,invasion and angiogenesis,and further explored its related molecular mechanism,to lay a theoretical foundation for the treatment of GBM patients with RhoJ-targeted antibodies or small molecule inhibitors.[Methods]1 Differential expression analysis of RhoJ in glioma tissue based on database mining1.1 TCGA database was used to analyze the gene expression of Rho GTPases family in GBM tumor and non-tumor tissues.The expression of RhoJ gene expression in GBM/LGG/non-tumor tissues was also analyzed by TCGA database.Kaplan-Meier method was used to analyze the correlation between RhoJ gene expression and survival of glioma patients in clinical data.1.2 CGGA database was used to analyze the gene expression difference of RhoJ in GBM/LGG and recurrent/primary GBM patients.The clinical data corresponding to the gene data in the CGGA database were sorted out.Kaplan-Meier method was used to analyze the correlation between RhoJ expression level and survival of all GBM patients/primary/recurrent GBM patients.1.3 Normal brain tissue sections and clinicopathological sections of gliomas of different grades were collected.Immunohistochemical staining was used to detect the difference of RhoJ expression in GBM tumor tissues and normal brain tissues,and western blot was used to detect the expression of RhoJ protein in GBM cell lines.2 Studies on RhoJ promoting proliferation,migration and invasion of GBM cells in vivo and in vitro2.1 SiRNA and shRNA were used to interfere or silence RhoJ expression,or plenti-RhoJ plasmid was transfected to overexpress RhoJ,the silencing or overexpression efficiency of RhoJ was verified by Q-PCR and Western blot.and the cell activity was detected by cell counting,cell activity was detected by CCK8,clonal formation experiment and tumor sphere formation experiment were performed to detect the effect of RhoJ on the proliferation function of GBM cells.2.2 Transwell migration and invasion assay and scratch healing assay were used to detect the effects of RhoJ on the migration and invasion of GBM cells.2.3 Established U87 cells expressing shNC or shRhoJ were subcutaneously or intracranially injected into nude mice to investigate the influence of RhoJ on the occurrence and development and intracranial metastasis of GBM in vivo.Cell cycle analysis in fixed cells was detected to investigate the effect of RhoJ knockdown on cell cycle arrest.2.4 The effect of knockdown or overexpression of RhoJ expression on tumor metastasis was detected by subcutaneous injection of melanoma cells A375 into lung metastasis model and caudal vein injection metastasis model by B16-F10 into lung metastasis model.3 Study on the molecular mechanism of RhoJ promoting the malignant proliferation,invasion and metastasis of GBM cells.3.1 Cell immunofluorescence assay and western blot were used to detect the effect of RhoJ knockdown on EMT.3.2 The upstream transcription factors of RhoJ were predicted online by using JASPAR and ALGGEN-PROMO databases.The double luciferase reporter assay using pGL3-RhoJ reporter plasmid and small molecule inhibitors were used to verify whether RhoJ was regulated by the expression of these transcription factors.3.3 Flag-RhoJ labeled plasmid was constructed and stably transfected into GBM cells.The key interaction proteins of RhoJ regulating the occurrence and development of GBM were found through mass spectrometry(MS)and bioinformatics analysis followed by co-IP assay using flag-tagged RhoJ vectors.Co-IP and IF were conducted to verify the interacting,and the downstream signaling proteins were explored by western blot and other techniques.4 Study on the role of RhoJ in GBM angiogenesis and related molecular mechanisms4.1 Transwell assay and angiogenesis experiments were used to detect the effects of RhoJ on the migration and angiogenesis of HUVEC cells.4.2 Expression changes of CD31,moesin,EpCAM in RhoJ overexpressed GBM tissues were detected by immunohistochemistry,immunofluorescence and other methods.4.3 VEGF stimulating or silenced RhoJ and JNK by shRNA targeting RhoJ and JNK in HUVEC cells.Western blot was used to detect the phosphorylation levels of related proteins VEGFR2/JNK/PAK/BRAF/ERK,as well as changes in RhoJ and moesin protein expression levels.[Results]1 Differential expression analysis of RhoJ in glioma tissue based on database miningTCGA and CGGA database mining analysis showed that expression of RhoJ in GBM was significantly higher than that of the non-tumor and LGG group,the expression of RhoJ in recurrent group was obviously higher than that of non-recurrence group,immunohistochemical staining found RhoJ was highly expressed in clinical GBM,and RhoJ expression levels were positively correlated with glioma malignant degree and the higher the glioma grading,malignant degree is higher,the expression of RhoJ also is higher.Western blotting also confirmed that RhoJ was highly expressed in GBM cell lines.Kaplan-Meier analysis showed that high RhoJ expression in GBM was significantly associated with poor prognosis in all patients with GBM/primary GBM/recurrent GBM,and was closely related to overall survival and disease-free survival of patients,suggesting that RhoJ was an important factor in poor prognosis of GBM.2 Studies on RhoJ promoting proliferation,migration and invasion of GBM cells in vivo and in vitroCCK-8 and cell proliferation experiments showed that silencing RhoJ expression inhibited the proliferation and activity of GBM cells,induced cell cycle G2/M phase arrest,and cyclin B1 was down-regulated accordingly.Clone formation experiments and tumor sphere formation experiments have demonstrated in vitro that RhoJ silence significantly inhibits the malignant proliferation of GBM cells.Subcutaneous tumor-bearing experiments and intracranial tumor-forming experiments have demonstrated that RhoJ knockdown inhibits the growth and intracranial metastasis of GBM cells in vivo,while overexpression of RhoJ promotes the malignant progression of GBM cells.Cell migration and invasion assay results showed that stable knockdown of RhoJ significantly inhibited invasion,migration and tumorigenicity of GBM cells.Both in situ injection of melanoma metastasis model and caudal vein metastasis model demonstrated that RhoJ promoted tumor metastasis phenotype.3 Study on the molecular mechanism of RhoJ promoting the malignant proliferation,invasion and metastasis of GBM cellsRhoJ knockdown affected cell morphology and EMT-like phenotype.EMT-related protein E-cadherin was significantly up-regulated,and mesenchymal cell protein Vimentin was significantly down-regulated.JASPAR and ALGGEN-PROMO database analysis found that there were three c-Jun binding sites in the upstream of RhoJ,and dual-luciferin reporting system verified that RhoJ expression in GBM cells could be regulated by the transcription factor c-Jun.The interaction protein moesin of RhoJ was found by mass spectrometry.RhoJ interacts with moesin,a protein of ERM family,and drives its downstream Racl-Pak-cofilin exchange,and promote the malignant proliferation,invasion and metastasis of GBM cells.4 Study on the role of RhoJ in GBM angiogenesis and related molecular mechanismsOverexpression of RhoJ significantly promoted tumor metastasis and GBM angiogenesis.Immunohistochemical results showed that CD31,moesin and EpCAM were up-regulated in GBM tissues with overexpressed RhoJ.After VEGF stimulation of HUVEC cells,the expressions of moesin and RhoJ were significantly up-regulated,and JNK,the upstream regulator of RhoJ,was also activated.After JNK silencing,the activities of VEGFR2 and its downstream effector molecules RhoJ-PAK2/PAK4 and BRAF/ERK were significantly down-regulated,while the activities of these effector proteins were significantly increased by VEGF stimulation.These results indicate that JNK-VEGFR2-RhoJ-PAK2/PAK4-BRAF/ERK signaling pathway plays an important regulatory role in the process of GBM angiogenesis.JNK-VEGFR2-RhoJ-PAK2/PAK4-BRAF/ERK signaling pathway plays an important role in the regulation of GBM angiogenesis.[Conclusions]1 RhoJ is generally highly expressed in GBM tumor tissues and cell lines,and the high expression of RhoJ is closely related to poor survival prognosis in GBM patients,and RhoJ is an important prognostic factor for GBM.2 It was verified in vitro and in vivo that RhoJ silencing could significantly inhibit the proliferation,migration,invasion and angiogenesis of GBM cells,and block the cells in the G2 phase.3 Silencing RhoJ expression can make the cells grow larger and inhibit the EMT process.RhoJ expression is regulated by transcription factor c-Jun.RhoJ interacts with ERM family protein moesin and drives its downstream Racl-Pak-cofilin signaling pathway activation,thereby promoting the malignant proliferation and metastasis of GBM cells.4 RhoJ promotes GBM angiogenesis.JNK/VEGFR2,as the upstream regulatory molecule of RhoJ,induces RhoJ expression and activates the downstream effector molecule PAK-BRAF-ERK pathway,which plays an important regulatory role in the process of GBM angiogenesis.Identifying the information or evidence of RhoJ regulating differential target molecular-mediated malignant phenotypes can provide novel molecular markers and intervention targets for molecular diagnosis and targeted therapy of glioma.RhoJ may also be a promising biomarker for GBM diagnosis and prognosis.