Mechanism Of XIST,G3BP2 And G3BP2 Regulating Glioblastoma Angiogenesis

Objective: Glioblastoma(GBM)is the most malignant primary intracranial tumor.Surgical resection combined with postoperative radiochemotherapy is a common treatment strategy for the tumors,but the median survival time for patients with GBM remains disheartening.Angiogenesis promotes GBM progression,which makes antiangiogenic therapy promising for GBM treatment.The blood–tumor barrier(BTB)restricts the delivery of most antitumor drugs to the brain tumor,which ultimately mitigates the effects of chemotherapy.Therefore,increasing BTB permeability in parallel with inhibiting glioma angiogenesis may be a more efficient treatment strategy for glioma.The proliferation,migration and tube formation of glioblastoma endothelial cells play vital roles in endothelial-depedent angiogenesis.Meanwhile,glioblastoma endothelial cells are the important structure foundation.Therefore targeting the behaviors of glioblastoma endothelial cells may contribute to inhibiting glioblastoma angiogenesis and increasing the permeability of BTB.Knockdown of long non-coding RNA inhibits the proliferation,migration and invasion,as well as promotes apotosis in glioblastoma stem cell.However it remains unclear whether XIST regulates the proliferation,migration,tube formation and BTB permeability.It has been reported that RNA binding protein G3BP2 mediates the behabiors of hepatocellular carcinoma cells and breast cancer cells.Similarly,it remains unknow whether G3BP2 regulates the behaviors of gliobalstoma endothelial cells.Numerous pro-angiogenic factors are expressed and secreted by glioblastoma cells,which created a powerful pro-angiogenic tumor microenvironment.Accordingly,targeting glioblastoma cells and reducing the secretion of pro-angiogenic factors may inhibit the glioblastoma angiogenesis.Recent findings have indicated that the expression levels of NFAT5 were aberrant in tumors.Moreover,growing publications demonstrated its potential roles in tumor angiogenesis and pro-angiogenic factors regulation.But the expression levels of NFAT5 in gliomas and its potential role in GBM angiogenesis still remain unclear.The present study aims to elucidate the expression levels of XIST and G3BP2,as well as explore the regulation of glioblastoma endothelial cell behaviors.Meanwhile the expression levels of NFAT5 in glioma and the role of NFAT5 in glioblastoma cell-driven angiogenesis are elucidated.Moreover,the present student may provide more novel strategies to combined GBM treatment.Methods: 1.The establishment of BTB model in vitro;2.The expression levels of XIST,mi R-137,FOXC1,G3BP2,HEIH,IRF6,CYR61 NFAT5,SBF2-AS1,EGFL7 were determined by Real-time PCR;3.The expression levels of FOXC1,ZO-1,ZO-2,occludin,CXCR7,NFAT5,EGFL7,ERK,p-REK,G3BP2,IRF6 and CYR61 were determined by Western blot;4.Glioblastoma endothelial cells were stably transfected with XIST knockdown,FOXC1 overexpression,FOXC1 knockdown,G3BP2 knockdown,HEIH overexpression,HEIH knockdown,IRF6 overexpression,IRF6 knockdown,CRY61 knockdown plasmids,and were transiently transfected with agomir-137 and antagomir-137.U87 and U118 cells were stably transfected with NFAT5 knockdown,SBF2-AS1 knockdown and EGFL7 overexpression(with or without 3' untranslated region)plasmids,and were transiently transfected with agomir-338-3p and antagomir-338-3p.5.The BTB integrity was determined by Transendothelial electric resistance(TEER)assay.6.The BTB permeability was determined by horseradish peroxidase(HRP)flux assay.7.The cell viability of GECs was determined by CCK-8 assay.8.The migration of GECs was determined by Transwell assay.9.The tube formation of GECs was determined by Matrigel tube formation assay.10.The binding sites of XIST and mi R-137,mi R-137 and FOXC1,ZO-2,SBF2-AS1 and mi R-338-3p,mi R-338-3p and EGFL7 were determined by dual-luciferase reporter assay.11.The binding of XIST,mi R-137,SBF2-AS1 and mi R-338-3p with Ago2,the binding of HEIH with G3BP2,the binding of HEIH and IRF6 m RNA with STAU1 were determined by RNA binding protein immunoprecipitation(RIP)assay.12.The association of FOXC1 and the promoter regions of ZO-1,occludin and CXCR7,the association of NFAT5 and the promoter region of SBF2-AS1 and the association of IRF6 and the promoter region of CYR61 were determined by Chromatin immunoprecipitation(Ch IP).13.Nude mouse xenograft model and microvessel density assay.14.In vivo Matrigel plug angiogenesis assay.Results: 1.XIST was upregulated in GECs.XIST inhibition significantly decreased the TEER values and increased the HRP flux,as well as inhibited the cell viability,migration and tube formation of GECs.Meanwhile knockdown of XIST significantly inhibited ZO-1,ZO-2,occludin and CXCR7 expression.Furthermore,the distribution of ZO-1,ZO-2 and occludin became discontinues on cell boundaries following XIST knockdown.2.Knockdown of XIST significantly upregulated mi R-137 expression in GECs.Meanwhile XIST targeted mi R-137,and were enriched in Ago2 immunoprecipitates with mi R-137.3.Overexpression of mi R-137 in GECs that stably knocked down XIST significantly promoted the decreases in TEER value,the increases in HRP flux and the inhibition in GECs cell viability,migration and tube formation induced by XIST knockdown alone,as well as promoted the inhibition in ZO-1,ZO-2,occludin and CXCR7 expression.On the contrary,downregulation of mi R-137 in GECs that stably knocked down XIST significantly revised the above effects.4.mi R-137 was downregulated in GECs.mi R-137 overexpression significantly decreased the TEER values and increased the HRP flux,as well as inhibited the cell viability,migration and tube formation of GECs.Meanwhile knockdown of XIST significantly inhibited ZO-1,ZO-2,occludin and CXCR7 expression.Furthermore,the distribution of ZO-1,ZO-2 and occludin became discontinues on cell boundaries following mi R-137 overexpression.5.mi R-137 targeted ZO-2 and FOXC1.Meanwhile,mi R-137 overexpression significantly inhibited FOXC1 expression in GECs.6.FOXC1 was upregulated in GECs.XIST inhibition significantly decreased the TEER values and increased the HRP flux,as well as inhibited the cell viability,migration and tube formation of GECs.Meanwhile knockdown of XIST significantly inhibited ZO-1,ZO-2,occludin and CXCR7 expression.Furthermore,the distribution of ZO-1,ZO-2 and occludin became discontinues on cell boundaries following FOXC1 knockdown.7.FOXC1 bound to the promoter regions of ZO-1,occludin and CXCR7 in a sequence-specific manner.8.Co-overexpression of FOXC1 and mi R-137 in GECs,significantly revised the the decreases in TEER value,the increases in HRP flux and the inhibition in GECs cell viability,migration and tube formation induced by mi R-137 overexpression alone,as well as promoted the inhibition in ZO-1,ZO-2,occludin and CXCR7 expression.9.G3BP2 was upregulated in GECs and G3BP2 inhibition reduced the cell viability, migration and tube formation in GECs.10.Knockdown of G3BP2 destabilize HEIH and downregulated HEIH in GECs.11.HEIH was elevated in GECs and HEIH overexpression promoted the cell viability,migration and tube formation in GECs.12.HEIH was involved in G3BP2-mediated GECs behaviors.13.HEIH inhibited IRF6 expression in a SMD manner.14.IRF6 was downregulated in GECs and IRF6 overexpression reduced the cell viability,migration and tube formation in GECs.15.HEIH promoted GECs angiogenesis by targeting IRF6 m RNA 3'UTR.16.IRF6 depressed CYR61 expression and bound to the promoted region of CYR61.17.CYR61 was upregulated in GECs and CYR61 inhibition reduced the cell viability,migration and tube formation in GECs.18.The combination of G3BP2 and HEIH knockdown inhibited the GECs angiogenesis in vivo.19.NFAT5 was upregulated in glioma samples and glioma cell lines,and was positively correlated with WHO grade.20.Knockdown of NFAT5 in U87 and U118 GBM cells significantly inhibited GBM cell-driven E cell viability,migration and tube formation of ECs.21.SBF2-AS1 was upregulated in glioma samples and glioma cell lines,and was positively correlated with WHO grade.Knockdown of NFAT5 in U87 and U118 GBM cells significantly inhibited GBM cell-driven cell viability,migration and tube formation of ECs.22.NFAT5 expression was positively correlated with SBF2-AS1 expression in gliomas.NFAT5 promoted SBF2-AS1 expression at transcriptional level in U87 and U118 cells.23.Co-downregulation of NFAT5 and SBF2-AS1 in U87 and U118 cells,significantly promoted the inhibition in GBM cell-driven ECs cell viability,migration and tube formation induced by NFAT5 knockdown alone.24.SBF2-AS1 bound to mi R-338-3p in a sequence-specific manner,and were enriched in Ago2 immunoprecipitates with mi R-338-3p.Meanwhile mi R-338-3p overexpression significantly revised the inhibition in GBM cell-driven ECs cell viability,migration and tube formation induced by SBF2-AS1 knockdown alone.25.mi R-338-3p inhibited EGFL7 expression and secretion by targeting EGFL7 3'-UTR.26.mi R-338-3p inhibited GBM cell-driven ECs cell viability,migration and tube formation induced by EGFL7 overexpression by targeting EGFL7 3'-UTR.27.Knockdown of NFAT5 and SBF2-AS1 significantly inhibited EGFL7 expression and secretion,respectively.28.Knockdown of NFAT5 and SBF2-AS1 significantly inhibited glioma xenograft growth and microvessel density,respectively. Furthermore,the combination of NFAT5 and SBF2-AS1 knockdown produced the strongest inhibitory effects.Conclusion: 1.XIST regulates BTB permeability and GBM angiogenesis by targeting mi R-137 in a sequence-specific manner,which inpaired FOXC1 and ZO-1 at post-transcriptional level.2.FOXC1 regulates BTB permeability by promoting ZO-1 and occludin expression at transcriptional level,and regulates GBM angiogenesis by promoting CXCR7 expression at transcriptional level.3.The XIST/mi R-137/FOXC1 pathway plays prominent roles in BTB permeability and GBM angiogenesis.5.G3BP2 promoted GECs angiogenesis by stabilizing HEIH.4.HEIH promoted GECs angiogenesis by downregulating IRF6 in an SMD manner,which impaired CYR61 at transcriptional level.5.IRF6 inhibited GECs angiogenesis by transcriptional depression of CYR61.6.G3BP2/HEHI/IRF6/CYR61 pathway played a vital role in GECs angiogenesis.7.NFAT5 knockdown inhibited GBM cell-driven angiogenesis by reducing SBF2-AS1 expression.8.SBF2-AS1 regulates GBM cell-driven angiogenesis by targeting mi R-338-3p in a sequence-specific manner,which impaied EGFL7 at post-transcriptional level.9.The NFAT5/SBF2-AS1/mi R-338-3p/EGFL7 pathway plays vital roles in GBM cell-driven angiogenesis.