Mechanisms Of Linc00707/miR-651-3p/SP2 And MiR-876-5p/BPTF Axis Regulating The Vasculogenic Mimicry Of Glioma Cells

Objective: Glioma is one of the most common primary malignant tumors of the central nervous system.The prognosis of patients is poor and the median survival time is less than 15 months.Glioma has a high degree of vascular invasiveness.Recent studies have found that the existence of vasculogenic mimicry(VM)limited the effect of anti-angiogenic therapy in glioma significantly.This study first clarified the endogenous expression of HNRNPD,ZHX2,linc00707,miR-651-3p,SP2,miR-876-5p,and BPTF in glioma tissues and cells,and further explored the possible regulatory mechanisms among the above factors and their effects on VM formation ability of glioma cells.We aimed to provide new ideas and new targets for the treatment of glioma from the perspective of anti-VM formation.Methods: Human glioma cells U87 and U251,human brain astrocytes and human embryonic kidney cells HEK293 T were cultured.qRT-PCR and Western blot were used to detect the expression of linc00707,miR-651-3p and miR-876-5p,and HNRNPD,ZHX2,SP2,BPTF in human normal brain tissues,glioma tissues,human brain astrocytes and glioma cells.Stably transfection of HNRNPD and linc00707 silencing plasmids,ZHX2 overexpression plasmids,SP2 and BPTF overexpression and silencing plasmids were used in glioma cells.Transiently transfection of miR-876-5p overexpression plasmids,miR-651-3p overexpression and silencing plasmids together with the corresponding negative control empty plasmids were also used.CCK-8,Transwell and in vitro three-dimensional tube formation experiments were applied to detect the proliferation,migration,invasion and VM formation ability of glioma cells.qRT-PCR and Western blot were used to detect the mRNA and protein expression level of MMP2,MMP9 and VE-cadherin.RIP assay was applied to verify the binding effect between linc00707 and miR-651-3p.The dual luciferase reporter gene analysis system was used to demonstrate the binding effects and binding sites between HNRNPD and ZHX2 mRNA,linc00707 and miR-651-3p,miR-651-3p and SP2 3’UTR,miR-876-5p and BPTF 3’UTR.Ch IP experiments were applied to verify the binding effects between ZHX2 and the promoter region of linc00707,along with SP2 and the promoter regions of MMP2,MMP9 and VE-cadherin.The effects of HNRNPD,ZHX2 and linc00707 in vivo were tested by experiments of subcutaneous transplanted tumors in nude mice and immunohistochemistry.Results: This study demonstrated that HNRNPD and linc00707 are highly expressed in glioma tissues and cells,while ZHX2 and miR-651-3p are lowly expressed.Silencing of HNRNPD,linc00707 and overexpression of ZHX2 and miR-651-3p could all significantly inhibit the VM formation ability of glioma cells.Knockdown of HNRNPD increased the stability of ZHX2 mRNA.ZHX2 bound to the promoter region of linc00707 and negatively regulate its expression.Linc00707 targetedly bound with miR-651-3p while miR-651-3p bound to the 3’UTR of SP2 mRNA and negatively regulate its expression.The transcription factor SP2 directly bound to the promoter regions of the VM formation related proteins MMP2,MMP9,and VE-cadherin and played a transcriptional promotion role.The combined application of HNRNPD,linc00707 knockdown and ZHX2 overexpression could significantly inhibited the growth of the transplanted tumors and significantly prolonged the survival period of nude mice.Our study also found that miR-876-5p is down-regulated in glioma cells.It targetedly bound with and negatively regulate BPTF and further inhibited the VM-forming ability of glioma cells.Conclusions:1.HNRNPD and linc00707 are highly expressed in glioma tissues and cells,while ZHX2 and miR-651-3p are lowly expressed.2.HNRNPD could reduce the stability of ZHX2 mRNA,further regulated the VM formation ability of glioma cells.3.ZHX2 inhibited the transcription of linc00707 through binding with its promoter region,and further inhibited the VM formation ability of glioma cells.4.Linc00707 acted as a molecular sponge by targeting miR-651-3p and inhibited the negative regulation of SP2 by miR-651-3p.5.The transcription factor SP2 played a transcriptional promotion role by directly binding to the promoter regions of the VM formation related proteins MMP2,MMP9 and VE-cadherin.6.miR-876-5p is lowly expressed in glioma cells,while BPTF is highly expressed in glioma cells.miR-876-5p targeted and negatively regulate the expression of BPTF,thereby inhibiting the VM formation ability of glioma cells.