ZRANB2/SNHG20/FOXK1 Axis Regulates Vasculogenic Mimicry Formation In Glioma

Objective: Glioma is globally recognized as the most common primary intracranial neoplasm.Despite the existence of various treatment methods including surgery,radiation and chemotherapy,the median survival time of patients suffering glioma is no more than 15 months.Although glioma tissue is characterized by angiogenesis and vasculogenesis,tumor treatment effects of anti-angiogenic drugs including bevacizumab are far from satisfaction.Vasculogenic mimicry(VM)formation was first discovered in 1999 and regarded as a new form of blood supply independent of blood vessels.The study of VM formation may bring light to the treatment of glioma.This study first examined the endogenous expression of ZRANB2,SNHG20 and FOXK1 in glioma tissues and U87 and U251 glioma cells,and studied the effects of ZRANB2,SNHG20 and FOXK1 on VM formation in glioma.Then we explored the probable molecular mechanism of ZRANB2,SNHG20 and FOXK1 in regulating VM formation.This study aims to provide new theoretical basis for the study of the mechanism of glioma development and provide new targets for the treatment of glioma.Methods: The human glioma cells lines(U87 and U251)and human normal astrocytes were purchased and cultured.qRT-PCR was used to detect ZRANB2 mRNA,SNHG20 and FOXK1 mRNA.Western blot was used to detect ZRANB2,FOXK1,MMP1,MMP9 and VE-Cadherin expression.U87 and U251 cells transfected with ZRANB2,SNHG20,FOXK1 knockdown and overexpression,STAU1 and UPF1 knockdown were constructed.Cell proliferation was detected by CCK-8 assay.Transwell was used to detect cell migration and invasion.In vitro VM tube formation assay was used to detect vasculogenic mimicry formation ability.Chromatin immunoprecipitation assay was used to confirm the binding between FOXK1 and the promoters of MMP1,MMP9 and VE-Cadherin.Luciferase reporter assay was used to confirm the binding between SNHG20 and FOXK1 mRNA.RNA immunoprecipitation was used to confirm the binding between ZRANB2 and SNHG20 as well as the binding between SNHG20 and FOXK1 mRNA.Nascent RNA capture and mRNA stability assay were used to detect the stability of RNA.CD34-periodic acid-schiff dual-staining was used to detect the vasculogenic mimicry formation ability in tissues.The effects of ZRANB2 and SNHG20 on the growth and vasculogenic mimicry in vivo was detected by xenograft mouse model.Results: 1.ZRANB2 is highly expressed in glioma tissues and cells.ZRANB2 knockdown inhibits the expression of MMP1,MMP9 and VE-Cadherin,and inhibits the proliferation,migration,invasion and vasculogenic mimicry of glioma cells.2.SNHG20 is highly expressed in glioma tissues and cells.Knockdown of ZRANB2 reduces the expression of SNHG20.Silencing of SNHG20 significantly inhibited the expression of MMP1,MMP9 and VE-Cadherin,and inhibits the proliferation,migration,invasion and vasculogenic mimicry of glioma cells.3.RNA-IP verified the binding of ZRANB2 with SNHG20.Overexpression of ZRANB2 prolongs the half-life of SNHG20.Knockdown of ZRANB2 and SNHG20 significantly inhibits the expression of MMP1,MMP9 and VE-Cadherin,and inhibits the proliferation,migration,invasion and vasculogenic mimicry of glioma cells.4.FOXK1 is down-regulated in glioma tissues and cells,and knockdown of ZRANB2 or SNHG20 significantly increases FOXK1 expression.Overexpression of FOXK1 significantly reduces the expression of MMP1,MMP9 and VE-Cadherin,and inhibits the proliferation,migration,invasion and vasculogenic mimicry of glioma cells.5.Dual luciferase assay demonstrates the binding of SNHG20 with FOXK1 mRNA via Alu sequence.RNA-IP experiments confirmed the binding of ZRANB2 with SNHG20,SNHG20 with STAU1 and FOXK1 mRNA with STAU1.Knockdown of SNHG20 significantly prolongs the half-life of FOXK1 mRNA.Silencing of SNHG20 and overexpression of FOXK1 significantly inhibits the expression of MMP1,MMP9 and VE-Cadherin,and inhibits the proliferation,migration,invasion and vasculogenic mimicry of glioma cells.6.FOXK1 binds to the promoter regions of MMP1,MMP9 and VE-Cadherin.Knockdown of ZRANB2 and SNHG20 significantly inhibits the growth of xenografts in nude mice,prolonged their survival,and reduced the VM formation in gliomas.Conclusions: 1.ZRANB2 and SNHG20 are highly expressed in glioma tissues and U87 and U251 cells,and silencing ZRANB2 or SNHG20 can inhibit the vasculogenic mimicry formation in glioma cells.FOXK1 is down-regulated in glioma tissues and U87 and U251 cells,and overexpression of FOXK1 can inhibit the formation of vasculogenic mimicry formation in glioma cells.2.ZRANB2 binds SNHG20 and increases its stability.3.SNHG20 acts on FOXK1 via the SMD pathway.SNHG20 increases the mRNA degradation of FOXK1.4.FOXK1 inhibits the transcription by binding to the promoters of MMP1,MMP9 and VE-Cadherin,thereby inhibiting vasculogenic mimicry formation in glioma cells.5.Knockdown of ZRANB2 reduces the stability and expression of SNHG20,reduces the degradation of FOXK1 mRNA,increases the expression of FOXK1 and reduces the expression of MMP1,MMP9 and VE-Cadherin,thus reducing the vasculogenic mimicry in glioma.