Mechanism Of Long Non-coding RNA LINC00662 Regulates Glioma-induced Vasculogenic Mimicry

Object: Gliomas are primary malignant brain tumors characterized by abnormal brain microcirculation hyperplasia.Researchers have attempted to inhibit tumor progression through anti-angiogenic therapy,but vasculogenic mimicry(VM)limits the therapeutic effects of anti-angiogenic therapy.Therefore,the combination of anti-angiogenesis and inhibition of VM formation therapy will be an effective strategy for glioma treatment.Long non-coding RNAs(LncRNAs)are a group of RNAs which are non-coding and more than 200 nucleotides.Studies have shown that lncRNAs play an important role in the progression of glioma.LINC00662 is widely expressed in the brain,ovary and the remaining 25 tissues,but the specific physiological functions are currently unknown.Similarly,it is not clear whether the expression level of LINC00662 in gliomas and whether it is involved in the regulation of glioma-induced VM formation.MicroRNAs(miRNAs)are short-chain non-coding RNAs that play important roles in regulating gene expression.MiRNAs mainly affect downstream gene expression via combining the 3'-untranslated region(3'-UTR)of the downstream gene mRNA.Downregulation of miR-874-3p expression levels has been shown to be associated with a variety of tumor progression.However,the role of miR-874-3p in gliomas and whether it is involved in the regulation of glioma-induced VM has not been reported.This study aimed to clarify the molecular mechanism by which LINC00662 regulates glioma-induced VM formation,thus providing a new therapeutic target for glioma treatment.Results: 1.CD34-PAS dual-staining was used to detect the VM in glioma tissue sections;2.Detection of expression of LINC00662 or miR-874-3p in glioma tissues and cell lines by qRT-PCR;3.U87 and U251 cell lines were stably transfected with LINC00662 knockdown plasmid;4.U87 and U251 cell lines were stably transfected with SDC1 knockdown plasmid;5.U87 and U251 cell lines were transiently transfected with agomir-874-3p or antagomir-874-3p;6.CCK-8 assay was used to evaluate glioma cell proliferation ability;7.Transwell assay was used to assess glioma cell migration and invasion ability;8.The VM formation assay was used to assess VM formation ability of glioma cells;9.Western blot was used to detect the expression level of SDC1,RhoA/ ROCK pathway protein,MMP-9 and MMP-2;10.Dual-luciferase reporter assay was used to identified the binding site between LINC00662 and miR-874-3p or between miR-874-3p and SDC1;11.RNA-binding protein immunoprecipitation assay was used to determine the binding of LINC00662 and miR-874-3p with Ago2.Conclusion : 1.LINC00662 was high-expressed in glioma tissues and cells and correlated with glioma-induced VM.Knockdown of LINC00662 inhibited gliomainduced VM formation by up-regulating the expression of miR-874-3p.2.Overexpression of miR-874-3p inhibited glioma-induced VM formation by targeting and down-regulating SDC1.3.Knockdown of SDC1 inhibited glioma-induced VM formation by inhibiting the RhoA/ROCK pathway and down-regulating the expression of VM-related factors MMP-2 and MMP-9.