SUMOylation Of IGF2BP2 Promotes Vasculogenic Mimicry Of Glioma Via Regulating OIP5-AS1/miR-495-3p And LINC00467/miR-4735-3p Axis

Objective:Glioma is the most common primary malignant tumor in human central nervous system,account for about 80%of primary brain tumors,with high mortality.Clinical studies have found that glioma is a kind of vascular-rich tumors,and the abundant blood supplies are conducive to the malignant growth of the tumor.Therefore,anti-angiogenic therapy has become a hotspot in the treatment of glioma.However,the presence of vasculogenic mimicry(VM)in glioma may be one of the main reasons for the poor effect of anti-angiogenic therapy in tumor.SUMOylation is a dynamic and reversible post-translational modification of proteins,which can affect cell function by regulating the stability of substrate proteins and subcellular localization.This study aims to explore the regulatory effects of SUMOylation,IGF2BP2,OIP5-AS1/LINC00467,miR-495-3p/miR-4735-3p and HIF1A/MMP14/CCND2 on the biological behavior of glioma cells and their related molecular mechanisms.Methods:1.Ni²⁺-NTA agarose bead pull-down assay and CO-IP assay verified the binding of endogenous or exogenous SUMO1 to IGF2BP2 and the effects of Ubc9 and SENP1 on the SUMOylation process,and clarified the SUMOylation changes of IGF2BP2 after the mutation of SUMOylation;2.q RT-PCR and Western blot were used to verify the effect of SUMOylation on the expression level of IGF2BP2 protein before and after translation;3.The degradation pathway of IGF2BP2 and the effect of SUMO modification on the degradation process were verified by Western blot.4.Western blot after nucleoprotein separation to verify the effect of SUMOylation modification on subcellular localization of IGF2BP2;5.q RT-PCR and Western blot were used to detect the expression levels of IGF2BP2,OIP5-AS1/LINC00467 and miR-495-3p/miR-4735-3p in glioma tissues and cell lines;CCK-8 assay,Transwell assay and in vitro tube formation assay were used to verify the role of the above factors in regulating the biological behavior of glioma cells.6.RNA binding protein immunoprecipitation experiment and double luciferase reporter gene analysis were used to verify the targeted binding between RBPs(IGF2BP2),lnc RNAs(OIP5-AS1/LINC00467),miRNAs(miR-495-3p/miR-4735-3p)anddownstreamtargetproteins(HIF1A/MMP14/CCND2),respectively;7.The in vivo experiment of xenografts tumor in nude mice verified the effect of SUMOylation of IGF2BP2 on the growth of glioma cells and the survival time of tumor-bearing nude mice.Results:1.IGF2BP2 binds to SUMO1 at lysine residues K497R,K505R and K509R,and SUMOylation occurs.Ubc9 enhances this modification,while SENP1 removes this modification.The SUMO modification inhibition effect of IGF2BP2 induced by three-site mutation(3KR,K497/K505/K509R)was the strongest,which significantly reduced the protein expression level of IGF2BP2.2.Three-site mutation had no effect on the m RNA expression level of IGF2BP2;3.SUMOylation blocked the degradation process of IGF2BP2 through ubiquitin-proteasome pathway and enhanced its stability;4.SUMOylation site mutation of IGF2BP2 did not affect the subcellular localization of IGF2BP2 in glioma cells,but significantly inhibited the proliferation,migration and invasion of glioma cells and VM formation;5.IGF2BP2,OIP5-AS1 and LINC00467were high-expressed in glioma tissues and cells.knockdown IGF2BP2,OIP5-AS1 and LINC00467 significantly inhibited the proliferation,migration and invasion,VM formation and related protein expression of glioma cells.6.IGF2BP2 targetedly binds to OIP5-AS1/LINC00467,and knockdown IGF2BP2 shorten the half-life of both.Knockdown IGF2BP2 and knockdown OIP5-AS1 or LINC00467 significantly inhibited glioma cell proliferation,migration and invasion,VM formation and related protein expression;Overexpression of OIP5-AS1/LINC00467 reversed the inhibitory effect of IGF2BP2 knockdown on malignant biological behavior and VM formation ability of glioma cells.7.miR-495-3p and miR-4735-3p were lowly expressed in glioma tissues and cells.Overexpression of miR-495-3p/miR-4735-3p significantly inhibited the proliferation,migration and invasion,VM formation and related protein expression of glioma cells,while knockdown miR-495-3p/miR-4735-3p significantly enhanced the proliferation,migration and invasion,VM formation and related protein expression of glioma cells.8.OIP5-AS1 targeted binding and inhibited the expression of miR-495-3p.Knockdown OIP5-AS1 and overexpression of miR-495-3p significantly inhibited the proliferation,migration,invasion,VM formation and expression of related proteins of glioma cells.LINC00467 targeted binding and inhibited the expression of miR-4735-3p.Knockdown LINC00467 and overexpression of miR-4735-3p significantly inhibited the proliferation,migration and invasion,VM formation and related protein expression of glioma cells.9.miR-495-3p targeted the 3'UTR region of HIF1A and MMP14,and overexpression of HIF1A and MMP14 reversed the inhibitory effect of miR-495-3p overexpression on malignant biological behavior and tube formation ability of glioma cells;miR-4735-3p targeted the 3'UTR region of HIF1A and CCND2,and overexpression of HIF1A and CCND2 reversed the inhibitory effect of miR-4735-3p overexpression on malignant biological behavior and tube formation ability of glioma cells.10.Three-sites mutation of IGF2BP2 SUMOylation significantly inhibited the growth of xenografts in nude mice and prolonged the survival time of tumor-bearing nude mice.Conclusions:1.IGF2BP2 binds to SUMO1 at sites K497R,K505R and K509R,and SUMOylation occurs.Ubc9 strengthens this modification,and SENP1 removes this modification.The SUMO modification inhibition effect caused by the mutation of three sites(3KR,K497/505/509R)was the strongest;SUMOylation increased the protein expression level of IGF2BP2 and blocked its degradation through ubiquitin-proteasome pathway,but did not change its subcellular localization.2.In glioma tissues and cells,IGF2BP2,OIP5-AS1 and LINC00467 were highly expressed,while miR-495-3p and miR-4735-3p were lowly expressed.Knockdown IGF2BP2,OIP5-AS1/LINC00467 and overexpression of miR-495-3p/miR-4735-3p significantly inhibited the proliferation,migration,invasion and VM formation of glioma cells.3.IGF2BP2 binds to OIP5-AS1/LINC00467 and enhances its stability;knockdown IGF2BP2 inhibits the expression of OIP5-AS1/LINC00467 and its targeted binding with miR-495-3p/miR-4735-3p,enhances the binding of miR-495-3p/miR-4735-3p to the 3'UTR region of downstream target gene(HIF1A/MMP14/CCND2)m RNA,reduces the protein expression level of HIF1A/MMP14/CCND2,and further inhibits the proliferation,migration,invasion and VM formation of glioma cells.4.SUMOylation of IGF2BP2 plays an important role in promoting vasculogenic mimicry of glioma cells by regulating OIP5-AS1/miR-495-3p and LINC00467/miR-4735-3p axis.