Mechanism Of SNORD17-Mediated KAT6B MRNA 2’-O-Methylation In Regulating Vasculogenic Mimicry In Glioblastoma Cells

Objective: Malignant glioblastoma(GBM)is a highly aggressive tumor of the central nervous system with an extremely poor prognosis for patients.Despite some advances in radiotherapy,chemotherapy,and surgery,the median survival time of patients with GBM remains less than 15 months.Vascular growth is the basis of the malignant biology of GBM.Vasculogenic mimicry(VM)is one of the major forms of vascular enrichment in malignant tumors,and VM is a pipeline of tumor cells whose presence limits the therapeutic efficacy of anti-angiogenic drugs.Studies have demonstrated that in GBM,VM is closely associated with poor prognosis of patients.Studies have shown that sno RNAs are dysregulated in tumors and can act as oncogenic or tumor suppressors,which are closely associated with clinical prognosis of tumor patients.SNORD17 is localized on human chromosome 20q11.23 and belongs to the C/D box sno RNA.Its member function is to mediate 2’-O-methylation modifications of target RNAs.2 ’-O-methylation is a methylation modification that occurs at the 2’ hydroxyl position of ribose and is abundant and highly conserved.FBL,also known as fibrillarin,can be directed to the target by SNORD17 and mediates the 2’-O-methylation modification of KAT6 B mRNA.KAT6B is a histone acetylase that can also mediate the acetylation of non-histone proteins.Protein acetylation refers to the process of adding acetyl groups to protein lysine residues in the presence of acetyltransferases.Studies have shown that acetylation can inhibit the degradation of proteins by inhibiting their ubiquitination and thus their ubiquitinproteasome pathway.It has also been shown that acetylation can promote the interaction of target proteins with ubiquitin ligases,thereby facilitating their degradation.ZNF384 is a C2H2-type zinc finger protein that transcriptionally regulates extracellular matrix genes.Studies have shown that ZNF384 may be a potential oncogene that is highly expressed in a variety of tumors.Studies have reported that ZNF384 promotes the proliferation of hepatocellular carcinoma cells and osteosarcoma cells.In this study,we take SNORD17 regulation of VM formation in GBM as an entry point to clarify the mechanism in which SNORD17 mediates 2’-O-methylation modification of KAT6 B m RNA,down-regulates KAT6 B expression and regulates VM formation in GBM.KAT6B-mediated acetylation modification of ZNF384 affects the expression of ZNF384 and thus regulates the mechanism in VM formation of GBM.This study aims to provide new targets for the progression and treatment of GBM by investigating the novel mechanism of VM in GBM.Methods: 1-2.Quantitative real-time reverse transcription-PCR(qRT-PCR)and western blot assay were performed to detect the expression levels of SNORD17,KAT6 B,ZNF384 in human brain astrocytes(HA),human glioblastoma cells-U251 and U373 cells.3.GBM cells with stable knockdown/overexpression of SNORD17,stable knockdown/overexpression of KAT6 B and stable knockdown/overexpression of ZNF384 were constructed by cell transfection.4.tumor cell proliferation ability was detected by CCK8 assay.5.tumor cell migration ability was detected by Hstudio M4 system.6.tumor cell invasive ability was detected by Transwell assay.7.tumor cell tube formation ability was detected by in vitro tube formation assay.8.Western blot assay was applied to detect VEGFR2 and VE-cadherin protein expression.9.The RTL-P assay was used to detect the 2’-O-methylation level of KAT6 B.10.Laser confocal microscopy was performed to observe the co-localization of KAT6 B with ZNF384 in tumor cells.11.The acetylation level of ZNF384 was detected by immunoprecipitation assay.12.The half-life of ZNF384 protein was detected by applying cycloheximide.13.Chromatin immunoprecipitation assay was verified the binding of ZNF384 to VEGFR2 and VE-cadherin promoter regions.14.The interaction and the binding sites between ZNF384 and VEGFR2,ZNF384 and VEcadherin were clarified by Dual-Luciferase reporter assay.15.The subcutaneous transplantation tumor assay in GBM nude mice was established to detect the tumorigenic ability of cells,the orthotopic transplantation tumor assay was established to detect the survival time in nude mice.16.The amount of VM in transplantation tumor tissues was measured by CD31-PAS staining.Results: 1.In this study,the expression of SNORD17 and ZNF384 were significantly higher in GBM cells than in HA cells,but the expression of KAT6 B was significantly lower than in HA cells(P<0.05).2.Compared with the NC group,knockdown of SNORD17 and ZNF384 and overexpression of KAT6 B significantly inhibited VM in GBM cells(P<0.01);3.Combined application knockdown of SNORD17,ZNF384 and overexpression of KAT6 B significantly inhibited the growth of transplanted tumors in nude mice,prolonged the survival time of nude mice,and reduced the number of VMs in transplanted tumor tissues(P<0.01).4.SNORD17 mediated 2’-O-methylation modification of KAT6 B and down-regulated KAT6 B protein expression,and knockdown of SNORD17 significantly increased KAT6 B protein expression;KAT6B acetylation modified ZNF384,reduced its protein stability and down-regulated its expression.ZNF384 directly bound to the promoter region of VEGFR2 and VE-cadherin,and the down-regulated ZNF384 attenuated the transcriptional promotion of VEGFR2 and VE-cadherin,decreased the expression of VEGFR2 and VE-cadherin,and thus inhibited the VM in GBM.Conclusion:1.High expression of SNORD17 in glioma tissues and cells mediated 2’-O-methylation modification of KAT6 B and down-regulated KAT6 B protein expression,and promoted VM in GBM.2.Low expression of KAT6B in glioma cells attenuated the acetylation modification of ZNF384,increased its protein stability,and upregulated the expression of ZNF384,and promoted VM in GBM.3.High expression of ZNF384 in glioma tissues and cells promoted the transcription of VEGFR2 and VE-cadherin,and promoted VM in GBM.4.The SNORD17/KAT6B/ZNF384 signaling pathway played an important regulatory role in regulating VM in GBM.