LINC01088 Inhibits Ferroptosis Through Upreglating SLC7A11 By Stablizing USP7/HLTF Axis In Glioblastoma Multiforme

Background: Glioblastoma multiforme(GBM)is the leading malignant tumour of central nervous system.Despite multidisciplinary treatment,which included surgical excision,radiotherapy and chemotherapy,the overall survival time was only about 15 months.Recently,Ferroptosis was recently discovered.Unlike apoptosis,necroptosis and pyroptosis,ferroptosis is a unique cell death pathway characterized by iron dependent lipid peroxidation.Recent studies have demonstrated that targeting cell death pathways is a promising therapeutic strategy for preventing GBM progression.Unfortunately,however,the results did not conclude the expected results,especially with regard to the regulation of ferroptosis in GBM.With the deepening of the understanding of lncRNAs,it is gradually demonstrated that the differential expression of lncRNAs play a key role in tumour proliferation.Currently,there has been no significant research progress in lncRNA and ferroptosis in GBM.Therefore,in order to further explore new effective treatment for GBM,we plan to combine lncRNA and ferroptosis to explore possible mechanisms in GBM and provide new possible treatment of GBM.Objective: 1)To explore the cause of high expression of LINC01088 in GBM tissues and its effect on malignant phenotype in GBM;2)To investigate the effects of LINC01088 on ferroptosis in GBM;3)To explore the specific molecular mechanism of LINC01088 regulating ferroptosis in GBM.Methods: 1)Ferroptosis related lncRNAs with differentially expressed in GBM were screened using multiple databases and literature reviews,and lncRNA LINC01088 was verified using the collected tumor tissues of GBM patients and brain tissues of non-tumor patients(traumatic brain injury).The causes of up-regulation of LINC01088 were performed by double luciferase reporting assay and Ch IP assay.2)The effects of LINC01088 on the proliferation of GBM were performed by CCK-8,Ed U,clone formation assay and cell cycle assay.The effect of LINC01088 on invasion and migration of GBM was performed by Transwell assay.3)The effect of LINC01088 on ferroptosis was detected by total iron content,ferrous content,MDA level,ROS level,transmission electron microscopy and GSSG/GSH.4)The subcellular localization of LINC01088 was performed by nucleoplasmal separation and FISH assay,and RNA binding protein HLTF was obtained by RNA pulldown,MS and RIP assay.5)WB,CO-IP and ubiquitination experiments explored the effect of scaffold LINC01088 on regulating USP7 as a deubiquitination enzyme of HLTF and stabilizing HLTF protein by reducing its ubiquitination proteasome degradation;6)Transcriptome sequencing was used to explore the differentially downregulated gene SLC7A11 after knockdowning HLTF,and dual luciferase reporting assay and Ch IP assay were used to explore specific binding sites.7)The effect of LINC01088/HLTF/ SLC7A11 axis on ferroptosis of GBM was verified by the rescue experiment;8)To investigate the tumorigenic ability of LINC01088 in GBM cells in intracranial and subcutaneous GBM xenograft model.Results: 1)The expression level of LINC01088 was up-regulated in GBM clinical samples and GBM cell lines,and SP1 can promote the transcription of LINC01088;2)LINC01088 can promote the proliferation,migration and invasion of two GBM cell lines.3)LINC01088 inhibited ferroptosis in U87 and U251 cell lines;4)LINC01088 was localized in the nucleus,and the 771-952 nt fragment of LINC01088 could bind to the binding domain(837-996aa)of HLTF;5)LINC01088 can be used as a scaffold platform for HLTF/USP7 interaction to protect HLTF from ubiquitin-proteasome degradation;6)HLTF inhibited ferroptosis by specifically up-regulating the expression of SLC7A11;7)The rescue experiments demonstrated that the interaction between LINC01088 and HLTF up-regulated the transcription of SLC7A11 and inhibited ferroptosis in GBM cells.8)LINC01088 improved the tumorigenicity of GBM cells in nude mice.Conclusion: 1)SP1 induced LINC01088 can promote the proliferation,migration and invasion ability of GBM cells and inhibit ferroptosis;2)LINC01088 acts as a scaffold to up-regulate the transcription of SLC7A11 by stabilizing the USP7/HLTF axis,thereby inhibiting ferroptosis in glioblastoma;3)LINC01088 improved the tumorigenicity of GBM cells in nude mice.