The Mechanism Of ATF4 Transcriptionally Regulate NLRP3 Expression To Promote Glioblastoma Migration And Invasion

Background:Glioblastoma（GBM）is the most malignant primary neuroepithelial intracranial tumor,with a poor prognosis and short median survival time.Clinically,the standard therapy for GBM is maximized surgical resection,followed by chemotherapy and radiotherapy.For GBM chemotherapy,temozolomide（TMZ）is the preferred medicine.Although medical techniques and treatment strategies for GBM are constantly evolving,its recurrence appears to be inevitable.The very invasive form of GBM is largely to blame for the dismal survival and high recurrence rates in GBM patients.The aggressive nature of GBM cells makes it challenging to completely remove them during surgery since they can diffusely infiltrate into nearby normal brain tissue.Within two years of surgery,patients frequently relapse,and recurrent tumors are more resistant to chemotherapy and radiotherapy.The highly aggressive mechanism of GBM hasn’t been fully understood,nevertheless,and there isn’t a targeted treatment being used in clinical.Therefore,identifying appropriate anti-invasion treatment targets and understanding the highly invasive molecular mechanism of GBM are essential issues in GBM therapy.The endoplasmic reticulum（ER）is a particularly sensitive organelle,and most of intracellular and extracellular stimuli,including chemotherapy,can affect the protein folding function of ER causing ER stress and activating unfolded protein response.Study have demonstrated that the ER stress marker protein BIP’s stability is crucial for controlling the epithelial-mesenchymal transition（EMT）of GBM cells and encouraging cell invasion.In addition,there is a significant correlation between the expression of PERK/e IF2α/ATF4 axis gene of the ER unfolded protein response branch and EMT-related genes in various tumor.EMT acts as a basis for epithelial tumors to develop capacity for invasion and migration.This suggests that PERK/e IF2α/ATF4 axis may be involved in the regulation of GBM invasion under ER stress.ATF4 is a key element of PERK axis and a powerful transcription factor that can transcriptionally regulate the expression of a variety of cell event-related genes.However,the regulatory network of ATF4 in GBM and its effect on GBM invasion remain unclear.ER is an important site for sphingolipid synthesis.Studies have shown that PERK/e IF2α/ATF4 axis may be involved in the regulation of sphingolipid metabolism under ER stress.One of the main varieties of sphingolipid is sphingosine-1-phosphate（S1P）which is involved in various cellular processes such as angiogenesis,cell survival,cell migration and invasion.S1P can be secreted extracellular as a paracrine factor to transmit stress signals to tumor cells in the secondary microenvironment,further enhancing the survival and invasion ability of these cells.The formation of S1P is catalyzed by sphingosine kinase 1（SPHK1）.Compared with the surrounding normal brain tissue,the expression of SPHK1 and the content of S1P in GBM is significantly upregulated,and is associated with GBM proliferation,mesenchymal phenotype and high aggressiveness.It is suggested that the association between PERK/e IF2α/ATF4 axis of ER stress and SPHK1/S1P signal may be an important clue to reveal the highly invasive molecular mechanism of GBM.Objective:To confirm the activation of ER stress PERK/e IF2α/ATF4 pathway in GBM under chemotherapy;To explore the transcriptional regulation of ATF4 on SPHK1 and its influence on GBM migration and invasion,providing theoretical basis for elucidating the molecular mechanism of high invasion and chemotherapy resistance of GBM cells and developing clinical therapeutic plan targeting GBM invasion.Methods:1.MTT assay was performed to detect the changes of GBM cells viability under various treatment situations.Western blot and flow cytometry were performed to detect Apoptotic protein expression and cell apoptosis changes to determine the time and concentration of TMZ.RNA-seq was performed to identify the significant change of gene transcription and pathways in LN229 cells with TMZ treatment for 3 days.Western blot and immunofluorescence were performed to detect the protein expression of PERK/e IF2α/ATF4 pathway and ATF4 localization to verify the activation of PERK/e IF2α/ATF4 pathway.Applying e IF2αagonists（Salubrinal）or e IF2αinhibitor（ISRIB）to interfere the e IF2αphosphorylation under TMZ treatment,western blot,clone formation and flow cytometry were performed to detect apoptotic protein expression,cell proliferation,and apoptosis changes to explore the effects of PERK/e IF2α/ATF4 pathway on chemotherapy stimulation.2.CHIP-seq was use to detect the genes of ATF4 binding to promoter region in LN229 cells with TMZ treatment,and combined with RNA-seq analysis to determine the genes transcriptionally up-regulated and the regulation of biological processes via ATF4.In order to analyze the role of ATF4 in GBM invasion and chemotherapy,the expression of ATF4 was interfered by si RNA,the migration and invasion ability of GBM cells were analyzed by wound healing assay and transwell invasion assay,the changes of GBM cell viability and apoptosis were analyzed by MTT and flow cytometry.3.Bioinformatics was used to analyze the expression content of SPHK1 in GBM tissues,the relationship between SPHK1 and the survival period of GBM patients,and the correlated expression between SPHK1 and ATF4.To determine the transcriptional regulation of ATF4 on SPHK1,CHIP-q PCR and double luciferase reporter gene were used to detect the binding of ATF4 to the SPHK1 promoter sequence in CHIP-seq results.After inhibited ATF4 expression by si RNA,western blot,q PCR,and immunofluorescence were performed to detect the SPHK1 expression and localization.4.In order to analyze the role of SPHK1 in GBM invasion and chemotherapy,after inhibited ATF4 expression by si RNA,wound healing assay and transwell invasion assay were used to analyze the changes of GBM cell migration and invasion ability.MTT and flow cytometry were used to analyze the changes of GBM cell viability and apoptosis.After ATF4 or SPHK1 was inhibited by si RNA in LN229 cells,ELISA was used to detect the S1P secretion;western blot and q PCR were used to detect the changes of EMT related gene protein and m RNA expression.Results:1.The vitality of GBM cells decreases across time and dose under TMZ treatment.Furthermore,the expression of apoptotic proteins was upregulated,the mitochondrial membrane potential decreased and apoptosis increased significantly after treated with TMZ.RNA-seq results showed that among the first 30 up-regulated genes,ER protein folding and Ca²⁺homeostasis related genes expression was up-regulated.And GSEA analysis revealed that the signaling pathway of ER protein process was significantly enhanced in LN229 cells with TMZ treatment.Under TMZ treatment,the expression of ER stress related proteins BIP and ATF4 in GBM cells were increased,as well as the phosphorylation of PERK and e IF2αand the nuclear localization of ATF4.Under TMZ treatment,the combined use of Salubrinal could significantly increase GBM cell viability,enhance cell proliferation and reduce apoptosis,while the combined use of ISRIB had the opposite result.2.It was found that the upregulated genes which ATF4 bind to its promoter region were mainly related to the positive regulation of cell migration when analyzed CHIP-seq combined with RNA-seq.And SPHK1 is one of the target genes regulated by ATF4.Under TMZ treatment,suppressing the expression of ATF4 by si RNA,further inhibit the migration and invasion of GBM cells,and increase apoptosis.3.Bioinformatics showed that SPHK1 was highly expressed in GBM,negatively correlated with the survival of GBM patients,and positively correlated with the expression of ATF4.CHIP-q PCR and Double luciferase reporter assay further verified that ATF4 could bind to the promoter of SPHK1.Under TMZ treatment,suppressing the expression of ATF4 by si RNA,could reduce the protein and m RNA expression of SPHK1.4.Under TMZ treatment,suppressing the expression of SPHK1 by si RNA could further inhibit the migration and invasion of GBM cells,and promote apoptosis.Suppressing the expression of ATF4 or SPHK1 by si RNA could both inhibit the secretion of S1P,inhibit the expression of mesenchymal related genes（Snail2,N-cadherin,Vimentin）and promote the expression of epidermal related genes（E-cadherin）in LN229 cells.Conclusion:1.One prominent GBM cell antagonism response to TMZ is activation of ER stress the PERK/e IF2/ATF4 axis.2.In GBM cells,ATF4 can participate in the regulation of various cell migration and motility associated biological processes and promote the transcription of genes involved to positive regulation of cell migration.3.ER stress induced by chemotherapy can interact with SPHK1/S1P signaling through the transcriptional regulation of ATF4,promoting EMT of GBM cells and enhancing GBM cell migration and invasion capabilities.