The Mechanism Of NF1 Mutation Promoting Epithelial Mesenchymal Transformation And Chemotherapy Sensitivity Of Glioblastoma

Background:The pathological grading of glioblastoma(GBM)mainly depends on the pathological morphology.However,GBMs with identical or similar histological characteristics often have different molecular genetic backgrounds,which leads to large prognostic differences between individuals with the same grade.This suggests that the patient's own molecular features play an important role in guiding treatment,evaluating efficacy and predicting prognosis,and directly affect the biological behavior of tumors.Metabolic reprogramming often occurs in tumor cells,and enhanced glycolysis promotes cell growth,invasion and migration,and reduced the chemosensitivity of tumor cells.Among the four molecular subtypes of GBM,interstitial GBM is often accompanied by mutations in the NF1 gene.As a tumor suppressor gene,NF1 down-regulates its encoded neurofibromin protein after mutation,thereby activating the RAS-RAF-MAPK signaling pathway to promote tumor proliferation and metastasis.However,the regulatory role of the NF1-mutated GBM cell energy metabolism pattern and its effect on the chemosensitivity of HDAC inhibitors remain unclear.The purpose of this study is to explore how NF1 gene mutation reshapes the energy metabolism pattern of GBM cells,and to reveal the molecular mechanism by which NF1 gene mutation increases the sensitivity of GBM to HDAC6 inhibitor chemotherapy,so as to provide new targets and strategies for precision chemotherapy.Methods:1.The expression of NF1 gene in glioma tissues of different grades and prognosis of patients were analyzed by tumor Genome Atlas(TCGA)and Chinese Glioma Atlas(CGGA)database.The expression of NF1 mRNA in glioma tissues was detected by qRT-PCR.2.The NF1 gene in U251 cells was knocked out by CRISPR-Cas9 gene editing technology,and U251 cell line with stable NF1 knockout was established by selecting single clones.At the same time,lentiviral vector with knockdown of NF1 gene was constructed and transfected into U251 and U87 cells,and GBM cell line with stable knockdown expression was established to simulate NF1 gene mutant cells.3.Different metabolites were detected by energy metabolomics.KEGG pathway enrichment analysis was used to screen the pathways that caused significant changes in NF1 knockout.Glucose and lactic acid in cell culture medium after NF1 gene knockout were detected by glucose and lactic acid detection kit.4.CP96 real-time unlabeled cell growth analyzer was used to detect cell proliferation in NF1 knockdown group and control group.The effect of NF1 knockdown on GBM cell invasion and migration was studied by scratch healing experiment and Transwell chamber experiment.The changes of EMT-related proteins(E-cadherin,N-cadherin and Vimentin)were detected by Western blot and immunofluorescence.5.Mitochondrial morphology and function detection:Stained with mitorTrack mitochondrial dye to observe the change of its morphology under confocal microscope;transmission electron microscope to observe the change of mitochondrial morphology;DCFH-DA kit to detect cellular reactive oxygen species;JC-1 kit to detect mitochondrial membrane potential Changes in ATP production;use ATP detection kit to detect ATP production.6.Energy metabolism detection:Seahorse XF24 extracellular flux analyzer was used to detect the effects of NF1 knockdown on glycolysis and mitochondrial oxidative phosphorylation in GBM cells;The expressions of Glut-1,HK2,PKM2,LDHA,HIF-la and mitochondrial proteins(ND1,SDHB,UQCRC2,MTCO2,ATP5A)were detected by WB and qRT-PCR.7.Protein interaction detection:Co-localization of NF1 and MFN1 and P53 was detected by cellular immunofluorescence.The interaction between NF1 and MFN1 and p53 proteins was verified by protein immunoprecipitation.8.Cell apoptosis detection:HDAC6 inhibitors(Tubastatin A and CAY10603)induced NF1 knockdown and control cells respectively,and the effect of HDAC6 inhibitors on GBM cell apoptosis was detected by drain cell assay.The expression of Bax,Bcl-2 and Cleaved-caspase 3 was detect by using Western blot.9.Animal experiments:glioma orthotopic transplanted tumor model was established by injecting U87-Luc cells into the basal ganglia region of the brain of nude mice.In vivo imaging and MRI of small animals were used to observe the effect of NF1 hypothermia on chemotherapy sensitivity of GBM cells by intritoneal injection of Tubastatin A(HDAC6 inhibitor).Finally,HE staining was used to observe the pathological morphology of orthotopic transplanted tumor,and the expression of Ki-67 was detected by immunohistochemistry in each group.Results:1.Transcriptome data analysis of glioma tissues and normal samples from TCGA and CGGA databases showed that the expression of NF1 mRNA was significantly reduced in glioma tissues,and the expression of NF1 mRNA was negatively correlated with the grade of glioma.Moreover,the expression of NF1 was significantly correlated with the prognosis of patients,and the higher the expression of NF1,the better the prognosis of patients.qRT-PCR also demonstrated a significant decrease in NF1 expression glioma tissues.2.Energy metabolomics analysis of NF1-knockdown U251 cells showed that the energy metabolites of NF1-knockdown U251 cells were mainly negatively correlated with glycolysis,pyrimidine metabolism,amino sugar and nucleotide sugar metabolism.3.CP96 real-time unlabeled cell growth analyzer,scratch healing experiment and Transwell experiment showed that NF1 knockdown inhibited GBM cell proliferation and promoted GBM cell invasion and migration.Immunofluorescence and Westernblot detection showed that NF1 knockdown epidermal marker E-cadherin protein expression was significantly decreased,mesenchymal marker N-cadherin and Vimentin protein was significantly increased,NF1 knockdown promoted the occurrence of EMT in GBM cells.4.DCFH-DA,JC-1 and ATP assay kits were used to find that the intracellular reactive oxygen species increased after NF1 knockdown;Mitochondrial membrane potential significantly decreased,and mitochondrial ATP production decreased.5.Using Seahorse XF24 extracellular flux analyzer,it was found that GBM cells with NF1 knockdown had enhanced glycolysis ability and reduced mitochondrial oxidative phosphorylation level.WB results showed that NF1 knockdown promoted the phosphorylation of AKT and mTOR,activated the AKT/mTOR signaling pathway,and induced the expressions of LDHA,C-MYC,HIF1a and PKM2 proteins.Decreased the expression of ND1,SDHB and UQCRC2.6.Transmission electron microscopy observed that NF1 gene knockout promoted mitochondrial division;Protein immunoprecipitation assay showed that NF1 inhibited mitochondrial division by regulating the expression of MFN1,which promoted its degradation through the ubiquitin-proteasome pathway.7.Bioinformatics analysis showed that HDAC6 expression was increased in GBM tissues and negatively correlated with NF1 expression.We also found that NF1 knockout enhanced GBM cells'chemosensitivity to HDAC6 inhibitors(Tubastatin A and CAY 10603),and promoted apoptosis by enhancing the expression of p53K120AC and p53K370AC.Co-IP results showed that NF1 had direct interaction with p53.8.In vivo,HDAC6 inhibitor(Tubastatin A)significantly inhibited the growth of orthodontic transplanted tumors in vivo,especially in NF1 knockdown group.Immunohistochemical results showed that the expression of Ki-67 was lower in the NF1 knockdown group treated with Tubastatin A.Conclusion:1.The significantly reduced expression of NF1 in GBM is significantly correlated with the prognosis of patients and can be used as a prognostic marker for GBM patients.2.NF1 knockdown promotes GBM cell invasion,migration and EMT.3.After NF1 gene mutation,the activation of AKT/mTOR signaling pathway promotes aerobic glycolysis in GBM cells,promotes mitochondrial fission through targeted regulation of MFN1 protein,and inhibits mitochondrial oxidative phosphorylation.Mutation of NF1 promotes EMT in GBM cells by enhancing aerobic glycolysis and mitochondrial fission.4.Through in vitro and vivo experiments,we found that HDAC6 inhibitors(Tubastatin A and CAY10603)significantly inhibited the proliferation of GBM cells and promoted their apoptosis by activating the mitochondrial apoptosis pathway.5.HDAC6 inhibitors enhanced the chemosensitivity of NF1-knockout GBM cells to HDAC6 inhibitors by targeting HDAC6 activity and stabilizing acetylation levels at lysine 382,120,and 370 residues of p53.