NFAT2-HDAC1 Signaling Contributes To The Malignant Phenotype Of Glioblastoma

Background and objectiveGlioblastoma(GBM)is the most common and fatal primary brain tumor in adults.GBM can be divided into mesenchymal(MES),classical(CL)and anterior nerve(PN).MES subtype has the most invasiveness and radiation resistance,which is related to poor prognosis.The molecular mechanism of MES differentiation is still unclear.Activated T nuclear factor(NFAT)is a transcription factor in T cells,which can regulate inflammatory response and immune response by interacting with histone deacetylase(HDAC)and nuclear factor NF-κ B pathway.NFAT molecules also play an important role in the occurrence and development of tumors.NFAT2,also known as NFATC1,is related to the acquisition of stem cell characteristics,self-renewal ability and promoting the transformation of tumor cell epithelium into mesenchymal(EMT),while EMT and dry acquisition can make cancer cells become highly invasive and lead to tumor metastasis,drug resistance and recurrence.Recent studies have shown that abnormal activation of NFAT2 signal has been found in colon,pancreatic and breast cancer,and NFAT2 has different functions in different tumors.In this study,we examined the effect of NFAT2 on the malignant phenotype of glioma stem cell(Glioma stem cell,GSCs(glioma stem cell)in vitro and in vivo,and discussed the downstream targets of NFAT2 and its mechanism.Methods1.Ethical reviewThis study was approved by the Institution Evaluation Committee of the first Hospital of China Medical University(approval No.81472360).All animal experiments are supervised by the Animal Ethics Committee of China Medical University(approval No.2019092).2.GSCs cultureFreshly resected clinical glioma tissues were isolated into single cells and cultured in5 CO2,37 degrees Celsius incubator with stem cell culture medium to grow glioma stem cells.3.TransfectionNFAT2,HDAC1 control and shRNA of plasmid vector were used for silencing.The cDNA of lentivirus vector NFAT2 and HDAC1 was used for overexpression.The effects of gene silencing and overexpression were detected by WB.4.Immunohistochemical(IHC)The paraffin-embedded sections of gliomas were stained with anti-NFAT2 and CD44 antibodies.Observe and take pictures under an optical microscope.5.Western blotting analysisWestern bolt was performed by protein extraction,protein quantification,electrophoresis,membrane transfer,milk blocking,first antibody,second antibody,luminescence and so on.NFAT2,YKL40,CD44,OLIG2,HDAC1,NF-κ B p65 and antiacetyllysine antibody were used to detect the expression of corresponding proteins.It was quantified by Image J software.6.ImmunofluorescenceThe cells were fixed by paraformaldehyde,permeated by Triton XMel 100,blocked by 3%BSA,the first antibody 4o C was overnight,and the fluorescence second antibody was observed under fluorescence microscope after 1 hour.CD133,CD44,nestin,GFAP,β-Ⅲ tublin,NFAT2 and HDAC1 antibodies were used to detect the localization and expression of the corresponding proteins in immunofluorescence assay.7.Q-PCRColumn RNA extraction reagent was used to extract total RNA,Prime-Script RT Master Mix to synthesize single-stranded cDNA,and then SYBR Green Master Mix was used to detect qPCR.Each sample was run repeatedly for 3 times,and β-actin was used as internal control.The 2-delta Ct method is used to calculate the relative multiple of expression.8.Limit dilution neuroball formation testThe self-renewal ability of neural stem cells was evaluated by a neurosphere formation experiment,which was dissociated and implanted into a 96-well plate at a rate of 50,100,500 or 1000 cells per well.After 7 days,the size of the sphere was observed and the spheres with a diameter of more than 50 μ m were counted.9.Stereoscopic invasion of sphereThe neurospheres were embedded into the invasion matrix composed of basement membrane proteins and cultured in 96-well plates.The invasive distance was measured at0 h and 48 h to indicate the invasive ability,and Image J was used to quantify the invasive ability.10.Cell viability assayThe cell survival rate was detected by 96-well plate according to the cell proliferation analysis kit,and the cell viability was measured and the curve was drawn every 0.2.4.6days.11.TranswellThe GSC cell spheres were made into single cell suspension and put into the upper chamber,and 10% serum was put into the lower chamber to invade the filter membrane coated with Matrigel for 20 hours.The invasive cells were photographed by microscope and counted by Image J software.12.TUNEL methodApoptosis was detected by TUNEL kit.The cells were fixed by paraformaldehyde,penetrated by Triton XMel 100,blocked by 3%H2O2 and incubated with TUNEL solution at 37 ℃ for 90 minutes.DAPI was used to stain the nuclei.The apoptosis rate was calculated by observing the samples under fluorescence microscope and taking pictures.13.Luciferase activity analysisThe bioinformatics of the Jaspar website predicts the NFAT2 binding sites of 312-812 base pairs upstream of the transcriptional initiation site of the HDAC1 promoter.The wild type and mutant luciferase vectors of NF-kappa B and HDAC1 promoters were constructed.Double luciferase report kit was used to detect luciferase activity.14.Orthotopic transplantation of intracranial gliomaThe transfected GSCs was injected into anesthetized female BALB/c nude mice with stereotactic apparatus.The symptoms and survival time of mice were observed,and the tumor growth was evaluated.The brains of mice were collected for analysis and HE staining was used to evaluate the size of the tumor.15.Statistical analysisAll experiments were repeated at least three times,and the data were expressed as mean ±standard deviation.Chi-square test,t-test or analysis of variance were used for comparison between groups.Access and process the TCGA tumor genome map database and the National Cancer Institute molecular brain tumor database(REMBRANDT database)through the Glio Vis online platform.Survival differences were analyzed by logrank test and Kaplan-Meier analysis.The relationship between NFAT2 and HDAC1 expression was analyzed by Pearson correlation.Different enrichment levels of NF-κ B pathway signals were detected by gene set enrichment analysis(GSEA).Double tail P <0.05 is considered to be statistically significant.SPSSv19.0 software was used for statistical analysis.Results1.NFAT2 was up-regulated in MES basement membrane and negatively correlated with survival rateThe high expression of NFAT2 was found in IV grade gliomas,MES subtypes and recurrent GBM.The high expression of NFAT2 is related to the low survival rate of gliomas and MES gliomas.The level of NFAT2 protein was positively correlated with MES markers YKL40 and CD44,and negatively correlated with PN labeled OLIG2.The expression of NFAT2 in GSCs with high expression of CD44 was higher than that of the subgroup with high expression of NFAT2.2.NFAT2 silencing inhibits the growth of MES-GSC rich tumor spheres in vitroSilencing NFAT2 resulted in decreased expression of CD44 and YKL40.NFAT2 gene knockout significantly inhibited cell proliferation and promoted cell apoptosis.The loss of NFAT2 expression reduced the invasive ability of GSC.The GSC sphere size of NFAT2 gene knockout decreased significantly.3.Overexpression of NFAT2 promotes MES transformation and growth of GSCs in vitroThe overexpression of NFAT2 increases the expression of CD44 and YKL40,suggesting the differentiation of MES.The upregulation of NFAT2 led to a significant increase in cell proliferation and a decrease in apoptosis.Overexpression of NFAT2 significantly promoted cell invasion,promoted the growth of GSCs spheres,and led to a significant increase in sphere size and sphere formation.4.Effects of NFAT2 on tumorigenicity and MES differentiation of GSCs in vivoSilence / overexpression of NFAT2 significantly inhibited / increased the growth of intracranial tumors in GSCs and prolonged / shortened the survival time of tumor models.IHC of tumors in vivo showed that knockout / overexpression of NFAT2 gene significantly decreased / increased the level of CD44 expressed in tumors in vivo.5.Regulation of HDAC1 expression in GSCs by NFAT2In TCGA database,the expression of NFAT2 was significantly correlated with that of HDAC1.The up-regulation of HDAC1 in MES subtype and recurrent GBMs was positively correlated with the expression of MES markers CD44 and YKL40.The high expression of HDAC1 was related to the poor survival rate in gliomas and gliomas.Similar to NFAT2,the expression of HDAC1 in GSCs with high expression of CD44 was higher than that of GSCs with low expression of CD44.NFAT2 silencing significantly inhibited the expression of HDAC1.Overexpression of NFAT2 significantly increased the expression of HDAC1.NFAT2 silencing / overexpression significantly decreased /increased the luciferase activity of G08 GSCs with wild type promoter,while the mutant had no change.The deletion of HDAC1 significantly decreased the expression of CD44 and YKL40,inhibited the ability of proliferation,invasion and self-renewal,and induced apoptosis.6.Saving HDAC1 in NFAT2 silent GSCs can partially restore tumor growthStable re-expression of HDAC1,in NFAT2-silenced GSC leads to the restoration of CD44 and YKL40 expression,cell proliferation,invasion,self-renewal ability,tumor growth in vivo,and partial recovery of CD44 expression in vivo.7.NFAT2-HDAC1 signal regulates the activity of NF-κ B pathwayNFAT2 silencing increased the level of acetylated lysine residues in p65.In transcriptional analysis using luciferase reported by NF-κ B binding site,NFAT2 silencing inhibited the activity of NF-κ B.However,rescuing HDAC1 in NFAT2 knockout GSC inhibited the hyperacetylation of p65 and restored NF-κ B-dependent transcriptional activity.ConclusionNFAT2-HDAC1 pathway may play an important role in maintaining the malignant phenotype of GBM by regulating the activity of NF-κB.Inhibition of the NFAT2/HDAC1/NF-κB axis is an attractive treatment method for the treatment of GBMS,especially the MES subtype.