NFAT1-regulated IL6 Signaling Contributes To Aggressive Phenotypes Of Glioma

1.Background and objectiveGlioma is a primary malignant tumor in central nervous system with the highest incidence and mortality rate.At present,the main treatment for glioma is surgery combined with chemoradiotherapy,but the treatment effect has not been satisfactory due to higher recurrence rate and shorter survival time.Antitumor immunotherapy based on the mechanism of tumor immunity is considered as the most promising cure for gliomas and other malignant tumors.With the development of the study of tumor immunology,immune microenvironment is becoming more and more important and plays a key role in promoting tumor growth,angiogenesis,invasion,anti-apoptosis and so on.The composition of glioma immune microenvironment is very complex,including glioma cells,infiltration of various types of immune cells,microglia,endothelial cells,parenchyma cells and several secreted cytokines,growth factors,chemokines.Therefore,the study of tumor immune microenvironment is helpful to identify the exact roles of each cell and factor in the development or occurrence of tumors,and even find potential targets for tumor immunotherapy.Interleukin 6?IL6?is an immunorelated gene screened by bioinformatics analysis in the China glioma genome mapping project?CGGA?and the Cancer Genome Atlas?TCGA?in our previous study.IL6 is highly expressed in glioma and glioblastoma,and can be used as a high risk factor affecting the prognosis of patients.Although several studies have reported the role of IL6 in the occurrence and development of glioma,neither the role of Interleukin 6 receptor?IL6R?in glioma nor participating in IL6promoting glioma growth and invasion has been mentioned.In addition,due to the heterogeneity of composition and molecular genetics in glioma,the traditional World Health Organization?WHO?could not accurately assess the specific characteristics of pathological grade of glioma.Therefore,molecular typing can effectively compensate for the shortcomings of the traditional WHO pathological grading.In this study,we took IL6R as the main object.Firstly,we observed whether there was difference expression of IL6R in any molecular typing of glioma.Then we observed the role of IL6R in promoting glioma occurrence,development,and whether it was involved in IL6regulating glioma.Activated T nuclear factor 1?nuclear factor of activated T cells 1,NFAT1?is a long-term glioma related gene in our research.As an immuno-regulatory transcription factor,it plays an important role in regulating glioma occurrence,invasion,tumor immune activation or inhibition.NFAT1 may be involved in the activation of T lymphocytes,promote the synthesis and secretion of IL2,IL4,IL17 and other cytokines.NFAT1 can also participate in the synthesis of IL13R?2 and other interleukin receptors in glioma,but there is no research about NFAT1 can regulate IL6 and IL6R.Therefore,we furtherly analyze the expression and regulation of IL6 and IL6R by NFAT1,which can improve the regulation role of IL6 and IL6R in the occurrence and development of glioma.2.Methods2.1 Bioinformatics analysisWe obtain the expression and clinical data of IL6,IL6R and NFAT1 in TCGA and CGGA datasets through the GlioVis tool?http://gliovis.bioinfo.cnio.es?,following t test to compare the differences expression and prognostic significance of IL6 and IL6R in various types of glioma and analyze the correlation between NFAT1 and IL6 or IL6R.We obtain the promoter sequences of IL6 and IL6R from UCSC database?http://genome.ucsc.edu/cgi-bin/hgGateway?and input those sequences in PROMO?http://alg gen.lsi.upc.es/cgi-bin/promo_v3/promo/promoini t.cgi?DirDB=TF₈.3?to predict whether there is any NFAT1 binding site.2.2 Glioma cell and patient derived stem cell cultureThe human glioma cell lines U87,U251,T98G,and SNB19 were cultured with DMEM high glucose medium containing 10%FBS in 37?and 5%CO₂ condition.We obtain the patient derived glioma cells from the fresh surgical resected clinical glioma specimens by type II collagenase,DNase 1,red blood cell lysate and culture in 2%B27,20ng/mL rh-bFGF and rh-EGF DMEM/F12 culture medium.Immunofluorescence assay was used to detect glioma stem cell markers,CD133,GFAP and?-?tubulin.qPCR was used to detect the expression level of the biomarkers in glioma stem cells and confirm the molecular typing.2.3 Lentivirus infection glioma cellslentivirus based IL6R and NFAT1 overexpression plasmid,IL6R shRNA1,IL6R shRNA2 and NFAT1 shRNA were designed and synthesized by Shanghai GeneChem Biotechnology Co.Ltd.We infect glioma stem cells M1,N2 and glioma cell line U87T98G to overexpress or knockdown the expression of IL6R or NFAT1,followed with western blot and qPCR to verify the infection efficiency.2.4 Western blotWe collect patient derived glioma stem cells and glioma cells with good growth status and extract total proteins of those cells with total protein extraction kit,following with BCA determination protein concentration,preparing protein sample buffer,4-20%gel electrophoresis,transmembrane,blocked with nonfat milk,incubation with primary,secondary antibodyies and finally expousured with ECL light to detect the expression of IL6,IL6R and NFAT1.2.5 qPCR experimentWe collect patient derived glioma stem cells and glioma cells with good growth status and extract total RNA by Takara RNA extraction kit.Then TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix was used to remove genomic DNA and construct cDNA.Finally the TransStart Tip Green qPCR SuperMix was used for quantitative PCR to detect the expression of IL6,IL6R and NFAT1.2.6 ImmunofluorescenceWe collect patient derived glioma stem cells and glioma cells with good growth status,inoculate into the confocal laser medium dish and culture for 24h.Fixed with 4%paraformaldehyde,permeated by trinton X-100,blocked with 5%BSA,incubation with primary,secondary antibodyies and Dapi.Finally,fluorescence microscope was used to observe the expression of CD133,GFAP and?-?Tubulin.2.7 Cell viability assayWe collect patient derived glioma stem cells and glioma cells with good growth status and inoculate into 96 well plates with 1000 cells per well,each repeated 4 well,5plates for 5 days.Then each well was added 20?l MTS solution and incubated at 37?for 3h.Finally,the absorbance valve at 495nm was detected to value the cell viability.2.8 Colony formation experimentWe collect patient derived glioma stem cells and glioma cells with good growth status and inoculate into 6 well plates,200 cells per well.After two weeks culturing,they are stained with 1%crystal violet and the clone formation number and clone formation rate are calculated under inverted microscope photographs.2.9 Wound healing assayWe collect patient derived glioma stem cells and glioma cells with good growth status and inoculate into 6 well plates with 1×10⁵cells per well.After the cell confluence reaches 100%,1ml pipette is used to scratch a straight line,following abandoning medium,cleaning off the shedding cells with PBS and culturing with serum-free DMEM medium for 24h.Finally,the inverted microscope is used to record the scratch vision of glioma cells after 0h and 24h and calculate the wound healing rate by Image J.2.10 Neurosphere formation assayWe collect patient derived glioma stem cells with good growth status and inoculate into 24 well plates with 24 cells per well for 7 days.The inverted microscope is used to photograph and calcuate the number and size of neurospheres.2.11 Transwell assayFirstly the matrigel is diluted at ratio of 1:8 with serum free DMEM and inoculate100?l diluted matrigel in the upper transwell chamber at the 37?for 30min.Then the patient derived glioma stem cells and glioma cells with good growth status are inoculate into the upper transwell chamber with 8×10⁴ cells per well.The lower transwell chamber is treated with 600?l 20%FBS DMEM and cultured for 20h.Finally the upper transwell chamber is stained by HE and photographed under the microscope to calculate the number of invasive glioma cells.2.12 Tunel assayWe collect patient derived glioma stem cells and glioma cells with good growth status and inoculate into confocal laser medium dish with 2×10⁴ cells for 24h.Then fixed with 4%paraformaldehyde,permeated by trinton X-100,and incubated with tunel reaction buffer and Dapi,fluorescence microscope is used to photograph and calcuate the apoptosis rate.2.13 ImmunohistochemistryParaformaldehyde is used to fix the fresh excised subcutaneous or in situ tumor of mice,followed with ethanol gradient dehydration,xylene permeable and paraffin embedded section.Then the sections is treated with xylene dewaxing,gradient alcohol rehydration,deionized water hydration,removal of endogenous peroxidase,antigen retrieval by sodium citrate solution,goat serum blocking solution.Then the primary,secondary and anti-streptavidin-peroxidase solution is incubated sequentially.Both DAB and hematoxylin staining is performed under microscope,followed with alcohol gradient dehydration,xylene transparent and neutral balata mounting.Finally,the positive expression rate is photographed and calculated under the microscope.2.14 HE stainingThe treatment of paraffin sections is similar to that of immunohistochemistry.After the deionized water hydration,it is stained with hematoxylin and eosin,each 2 minutes.Finally,it is treated by alcohol gradient dehydration,xylene transparent,neutral balata mounting and photographed by microscope.2.15 Enzyme linked immunosorbent assayThe 100?L Assay Diluent RD1W is added to 96 well plates at first.Then the collected supernatant of patients derived glioma stem cells and glioma cell lines with good growth state and the diluted IL6 standard is added to 96 well plates,incubated with biotinylated IL6 antibody,peroxidase labelled reaction solution the stop solution.Finally,the microplate reader is used to detect the absorbance valve and calculate the IL6concentration of each sample.2.16 Dual luciferase reporter gene assayWe collect patient derived glioma stem cells and glioma cells with good growth status and inoculate into 96 well plates.After the confluence degree reaching 70-90%,pGL3-IL6-mt,pGL3-IL6-wt,pGL3-IL6R-mt,pGL3-IL6R-wt and pRL-TK plasmid are co-transfected into those cells for 48h.Then,the fluorescein intensity is detected by spectrophotometer through Dual-Glo Luciferase and Assay System.2.17 Chromatin immunoprecipitationWe collect patient derived glioma stem cells and glioma cells with good growth status and perform formaldehyde cross-linking,cell lysis,ultrasonic shear of genomic DNA.20?l samples is saved for input and other samples is incubated with NFAT1antibody at 4?overnight,elution,crosslinking removal and DNA purification.Finally,qPCR is used to detect the regulation of NFAT1 on IL6 and IL6R.2.18 Subcutaneous tumorigenesis of nude miceWe collect patient derived glioma stem cells and glioma cells with good growth status and inject 200?l of 1×10⁸/ml cells into the neck back of nude mice,each group with 5 mice.Mice are observed every five days and the long and short diameter of subcutaneous tumor are measured and calculated according to this formula:tumor volume V=?D*d²?/2 mm³,D is long diameter and d is short diameter.All nude mice are sacrificed by cervical dislocation after 35days and compare the difference of each tumor weight.2.19 Intracranial tumorigenesis of nude miceThe nude mice are anaesthetized by 0.75%pentobarbital sodium.We collect patient derived glioma stem cells and glioma cells with good growth status and inject 3?l of 1×10⁸/ml cells into the right sagittal suture 2mm and anterior fontanel 2mm at the speed of0.5L/min.After injection and waking up,we observe the status of mice daily and calculate the survival rate.When the mice died,the brain tissue is immediately excised,fixed,sliced and stained with immunohistochemical and HE to calculate the intracranial tumor volume.3.Results3.1 The expression of IL6 and IL6R in different genotyping gliomasIn TCGA glioblastoma database,the expression of IL6 and IL6R in mesenchymal type is significantly higher than that of the classical,proneural and neural type,while there is no difference of IL6 and IL6R among classical,proneural and neural type.In the TCGA glioma database,the expression of IL6 and IL6R was higher in IDH1 wild type than that of IDH mutant type.In the TCGA glioma database,TCGA glioblastoma data and Rembrandt glioma database,Kaplan-Meier survival analysis showed that the average survival time of patients with higher expression of IL6 and IL6R live shorter than that of patients with lower expression.Among the eight glioma stem cells with different molecular types,western blot and qPCR showed IL6 and IL6R express the highest in the mesenchymal type stem cells M1 and M2 and lowest in in the proneural type glioma stem cells P1 and P2.What's more,among the common glioma cell lines U87,U251,SNB19,LN229 and T98G,western blot showed IL6 and IL6R also expressed the highest in U87 with highest degree of malignancy and invasion,while expressed the lowest in T98G with less invasiveness and malignant degree.3.2 Regulation of IL6R on the proliferation of glioma.MTS,colony formation assay and neurospheres formation assay were performed to detect the proliferation of glioma.MTS results showed the absorbance values of M1 and U87 decreased after IL6R silencing,while the absorbance value of N2 increased after IL6R overexpression.Colony formation assay showed the colony forming rate of M1 and U87 decreased after IL6R silencing,while the colony forming rate increased after IL6R overexpression in N2.Furtherly,neurospheres formation assay showed the size and number of neurospheres formed less in M1 after IL6R silencing,while increased after IL6R overexpression in N2.3.3 Regulation of IL6R on the migration and invasion of glioma.Both the transwell assay and wound healing assay showed the invasive cell number and wound healing rate decreased in M1 and U87 after IL6R silencing,while increased after IL6R overexpression in N2.3.4 Regulation of IL6R on apoptosis of glioma.Tunel assay showed the number of apoptotic cells and apoptotic rate increased in M1 and U87 after IL6R silencing,while decreased after IL6R overexpression in N2.3.5 The regulation of IL6R on the tumorigenesis.Glioma stem cell M1 and N2 with IL6R knockdown,overexpression and control were injected into the subcutaneous and in stiu of nude mice.The survival rate of nude mice was significantly prolonged,and the volume and weight of tumor tissue decreased after IL6R knockdown in M1,while the opposite results were all got after IL6R overxpression in N2.3.6 IL6R mediated IL6 regulation on the proliferation of glioma.Recombinant human IL6 stimulation showed the absorbance valve increased followed with higher IL6 concentration and 20ng/ml was chosen as the best treatment concentration in the following experiments if there was no special instruction.The absorbance valve and colony formation rate didn't increase after IL6 treatment in IL6R knockdown M1 or IL6 untreated IL6R shRNA control M1,while increased in IL6 treated shRNA control M1.Optical microscope found the adherent M1 cells showed faster proliferation and higher confluence rate in IL6 treated shRNA control M1 while there was no obviously increase in IL6 treated IL6R knockdown M1 or IL6 untreated IL6R shRNA control M1.3.7 IL6R mediated IL6 regulation on the migration and invasion of glioma.Both the transwell assay and wound healing assas showed the invasive cell number and wound healing rate increased in IL6 treated shRNA control M1 while there was no obviously increase in IL6 treated IL6R knockdown M1 or IL6 untreated IL6R shRNA control M1.3.8 IL6R mediated IL6 regulation on the apoptosis of glioma.Tunel assay showed the apoptotic cells and apoptosis rate decreased in IL6 treated shRNA control M1,while there was no obviously decrease in IL6 treated IL6R knockdown M1 or IL6 untreated IL6R shRNA control M1.3.9 NFAT1 regulates the expression of IL6 and IL6R in glioma.Firstly,we detected the expression of IL6,IL6R and NFAT1 in the TCGA glioma database and found there are significant correlations between the expression of NFAT1and IL6 or IL6R.Western blot showed the expression of IL6 and IL6R obviously decreased after NFAT1 silencing in M1 and U87,and increased after NFAT1overexpression in N2 and T98G.ELISA also showed the secretion of IL6 in the supernatant decreased after NFAT1 silencing and increased after NFAT1 overexpression.We further analyzed the promoter regions of IL6 and IL6R by bioinformatics analysis.There was a certain NFAT1 binding site in both IL6 and IL6R promoter regions.The following dual luciferase reporter gene assay and ChIP experiments were designed and confirm that NFAT1 can participate in transcriptional regulation of IL6 and IL6R.3.10 NFAT1 regulates the occurrence of glioma.Based on the above results,we further explored the effect of NFAT1 on tumorigenesis.The results of subcutaneous tumor formation in nude mice showed the volume and weight of tumor decreased obviously in NFAT1 silenced M1.4.Conclusion4.1 Both IL6 and IL6R express higher in mesenchymal and IDH wild type glioma,and the average survival time of glioma patients decreased with the increasing IL6 and IL6R expression.The expression of IL6 and IL6R is associated with the invasion phenotype of glioma.4.2 IL6R overexpression can promote the proliferation,migration,invasion and tumorigenesis and inhibit apoptosis in glioma.4.3 IL6R can play a key role in IL6 regulated glioma occurrence and development and can be an important target for glioma treatment.4.4 NFAT1 can directly transcriptional regulate the expression of IL6 and IL6R and promote the occurrence and development of glioma.Its biological function is union with IL6 and IL6R and improve the NFAT1-IL6/IL6R signal pathway.NFAT1 may be an important target in the treatment of glioma.