| Objective:Glioblastoma?GBM?is the most common and aggressive type of malignant tumors of central nervous system in human adults.Despite the investment of considerable effort in improving the diagnosis and treatment for GBM,it remains one of the most deadly malignancies worldwide.The current treatment strategy for GBM patients is surgical resection followed by combined chemo-radiotherapy,however prognosis for GBM has not seen any meaningful improvement.The unfavorable efficacy is mainly due to the high proliferation,invasiveness and therapeutic resistance of GBM.Hence,there is an urgent need to understand the molecular mechanisms involved in malignant behaviors and explore novel targets for GBM treatment.In recent years,many groups have performed integrated analyses based on high-throughput profiling of genome and transcriptome to comprehensively explore the molecular mechanism of GBM.As the most representative scheme of molecular pathological subclassification,the integrated genomic analysis by Verhaak et al.has classified GBM into four subtypes:proneural?PN?,neural?NE?,classical?CL?and mesenchymel?ME?.Among them,PN subtype and ME subtype have been identified consistently in various classification systems and indicated to have different biological behaviors and distinct clinical prognoses between each other.ME subtype GBM have been proved to have more aggressive phenotypes leading to therapeutic failure and poor prognosis,such as radio-and chemo-resistance,increased invasiveness,and reduced cell stiffness,comparing with PN subtype tumors.On the other hand,similar to tumor cells undergoing epithelial-mesenchymal transition?EMT?in order to obtain a more aggressive property,the shift towards the ME subtype appears to be a common pattern during the malignant progression of GBM.Furthermore,revealing an aggressive phenotype,the ME subtype GBM has been identified to typically express neural stem cell markers,and EMT is an important inducer of the cancer stem cell properties.Taking account of the extremely aggressive behavior of ME subtype GBM,it is necessary to clarify the molecular mechanism that activating and transition of the mesenchymal phenotype in these malignant brain tumors.Accumulating evidences have indicated that microRNAs?miRNAs?play important roles in the tumorigenesis of GBM,by functioning as both oncogenic promoters and tumor suppressors.These specific miRNAs can serve potentially as effective biomarkers for improving diagnostic and prognostic accuracies,or as targets for novel treatment strategies against for GBM patients.However,the mechanism of miRNAs in regulation of mesenchymal transition in GBM remains unclear.Previously,we have firstly reported that miR-504 is down-regulated both in tumor tissues and cell lines and correlated with poor prognosis in high-grade glioma,including GBM.By statistically evaluating the expression relationship between miRNAs and mRNAs,we found that expression of miR-504 negatively correlated with the expression of ME markers in GBM.Furthermore,our recent study demonstrated that mi R-504 could inhibit cell proliferation and promote apoptosis by targeting FOXP1 in glioma cells.These findings suggested the possibility that mi R-504 might take an important role in regulating mesenchymal phenotype transition in GBM.In the present study,we aimed to clarify the function and mechanism of miR-504 in biological regulation of malignant mesenchymal phenotypes.Methods:1.Processed miRNA and mRNA expression data?level 3?as well as the clinicopathological annotations were downloaded from The Cancer Genome Atlas?TCGA?portal.A total of 517 GBM samples and 10 non-neoplastic tissues?NBT?were enrolled in this study.2.Principal components analysis?PCA?analysisPCA was performed by using R programing language to access the expression patterns of grouped people.3.To identify biological processes associated with miR-504 in GBM,top 200negatively related genes and top 200 differentially up-regulated genes were analyzed using the DAVID web tool?https://david.ncifcrf.gov/?.4.Gene set enrichment analysis?GSEA?was carried out to explore whether the identified sets of genes displayed statistical differences between two groups.Normalized enrichment score?NES?and false discovery rate?FDR?were used to determine the statistical significance.5.Following the experimental design,commercially available lentiviral vectors were used to construct the LV-hsa-miR-504 vector.This structure was used to overexpress miR-504 in U87,U373,GSCs-U87,GSCs-1295 cells after being verified by DNA sequencing.The LV-empty vector?miR-NC?acted as a negative control.Lentiviral vector containing FZD7 coding sequence was used to overexpress FZD7 in U87,U373 cells stably expressing miR-504.LV-NC acted as a negative control.6.Wound healing assay was used to evaluate the migration rate of glioma cells.7.Transwell assay was performed to assess the invasion capability of glioma cells.8.Total RNA of the glioblastoma cells was extracted using TRIzol Reagent?Ambion,USA?,according to the manufacture's instruction.Mir-X miRNA First-Strand Synthesis Kit?Takara,Japan?and Prime-Script RT reagent Kit?Takara,Japan?were used to synthesis cDNA for miRNA and mRNA respectively.Subsequently,expression levels of miR-504 and FZD7 were quantified by qRT-PCR using SYBR Premix Ex TaqII?Takara,Japan?.Expression of U6 was used as an endogenous control for miR-504,while GAPDH was used as an internal control for FZD7.The 2-??Ctmethod was used to calculate relative expression changes.9.Total protein was extracted using RIPA buffer according to the manufacturer's protocol and the protein concentration was determined using a BCA protein assay kit?Beyotime Biotec,China?.Protein samples were fractionated using 8–15%SDS–PAGE and transferred to PVDF membrane?Millipore,NY,USA?.The membranes were blocked with 5%nonfat milk in TBS-T and incubated overnight at 4°C with the following antibodies:FZD7?Abcam,USA?;E-cadherin,N-cadherin Vimentin,Snai2,CD44,CD133,Nestin,SOX2,GSK3?,,?-catenin?Proteintech,USA?,p-GSK3??S9??Wanleibio,CHINA?,GAPDH ?Proteintech,USA?was used as endogenous control.The immune complexes were detected using the enhanced chemiluminescence?ECL?method.10.Immunofluorescence staining was done to detedct the protein expression level of Vimentin,CD133,GFAP and FZD7 in glioma cells.11.Spheroid formation assay was used to assess the selfrenew capability of GSCs.12.Tumor xenograft mode was used to evaluate the tumorigenic ability of GSCs.13.Immunohistochemistry were performed to detect the protein expression level in xenografts..14.The dual luciferase reporter gene assay was conducted to confirm whether FZD7 was the target gene of the miR-504.16.Statistical analyses were performed by using Microsoft Excel 2010,GraphPad Prism 5 and SPSS 19and R software.Student's t-test was used to calculate the difference between two groups.Pearson correlation was used to evaluate the linear relationship between the expression levels of different genes.The chi-square test was used to evaluate the association between miR-504/FZD7 ratio and GBM molecular subtype.Kaplan-Meier survival analysis was applied to estimate the survival distributions and the log-rank test was used to assess the statistical significance.Univariate and multivariate Cox regression analyses were performed to identify independent prognostic factors.All experiments were performed in triplicate and the data are expressed as mean±standard deviation?SD?.Differences were considered significant at P<0.05.Result:1.We analyzed miR-504 expression levels by qRT-PCR in 50 GBM tissues,in 5commonly used glioblastoma cell lines?U87,U373,T98G,LN18,GSCs-12595?.We found miR-504 was singifcantly down-regulated in GBM tissues and glioblastoma cell lines compared to normal brain tissues?P<0.05?.The result was validated by TCGA dataset.2.Down-regulation of miR-504 correlates with ME subtype GBM.GBM patients in ME subtype showed significant low-level of miR-504 expression than that in PN subtype?P<0.05?.Principal components analysis?PCA?based on the miR-504related genes showed a distinct distribution pattern,between GBM patients in ME and PN subtype,indicating different expression of miR-504 and its related genes among these two different molecular subtypes.In addition,ME and PN signature genes exhibited distinct different expression patterns corresponding to miR-504 expression.Expression of miR-504 negatively correlated with ME score based on the mean expression level of206 ME signature genes in GBM patients?R=-0.258 and P<0.001?.3.GO analysis was performed based on genes that differentially up-regulated in miR-504 low-expression tumors,and genes showed negative correlation with miR-504,respectively.Our results showed that mesenchymal phenotypes associated biological processes including cell adhesion,angiogenesis,cell migration,were significantly enriched.Consistently,GSEA showed enrichment of mesenchymal phenotypes in GBM with low-miR-504 compared to that with high-miR-504,indicating the involvement of miR-504 in mensenchymal transition related process in GBM.4.Based on ectopic mi R-504 expression,tumor cells displayed a remarkable morphological change from the typical spindle-like shape to an epithelial-like shape.Subsequently,results from wound healing assay and transwell invasion assay showed that exogenous miR-504 overexpression significantly suppressed the migration and invasion of glioblastoma cells,respectively.Western blot analysis revealed that miR-504 overexpression remarkably decreased expression of mesenchymal markers N-cadherin,Vimentin,Snai2 and CD44,both in U87 and U373.On the contrary,epithelial marker E-cadherin showed significant upregulation compared with controls.Moreover,immunofluorescence assay confirmed this observation by showing that cell lines with miR-504 overexpression displayed weaker Vimentin staining than negative controls.5.To determine whether miR-504 regulates self-renewal ability of GSCs,sphere-forming assay was performed,and the results indicated that overexpression of miR-504 in GSCs suppressed their capability of tumor spheres formation.In addition,with miR-504 overexpression,both GSCs-U87 and GSCs-1295 showed significantly decreased expression of widely-identified neural stem cell markers:CD133,Nestin,Sox2and CD44,by western blot analysis.GSCs-U87 cells stably transfected with miR-504 or negative-control miRNA?miR-NC?were injected into the flank regions of nude mice.At6 weeks post-inoculation,mice with GSCs-87-miR-504 tumors had smaller tumor size and less tumor weight.In addition,immunohistochemistry confirmed that neural stem cell markers CD133 and KLF4 protein levels were reduced in xenografts generated with GSCs-U87-miR-504 cells.6.We predicted putative targets of miR-504 using 3 different publicly available online algorithms?TargetScan,miRDB,and PITA?and identified common candidate genes predicted by all 3 methods.Meanwhile,200 genes that showed significantly negative correlation with mi R-504 were selected by Pearson correlation test based on TCGA data.By overlapping these two gene sets,FZD7 was identified as the only candidate target gene of miR-504.FZD7 showed a significantly negative correlation with miR-504 in TCGA.Luciferase reporter assay showed that miR-504 derectly binding to FZD7 3'UTR.Furthermore,our results from qRT-PCR assay,western blot analysis and immunofluorescence assays demonstrated the negative regulation of FZD7 by miR-504 in GBM.Moreover,immunohistochemical staining in GSCs-U87 xenograft tumors displayed the similar protein downregulation of FZD7 with mi R-504overexpression.7.We performed wound healing and transwell invasion assays with the treated tumor cells and found that the inhibition of cell migration and invasion caused by miR-504 was partially reversed with FZD7 overexpression in U87 and U373 cells.Consistently,up-regulation of the epithelial marker E-cadherin and down-regulation of the mesencymal markers Vimentin and CD44 by miR-504 were also reversed by FZD7overexpression.Immunofluorescence staining of U87 cells showed that the accumulations of?-catenin in nucleus were remarkably attenuated by ectopic miR-504.Western blotting analysis demonstrated that,expression of phosphorylated GSK-3??p-GSK-3??and?-catenin significantly decreased with mi R-504 transfection,and such down-regulation could be totally reserved by FZD7 overexpression,both in U87 and U373 cells.8.We comprehensively evaluated the potential of the combination of miR-504 and FZD7 in predicting the prognosis of GBM.The result showed that GBM patients with low miR-504/FZD7 ratio had shotter overall survival than that with high ratio.Additionally,we performed univariate and multivariate Cox regression analysis and determined that low ratio of miR-504/FZD7 was an independent risk factor of poor prognosis for GBM patients.Moreover,we found that low miR-504/FZD7 ratio closely correlated with survival disadvantage both in GBM patients receiving chemotherapyand radiotherapy,indicating that low miR-504/FZD7 ratio as an indicator of chemo-and radiotheraputic resistance,for GBM patients.Conclusions:1.MiR-504 is down-regulated in GBM tissues and glioblastoma cell lines.2.Down-regulation of miR-504 correlates with ME subtype GBM.MiR-504 is involved in mesenchymal phenotype associated processes.3.MiR-504 inhibits mesenchymal properties,EMT process and GSCs activity in GBM.4.FZD7 is identified as a direct target of miR-504 in GBM.5.MiR-504 attenuates mesenchymal signatures by suppressing FZD7 mediated wnt/?-catenin pathway in glioblastoma.6.MiR-504/FZD7ratio is a prognostic and chemo-and radio-therapeutic indicator for GBM patients. |
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