The Prognostic Value And Functions Of LncRNAs In Glioblastoma

Section I Identification of a IncRNA signature to predict the prognosis of glioblastomaObjective:To identify IncRNAs associated with the clinical outcome of glioblastoma(GBM).Methods:Gene expression microarray and clinical data of GBM were downloaded from public available database.LncRNAs were re-annoated and its expression value was obtained by using the updated microarray annotation files.Survival-related IncRNAs were screened by univariate Cox regression analysis using Biometric Research Branch-Array Tools in a trainning set of 108 GBM patients.Hierarchical clustering analysis was performed to divide patients based on IncRNA expression profiles.The survival-related IncRNAs were then integrated into a prognostic signature by risk scoring method and divieded patients into high-risk and low-risk groups with the median survival risk score of the training set.Survival analysis was performed by Kaplan—Meier method with log-rank test.Time-dependent receiver operating characteristic(ROC)curve was depicted by R-package "survivalROC" to assess the sensitivity and specificity of the prognostic methods.The prognostic value of the IncRNA signature was further confirmed in additional cohorts and assessed by multivariate Cox regression analysis and data stratification analysis to determine whether it was an independent prognostic indicator.In addition,the correlation of the IncRNA expressions and mutation status of IDHI and EGFR were investigated.Results:We identified four IncRNAs that were significantly associated with the overall survival in a training-set of 108 GBMs(P<0.005),which were also aberrantly expressed in GBM.A hierarchical clustering analysis of the patients based on the expression intensities of these four IncRNAs showed two distinct clusters,whose median survival were remarkably different.The IncRNAs were then integrated into a prognostic signature which classified patients of the training-set into high-risk and low-risk groups with significantly distinct survival(HR = 2.72,95%Cl=1.71-4.31;P<0.001).The prognostic value of the four-IncRNA signature was confirmed in additional cohorts and independent of age,Kanofsky performance score(KPS)and extent of surgery.Moreover,the four-IncRNA signature was correlated with the mutation status of isocitrate dehydrogenase 1(IDH1)and epidermal growth factor receptor(EGFR)and able to predict the clinical outcome of glioblastomas carrying IDH1 mutation.Conclusion:A novel four-IncRNA signature was identified for risk stratification and survival prediction of GBM patients.Section Ⅱ Expression profiles and functional network of IncRNAs in GBMObjective:To investigate the expression profiles and functional network of IncRNAs in GBM.Methods:LncRNA microarray was adopt to investigate the expressions of IncRNAs in GBM tissues,follow by a literature systematic review of the IncRNA profile studies of GBM.The differently expressed IncRNAs we identified were measured by quantified real-time PCR(qPCR).The IncRNAs conservation analysis,correlation analysis of IncRNA expressions to genomic copy number variation(CNV)and promoter methylation level as well as gene co-expression network analysis were also performed.Results:3989 lncRNAs were identified differently expressed in GBM compared with normal brain tissue(P<0.05 and fold change>2),including TP73-AS1 and other three survival-related lncRNAs discovered in Section I.39 out of the lncRNAs found in the literature systematic analysis overlapped with our microarray study as significantly deregulated in GBM(fold change>5),all of which were evolutionarily conserved.Further analysis found that the expression levels of HOTARM1 and other lncRNAs were related to CNV,CRNDE and others related to promoter methylation levels.In addition,gene co-expression analysis revealed that these IncRNAs were associated with tumor signaling pathways such as cell cycle and apoptosis.Conclusion:There a wide range of different expressions and regulation networks of lncRNAs in GBM.Sectoin Ⅲ Mechanism of TP73-AS1 in proliferation and invasion of glioma cellsObjective:To investigate the effect of TP73-AS1 on the proliferation and invasion of glioma and its potential molecular mechanism.Methods:The expression of TP73-AS1 in different grade glioma and glioma cell lines was detected by qPCR.CCK8 was used for proliferation measure,and wound healing and transwell assay were performed to detect migration and invasion of glioma cells after knockdown of TP73-AS1 by siRNA.Luciferase reporter gene method was adopted to detect the binding of TP73-AS1 and miR-200a.Western blot and immunofluorescence were implemented to detect the expressions of ZEB1,ZEB2 and epithelial-mesenchymal transition(EMT)markers.Results:Up-regulation of TP73-AS1 was observed in GBM tissue and glioma cell lines U87 and U251.Bioinformatics revealed that TP73-AS1 was associated with EMT and predicted to bind with miR-200a.Knockdown of TP73-AS1 by siRNA could inhibit cell proliferation,migration and invasion of glioma cells.Luciferase reporter gene assay revealed that miR-200a specifically binded to the predicted site of TP73-AS1.Furthermore,overexpression of miR-200a reduced the expression of TP73-AS1.In addition,Knockdown of TP73-AS1 down-regulated the expressions of ZEB1,ZEB2 and mesenchymal markers as well as up-regulated the expression of epithelial markers.Conclusion:TP73-AS1 affect the proliferation,invasion and EMT of glioma cell via mir-200a-ZEB1/2 signaling.