| 1.IntroductionGlioma is the most prevalent and lethal primary brain tumor,accounting for nearly 30%of all cases.Patients with glioma—especially glioblastoma multiforme（GBM）—have poor prognosis,with a median survival of approximately 14 months,even with treatment（surgery,radiotherapy,and adjuvant chemotherapy）.In the effort to facilitate the development of more effective target therapies,large-scale genomic projects on patient specimens have adopted to identify GBM molecular subtypes associated with prognostic values.Ultimately,GBM was classified into at least four molecular subgroups namely proneural（PN）,neural（NL）,classical（CL）and mesenchymal（MES）.The MES subtype was the most aggressive and strongly associated with a poor prognosis compared to PN subtype.In addition,a shift from PN to MES subtype occured in patients following radiotherapy and chemotherapy.Therefore,clarifying the mechanism inducing PMT is important to improve treatment outcome and survival prognosis of glioma patients.During last few years,more attention was paid to a new kind of ncRNA named long noncoding RNA（lncRNA）which was larger than 200 bases.Currently,increasing evidence demonstrated that lncRNAs were dysregulated in different human cancer and played a great role in tumorigenesis.Results from previous studies identified some dysregulated oncogenic and tumor-suppressive lncRNAs in glioma.Some lncRNAs were found to be related to mesenchymal transition in glioma.For example,LINC00152 could activate NF-κB pathway to induce proneural-to-mesenchymal transition in glioma cells.Therefore,investigating long non-coding RNAs related to proneural-to-mesenchymal transition was of great significance for the diagnosis,treatment and prognosis of gliomas.Here,we used public expression profiles of gliomas to analyze and screen lncRNAs related to mesenchymal transition of gliomas,and further explored the clinical features and functional roles of these lncRNAs in gliomas,aiming to provide new strategy for the treatment of gliomas.2.Materials and Methods2.1 Materials2.1.1 Patient samples and public datasetsGlioma tissue samples（n=28）for qRT-PCR were collected at the First Hospital of China Medical University.The histological diagnoses of the samples were independently verified by two experienced neuropathologists based on 2010 WHO classification guidelines.Four non-neoplastic brain tissue specimens from cranial injury internal decompression patients served as the negative control.The present study was approved by the Ethics Committee of the First Hospital of China Medical University.RNA-seq data,including lncRNA expression of glioma samples were acquired from TCGA database,CGGA database,Rembrandt and GSE16011.2.1.2 Glioma cell linesU87,LN229,U373,U251,and T98G human glioma cell lines、THP-1 cell and normal human astrocytes were purchased from American Type Culture Collection.DGCs were derived from freshly resected GBM patient specimens.2.2 Bioinformatics Analysis Method2.2.1 Calculation of glioma microenvironment cell subsets and related gene setscoresWe used ssGSEA in R language to calculate microenvironment cell subsets and related gene set scores.PMT score was calculated using the formula:PMT socre=mesenchymal score–proneural score.2.2.2 GO AnalysisGO analysis was conducted mainly through ClueGO in Cytoscape software,Metascape or DAVID website.2.2.3 Gene Set Enrichment Analysis（GSEA）Gene set enrichment analysis（GSEA）software was used to analyze the difference of gene set enrichment between the low and high groups.2.2.4 Construction of lncRNA–mi RNA–mRNA networkmiRNAs potentially binding lncRNAs were identified from DIANA tools and LncBase Predicted v.2.LncRNA–mi RNA pairs were determined from the intersection of miRNAs predicted to bind lncRNAs and those downregulated in GBM compared with normal brain tissue（P<0.01）.Target genes of overlapping miRNAs were predicted with star Base v.3.0,which includes seven prediction algorithms（PITA,RNA22,mi Rmap,microT,miRanda,PicTar,and TargetScan）.miRNA–mRNA pairs were identified by overlapping target genes predicted by at least three algorithms with up-DEGs in the high vs.low group or signature-related genes.lncRNA–miRNA and miRNA–m RNA pairs were used to construct a lncRNA–mi RNA–mRNA network using Cytoscape v.3.6.1 software.2.2.5 WGCNAWGCNA was conducted by the WGCNA R package.2.3 Biological experiment method2.3.1 Cell transfectionGlioma cell lines were tranfected by siRNAs with lipo3000.After 48h transfection,silencing efficacy was verified by qRT-PCR.2.3.2 qRT-PCRTotal RNA was extracted from samples using TRIzol reagent,and cDNA was synthesized using an RNA PCR kit.qRT-PCR was performed using TB Green Premix Ex Taq II,according to the manufacturer’s protocol,with the 18S gene sequence serving as the internal standard.To evaluate miR-129-5p and miR-495-3p levels,cDNA was generated with the Taqman miRNA Reverse Transcription kit and qRT-PCR was performed with a TaqMan miRNA assay using TaqMan Universal Master Mix II;the U6 gene served as an endogenous control.Relative expression levels were calculated with the 2-ΔΔCt method.2.3.3 Protein isolation and western blottingGlioma cells were lysed by prepared protein lysis.The lysate was crushed on ice for 30 minutes,then centrifuged at 4℃and 14 000 rpm for 15 minutes,and the supernatant was taken for protein quantification to prepare 3ug/ul WB sample.Western blot:Gel electrophoresis,transmembrane,blocked with nonfat milk,incubation with primary and secondary antibodies and finally exposure with ECL light.2.3.4 In vitro chemosensitivity assayTMZ was supplied by Tasly Pharmaceutical（Tianjin,China）and dissolved in dimethyl sulfoxide to a concentration of 100 mM and diluted in cell culture medium to the appropriate final concentration.Approximately 5×103 cells per well were seeded in a 96-well plate and incubated at 37°C for 24 h.TMZ was added at concentrations ranging from 62.5 to 4000μM.Cell viability was assessed with the CCK-8 kit.A dose–response curve was plotted,and IC50 values were calculated by non-linear regression（curve fit）with Prism v.7.0a software（GraphPad,La Jolla,CA,USA）.2.3.5 Dual luciferase reporter assayThe pmirGLO luciferase vector with putative hsa-miR-495-3p and hsa-miR-129-5p binding sites in HOTAIRM1 and their mutated forms were purchased from Promega（Madison,WI,USA）.The plasmid and miRNA mimic were co-transfected into HEK-293T cells,which were lysed 48 h later and tested for firefly and Renilla luciferase activities using the Dual-Luciferase Reporter Assay System（Promega）.Renilla luciferase activity served as an internal control.2.3.6 Cell proliferation,migration,and invasion assaysProfliferation:After 48h transfection,2×10 3 cells/well were seeded into96-well plates and cultured for 0,1,2,3,4 and 5 days.10μl CCK-8 solution was added to each well,and cultured for 1 h at 37°C.Then the absorbance at 450 nm was measured.Migration and invasion assays:Cells were seeded into the upper chamber of the Matrigel transwell chamber with or without Matrigel.Add 600μl of DMEM containing 20%FBS or conditioned medium in the lower chamber.Cells that migrated to the lower surface of the membrane were fixed and stained with 0.5%crystal violet.2.4 Statistical analysis methodsStatistical analysis was carried out using software of GraphPad Prism 7 and R3.5.0.Student’s or Welch t-test,Wilcoxon rank sum test was applied to evaluate differences among two groups,and Pearson correlation analyses were conducted to evaluate relations among groups.Survival distribution was assessed via Kaplan-Meier survival analyses,and the log-rank test was used to estimate significance between stratified groups.All statistical computations and figures were built with R.Statistical significance was defined as a 2-tailed p-value<0.05.3.ResultsPart 1:PMT score was positively associated with malignancy in glioma.Ten lncRNAs（LINC01057,TP73-AS1,AP000695.4,LINC01503,CRNDE,OSMR-AS1,SNHG18,AC145343.2,RP11-25K21.6,RP11-38L15.2）with powerful prognostic value were identified to be significantly correlated with PMT.A PMT-related lncRNA risk signature based on these lncRNAs could divide cases into two groups with distinct prognosis,being validated in CGGA dataset.Multivariate Cox regression analyses indicated that the signature was an independent prognostic factor for high-grade glioma.High-risk cases were more likely to be classified as mesenchymal subtype,conferring enhanced immunosuppressive status by recruiting macrophages,neutrophils,and regulatory T cells.Additionally,six lncRNAs of the signature might act as competing endogenous RNAs to promote PMT in glioblastoma.Part 2:Expression level of LINC01503 was related to WHO grade and molecular subtype of glioma.LINC01503 expression was signicantly higher in WHO IV grade samples than WHO III and WHO II samples.We also found that the mesenchymal glioma had a higher LINC01503 expression,whereas the proneural contained a lower LINC01503 expression.In addition,high LNC01503 expression can promote proliferation,migration and invasion of glioma cells.Meanwhile,LINC01503 can promote the proneural–mesenchymal transition by activating the NF-κB pathway in glioblastoma.Furthermore,glioblastoma with high LINC01503 was enriched with abundant immunosuppressive cells such as macrophages,neutrophils and regulatory T cells.Finally,knockdown of LINC01503 could suppress migration of THP-1-derived macrophages.Part 3:RNA sequencing data for 946 glioma samples from The Cancer Genome Atlas and the Chinese Glioma Genome Atlas databases were analyzed to evaluate the prognostic value and function of homeobox A transcript antisense RNA myeloid-specific（HOTAIRM）1.HOTAIRM1 expression was associated with clinical and molecular features of glioma:patients with high HOTAIRM1 expression were more likely to be classified as malignant cases,and elevated HOTAIRM1 level was associated with shorter survival time in subgroups stratified by clinical and molecular features.A multivariate Cox regression analysis showed that HOTAIRM1was an independent prognostic factor for patient outcome.In vitro experiments revealed that HOTAIRM1 knockdown suppressed the malignant behavior of glioma and increased tumor sensitivity to temozolomide.The results of an in silico analysis indicated that HOTAIRM1 promotes the malignancy of glioma by acting as a sponge for microRNA（miR）-129-5p and miR-495-3p.HOTAIRM1 overexpression was also associated with immune activation characterized by enhanced T cell-mediated immune and inflammatory responses.4.ConclusionPart 1:We profiled the PMT status in glioma and established a PMT-related ten-lncRNA signature for glioma,which could independently predict glioma survival and trigger PMT to enhance immunosuppression.Part 2:LINC01503 could promote proliferation,migration and invasion of glioma cells.Meanwhile,high LINC01503 expression could enhance proneural-to-mesenchymal transition by activating NF-κB pathway,and then recruit more macrophages to enhance the immunosuppressive status in gliblastoma.Part 3:We found that HOTAIRM1 could serve as a clinical signature in glioma.It has great influences on facilitating malignant behavior,immune response and inflammatory response,indicating its potential as therapeutic target for glioma patients... |
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