The Regulation Of Malignant Phenotype Of Glioma By Epithelial To Mesenchymal Transformation-related Long Non-coding RNA LINC00339

Objective: Glioma is the most common and most malignant primary brain tumor in adults.Currently,the standardized treatment for glioma is surgical maximal resection combined with chemotherapy and radiotherapy,but the therapeutic effect is not satisfactory.Epithelial to mesenchymal transition(EMT)is the process that epithelial cells lose connection with basal lamina,reduction of intercellular adhesion and increase of migration ability,and transform into mesenchymal cells that characterized by a high level of migratory and invasive ability.Epithelial to mesenchymal transition is an important biological process for glioma cells to obtain migration and invasion ability.Further study of the molecular mechanism of epithelial to mesenchymal transition is beneficial for us to deeply understand the occurrence,development,invasion and migration process of glioma.We used glioma public expression profiling data to analyze and screen long non-coding RNA associated with glioma epithelial to mesenchymal transition and used bioinformatics methods to investigate the application and significance of these long non-coding RNA in glioma subtype classification and prognosis evaluation.LINC00339 was further selected from these long non-coding RNA.LINC00339 is long intergenic non-coding RNA located on chromosome 1p36 and is originally found to be one of the endometriosis risk factors.LINC00339 can affect tumor development through ce RNA networks in some kind of tumors.It has been found that LINC00339 is up-regulated in glioma tissues and cell lines,however the potential clinical significance of abnormal expression and the specific biological mechanism of LINC00339 in glioma are not clear.In this study,we revealed the effects of LINC00339 on the proliferation,invasion,migration,cell cycle and EMT process of glioma cells by bioinformatics analysis and a series of experiments,and then confirmed the role of LINC00339/mi R-377-3p/HOXC6 axis in epithelial to mesenchymal transition of glioma,providing new ideas and methods for glioma treatment.Methods: In this study,the common expression profile data of gliomas in The Cancer Genome Atlas(TCGA)were used to screen out the lnc RNA related to EMT in glioma.Using bioinformatics methods to explore the application and significance of these lnc RNA in the classification of glioma subtypes and clinical prognosis evaluation.Principal component analysis and heat map were performed by R software to evaluate the distribution pattern among the different groups.The EMT-related lnc RNA was divided into two groups by consensus clustering.Heat map,sankey diagram,and stacked diagram were used to evaluate the correlation of clinical indicators and molecular characteristics between the two groups by R software.An EMT-related lnc RNA risk score model was constructed,and then the difference of clinical indicators and molecular characteristics between high and low EMT-related lnc RNA risk scores was assessed.Chi-square test was used to evaluate the relationship between high and low EMT-related lnc RNA risk scores and tumor grade and texture analysis parameters.Kaplan-Meier survival curves were plotted using the R software package to assess differences in survival between high risk score and low risk score in stratified cohorts of overall patient and clinical and molecular characteristics.The function annotation was performed using gene ontology(GO)function enrichment analysis and gene enrichment analysis(GSEA)analysis.The validation was performed using data from the Chinese Glioma Genome Atlas(CGGA)database.Further,LINC00339 was screened out from these EMT-related lnc RNA,and the significance and function of LINC00339 in glioma were evaluated using bioinformatics methods.Principal component analysis,heat map,ROC curve,correlation coefficient matrix diagram,scatter diagram and Kaplan-Meier survival curve were drawn by R software.The function annotation of LINC00339 was performed using gene ontology analysis(GO)and gene set enrichment analysis(GSEA).U87 and U373 glioma cells were selected for experimental verification in the knock-down LINC00339 group and the control group or the overexpressed LINC00339 group and the control group.First,the effect of LINC00339 on the proliferation of glioma cells was detected through CCK-8 assay,EDU assay and colony formation assay.Then the effects of LINC00339 on the migration and invasion of glioma cells were detected by Transwell migration assay and wound healing test.The effect of LINC00339 on cell cycle was observed by flow cytometry.The Western blot was conducted to observe the effect of knockdown or overexpression of LINC00339 on the expression of migration-related proteins and EMT markers.To evaluate the effect of LINC00339 on the tumorigenesis of glioma cells by establishing the nude mouse subcutaneous xenograft model.LINC00339 was predicted to bind to mi R-377-3p by website prediction and literature review.The binding of LINC00339 to mi R-377-3p was confirmed by luciferase reporter assay.q RT-PCR experiments verified that the relative expression of mi R-377-3p increased in the LINC00339 knockdown group.The rescue assay was performed by wound healing assay,Transwell assay and Western blot and showed the absence of the effects of LINC00339 knockdown was reduced after mi R-377-3p inhibitor added.As a result,LINC00339 promoted the migration,invasion and EMT process of glioma by regulating mi R-377-3p.HOXC6 was a direct and functional target of mi R-377-3p in glioma cells through website prediction and literature review.The expression of HOXC6 at both RNA and protein levels was determined to be regulated by LINC00339/mi R-377-3p using q RT-PCR and western blot.HOXC6 kowndown reversed such adverse effects by mi R-377-3p inhibitor.Patients in the TCGA and CGGA databases were divided into the LINC00339high-expression and HOXC6 high-expression groups or the other groups,and the survival effects between the above groups in patients,especially in patients after chemotherapy or radiotherapy,were analyzed using multivariate COX analysis and Kaplan-Meier survival curve analysis.Results:1.In this study,EMT-related lnc RNA were screened out from TCGA database,glioma patients were classified according to EMT-related lnc RNA expression and it was found that Cluster1 had differential expression and poor prognosis in different molecular subtypes of glioma.Further,an EMT-related lnc RNA risk score model was constructed,it was found to be an independent risk factor for glioma and was correlated with clinical characteristics,molecular characteristics,and magnetic resonance texture analysis characteristics.EMT-related lnc RNA and the risk score could be used as prognostic predictors of survival in glioma.2.Analysis of public database data revealed that the expression level of LINC00339 was positively correlated with the malignant progression of glioma and correlated with the poor prognosis of glioma.Then functional analysis predicted that LINC00339 could regulate glioma progression through the EMT process in glioma.3.Knockdown LINC00339 expression can reduce the proliferation of glioma cells;arrest the cell cycle in G0/G1 phase;reduce the invasion and migration of glioma cells,and reduce the expression of migration and EMT-related proteins;a subcutaneous xenograft model revealed that silencing of LINC00339 decreased the tumorigenic ability of glioma cells;overexpress LINC00339 exoression can increase the migration and invasion of glioma cells and increase the expression of migration and EMT-related proteins.4.After luciferase reporter assay revealed that LINC00339 regulated the above effects via binding to mi R-377-3p,the rescue assay was perfected to verify that the LINC00339/mi R-377-3p axis affected the migration,invasion and EMT process of glioma cells.5.The target gene of mi R-377-3p was found to be HOXC6 by website prediction and literature review,and improved rescue experiments revealed that the mi R-377-3p/HOXC6 axis affected the migration,invasion and EMT process of glioma cells.6.Survival curve and COX analysis revealed that high expression of LINC00339/HOXC6 indicated poor prognosis of glioma patients,and reduced the efficacy of radiotherapy and chemotherapy.Conclusion:1.Epithelial-mesenchymal transition associated lnc RNA risk score is an independent risk factor for glioma.2.High LINC00339 expression is associated with malignant progression and poor prognosis of gliomas.3.LINC00339 is able to arrest the cell cycle and promote the proliferation,invasion,migration,and EMT of glioma cells.4.LINC00339 regulates the expression of HOXC6 by binding mi R-377-3p thereby affecting the malignant behavior of gliomas.5.The simultaneous high expression of both LINC00339 and HOXC6 indicates a poor prognosis for glioma patients,and reduces the efficacy of radiotherapy and chemotherapy,which can be used as a potential therapeutic target for glioma.