The Mechanism Of LncRNA HOTAIR Promotes Temozolomide Resistance In Glioma

Objective:Temozolomide（TMZ）is a first-line chemotherapy agent in the standard treatment strategy for newly diagnosed glioblastoma（GBM）patients.Acquired TMZ resistance in glioma patients is a major cause of treatment failure.Lnc RNA HOTAIR has been confirmed to play a role in epithelial-mesenchymal transition,angiogenesis to participate in the occurrence and development of glioma.However,the research of HOTAIR on GBM TMZ resistance and its mechanism is insufficient.The aim of this study is to explore not only the clinical prognostic potential of HOTAIR but also the relationship and molecular mechanism of HOTAIR and TMZ resistance.Methods:Transcriptome data on TMZ-resistant and TMZ-sensitive GBM cells,as well as transcriptome data from three pairs of primary and recurrent GBM patients samples（GSE100736,GSE113510,and GSE7697）,were collected to identify differentially expressed lnc RNAs.The patients in the TCGA,CGGA and GSE16011 datasets were reclassified according to the fifth edition of World Health Organization（WHO）central nervous system tumor classification guidelines.The relationship between HOTAIR expression and the clinical and molecular pathological features of glioma patients was explored.The TMZ-resistant cell line U251R was constructed,and the expression of HOTAIR was compared with the parent cell line U251.The transfection efficiency was verified by transfection of HOTAIR overexpression plasmid in U251 cells and si-HOTAIR in U251R cells,q PCR to verify transfection efficiency,and to detect changes in cell IC50,cell cycle and apoptosis after TMZ treatment.The correlation between HOTAIR and MGMT expression in glioma patients was analyzed,and the expression and regulation relationship of HOTAIR to MGMT was further verified in cell experiments.The gene set variation analysis（GSVA）method was used to predict the associated functional characteristics and validated by western blotting.In U251R cells,the relevant pathway modulator and TMZ were combined to detect the changes in cell cycle and apoptosis,and to validate the molecular mechanism of HOTAIR leading to TMZ resistance.Besides,to investigate the mechanism of the activatedβ-catenin/MGMT and HOTAIR,the western blotting after transfection of mi R-214-3p mimic in U251R cells was examined to detect the protein changes ofβ-catenin and MGMT.Finally,bioinformatics technology was used to screen out potential therapeutic drugs for high HOTAIR expression GBM patients,and further verified in cell experiments.Results:1.HOTAIR was significantly up-regulated in the TMZ-resistance GBM cells and recurrent GBM tissues by bioinformatics methods.GBM patients exhibited a significantly higher expression than those with lower-grade gliomas（Grade 2 and 3）.Moreover,survival analysis showed that patients with high HOTAIR expression had a poor prognosis and worse reactions on TMZ treatment.2.U251R cells exhibited a significantly higher expression of HOTAIR than the parental U251 cells.Silencing HOTAIR in U251R cells(U251R^si-HOTAIR)increased TMZ sensitivity in comparison to control si RNA,demonstrated by a decreased IC50 and an increased proportion of G2/M arrest and apoptosis after TMZ treatment.It indicates that HOTAIR silencing could partially restore the sensitivity of TMZ.3.The U251 cells overexpressing HOTAIR(U251^HOTAIR)exhibited a higher IC50 than those transfected with vector and caused a significant decrease proportion of G2/M cell cycle arrest and cell apoptosis after TMZ treatment,suggesting that increased HOTAIR expression confers TMZ resistance in U251 cells.4.A positive correlation between HOTAIR and MGMT m RNA expression was analysed in TCGA,CGGA and GSE16011 cohorts by Spearman correlation analysis.The expression of MGMT at the m RNA and protein levels in U251R^si-HOTAIR cells was significantly reduced,whereas increased in the U251^HOTAIR cells,indicating that HOTAIR may have a positive regulatory effect on the expression of MGMT.5.The Wnt/β-catenin signaling pathway was enriched in high HOTAIR expression group through GSVA pathway enrichment analysis.Compared with the control group,theβ-catenin expression levels of total protein as well as in the cytoplasmic and nuclear fractions in U251R^si-HOTAIR cells were significantly reduced,whereas increased in U251^HOTAIR cells,indicating thatβ-catenin is a downstream effector of HOTAIR.6.Treatment ofβ-catenin agonist SKL2001 reduced the sensitivity to TMZ and attenuated TMZ-induced G2/M cell cycle arrest and cell apoptosis in U251R^si-HOTAIRcells,but it induced an increase in the protein levels of MGMT,indicating that HOTAIR modulates TMZ resistance viaβ-catenin/MGMT pathway.7.It was found that mi R-214 had binding sites with HOTAIR andβ-catenin through website predictive analysis.U251R cells exhibited a lower level of mi R-214 compared to U251 cells.Silencing HOTAIR in U251R cells increased mi R-214 expression,whereas overexpressing HOTAIR in U251 cells decreased mi R-214 expression.In U251R cells,mi R-214-3p mimic not only increased the sensitivity to TMZ treatment but also decreased intracellularβ-catenin and MGMT,indicating the involvement of mi R-214 in HOTAIR-mediated TMZ resistance.8.The 8 potential therapeutic compounds were screened out for those high HOTAIR expressions GBM patients by bioinformatics analysis.An FDA-approved cancer treatment drug,methotrexate（MTX）,was selected for further verification due to its ability to cross the blood-brain barrier.MTX played a cytotoxic effect on U251R cells and reduce the expression of HOTAIR andβ-catenin in U251R cells.It suggests that MTX combined with TMZ may be an effective treatment strategy for patients with HOTAIR-high expression glioblastoma.Conclusion:1.HOTAIR was up-regulated in TMZ-resistant GBM cells,and patients with high HOTAIR expression had a poor prognosis and a poor response to TMZ treatment,suggesting that HOTAIR can be used as one of the biological markers of GBM patients.2.HOTAIR confers TMZ resistance in GBM cells via mi R-214-3p/β-catenin/MGMT axis.3.For TMZ-resistant GBM patients with high expression of HOTAIR,it is recommended to combine MTX treatment,MTX could reduce the expression of HOTAIR andβ-catenin to enhance the drug sensitivity of GBM cells to TMZ.