| Background and objective:Gliomas are the most notorious form of primary brain tumor in the adult central nervous system.Even with aggressive surgery,radiation,and chemotherapy,the five-year survival rate of glioma patients is the most inferior.Moreover,survival time of patients with glioblastoma multiforme(GBM)averages only about 15 months after diagnosis.As secreted amine oxidases,the main function of LOX family members is to covalently cross-link elastin and collagen in the extracellular matrix(ECM),which are indispensable to maintain the structural integrity of many tissues.LOXL2 is believed to perform an analogous function to LOX by promoting the cross-linking of collagen and elastin in the ECM.It has also been confirmed that LOXL2 can regulate signaling pathways inside or outside the cell.Disorders of LOXL2 have been related to a number of diseases,such as fibrosis and heart disease.The expression and function of LOXL2in tumor progression depends on tissue type.Although decreased expression level has been informed in ovarian tumors,LOXL2 is overexpressed and linked with unfavorable outcomes in patients with colon and esophageal tumors,squamous cell carcinoma in oral,head and neck,or laryngeal.In addition,over-expression of LOXL2 has been verified to promote tumor metastasis.However,the function and specific mechanisms of LOXL2 in glioma have not been fully elucidated thus far.TMZ is a first-line medicine used in the treatment of patients with glioma.Theultimate efficacy of TMZ varies according to intrinsic and acquired resistance,which greatly undermines its use in clinical treatment.Therefore,TMZ resistance is a primary handicap to the treatment of glioma,and research on TMZ resistance is vital to restore therapeutic efficacy and relieve the suffering of patients.Autophagy is an extremely conservative mechanism of lysosome-mediated protein and organelle degradation that has an important effect on survival,differentiation,development,and homeostasis.In cancer,autophagy not only suppresses tumorigenesis via its quality control function,it also withstands microenvironmental stress and promotes malignant phenotype.In glioma,autophagy not only reduces TMZ sensitivity,it also modifies other processes including EMT,increasing the degree of malignancy.However,the specific regulatory mechanism of autophagy on TMZ sensitivity and EMT in gliomas has not been fully explained.Here,we first explored the expression and prognostic efficacy of LOXL2 in The Cancer Genome Atlas(TCGA),Chinese Glioma Genome Atlas(CGGA),GSE16011and the Repository for Molecular Brain Neoplasia Data(REMBRANDT),then detected its functions in tumorigenesis,EMT,and sensitivity to TMZ in glioma cells.The results suggested that high expression of LOXL2 is an important cause of glioma pathogenesis and TMZ resistance in glioma cells that might become a new target for glioma treatment.Methods:1.Gene expression analysis using online databasesGene expression data and clinical characteristics(tumor grade,age at diagnosis,Karnofsky Performance Status[KPS],survival time,censored status,and treatment history)from TCGA for patients with GBM and low-grade glioma(LGG)were accessed through the c Bio Portal for Cancer Genomics.Kaplan-Meier curves were generated using Graph Pad Prism 7(Graph Pad Software Inc.,San Diego,CA,USA),comparing overall survival rates between patients with and without the genetic alterations of interest.Survival differences were assessed using the log-rank test.Biospecimen and clinical data were collected and processed as described in TCGA publications.Progression-free survival data regarding GBM were obtained from Affymetrix arrays.mRNA expression data and clinical material from the CGGA database(http://www.cgga.org.cn)were also used in this study.Data from m RNAseq?693 were used for chemotherapy-related survival analyses.Other analyses were implemented using m RNAseq?325 data.GSE16011 gene expression data and clinical information were obtained through the Gene Expression Omnibus(GEO)database.Raw data were processed using the affy package,and the robust multi-array analysis(RMA)method was used for background correction and normalization.Biospecimen and clinical data from related research24were used as supplements.The gene expression data and clinical information from REMBRANDT were downloaded from the Glio Vis portal(http://gliovis.bioinfo.cnio.es).GSE43107 patient samples were collected in a Phase III randomized clinical trial,EORTC26951,that investigated adjuvant procarbazine,CCNU(lomustine),and vincristine(PCV)chemotherapy in anaplastic oligodendroglial tumors25.A total of 140patients out of the original 368 underwent expression profiling.Of these,45 had profiling from HU133plus 2.0 arrays and 95 from exon arrays(Hu Ex?1.0?st arrays).Because of the significant differences between these two platforms,we included samples only from exon arrays.Raw gene expression data and clinical information were obtained through the GEO database.Raw data were processed using the Oligo package,and only the core probe set was analyzed for gene expression.The RMA method was used for background correction and normalization.Gene expression was summarized with a Brain Array custom CDF file.2.Specimens and patient dataThe protocol for this study was approved by the Medical Ethics Committee of the Medical Ethics Committee of the First Affiliated Hospital of China Medical University.Written informed consent was obtained from all patients.Fifty-six clinical samples from glioma patients were collected from 2016 to 2018 in the Medical Ethics Committee of the First Affiliated Hospital of China Medical University.Samples from six additional patients without any previous neurological diseases,who suffered severe brain trauma and underwent surgery immediately thereafter were collected for the control group during the same period.All enrolled patients underwent curative surgical resection without prior chemotherapy or radiation therapy.3.Cell culture and reagentsNormal human astrocyte cell line(NHA)and the human malignant glioma cell lines,U373,U251,and U87 were obtained from the Chinese Academy of Sciences Cell Bank(Shanghai,China).T98 was purchased from the American Type Culture Collection(ATCC,Manassas,VA,USA).LN229 cells were provided by Professor Tao Jiang(Department of Molecular Neuropathology,Beijing Neurosurgical Institute).All of the cell lines were maintained as monolayer cultures in Dulbecco's Modified Eagle's Medium(DMEM;Hyclone,Logan,UT,USA)supplemented with 10%fetal bovine serum(FBS),penicillin(100 U/m L),and streptomycin(100 U/m L)and incubated at37°C in a humidified atmosphere with 5%carbon dioxide.4.Quantitative real-time PCRTotal RNA was isolated using TRIzol Reagent(Invitrogen,Waltham,MA,USA)according to the manufacturer's protocol.Prime-Script RT Master Mix(Takara Bio Inc.,Shiga,Japan)was used to synthesize first-strand c DNA,followed by q PCR detection(PCR Light Cycler(?)480;Roche Diagnostics Ltd.,Basel,Switzerland)using SYBR Green Master Mix(Ta Kara).Each sample was tested in triplicate.Melting curve analysis of each sample was used to assess amplification specificity.Relative gene expression levels were calculated using the 2-??Ct method.5.Migration and invasion assaysThe migratory ability of cells was determined via wound healing assays.The rate of wound closure was monitored at indicated time points under a microscope and quantified using Image J(National Institutes of Health,Bethesda,Maryland,USA).Invasion potential was determined using collagen-coated transwell assays(Corning,8?m;Corning,NY,USA).DMEM containing 0.1%FBS was added to the upper wells and DMEM containing 10%FBS was added as a chemoattractant to the lower wells.Cells were allowed to invade Matrigel-coated filters in the lower compartment for 20?h at 37°C.Cells that reached the lower surface of the filter were fixed,stained,and counted using a microscope.A total of 10 fields were counted for each transwell filter.6.Western blot assaysCells were harvested and lysed with a protein extraction agent(Beyotime,Beijing,China).Total protein was quantified using the bicinchoninic acid(BCA)method.In each lane,25–50?g protein per sample was loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and transferred onto PVDF membranes(0.45?m,Millipore).After incubation with 5%skim milk blocking solution for 1 h,the membranes were incubated with the indicated antibodies at 4°C for 16 h.The membranes were then incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies at 20°C for 1 h.Immunoreactive proteins were visualized and quantified using an enhanced chemiluminescence reagent(Beyotime)with a Chemi Doc?Touch detection system(Bio-Rad Laboratories,Hercules,CA,USA)and Image J software.7.Colony formation,cell proliferation,and cytotoxicity assaysCells were seeded into 6-well plates(1000 per dish)and incubated for two weeks.Plates were washed with phosphate-buffered saline(PBS)and stained with crystal violet staining solution before the number of colonies with>50 cells were counted.Cell proliferation and cytotoxicity were assessed using Cell Titer 96(?)AQueous Non-Radioactive Cell Proliferation Assay kits(Promega,Madison,WI,USA)according to the manufacturer's instructions.Cells were seeded into five 96-well plates at 1,000cells/100?L per well and evaluated daily for 5 d.For cytotoxicity assays,5,000cells/100?L per well were seeded and cultured for three days.Absorbance was measured at 490 nm using a microplate reader.Cell viability was calculated as the percentage of the optical density value of the control group for each time point.8.Transmission electron microscopyCells were fixed for 2 h in 2%glutaraldehyde,post-fixed in 1%osmium tetroxide,dehydrated in graded ethanol,embedded in epoxy resin,cut into 50-nm ultrathin sections,stained with lead citrate and viewed using an H-7650 transmission electron microscope(Hitachi,Japan).9.Apoptosis assayApoptosis was determined using an Annexin V-PE/7-AAD double staining apoptosis detection kit(BD Pharmingen Inc.,San Diego,CA,USA)according to the protocol provided.In brief,3×105 cells were harvested,washed twice with cold PBS,and resuspended in 1×binding buffer.Aliquots of 105 cells were stained with 5?L of Annexin V/PE and 10?L of 7ADD.Stained cells were analyzed via flow cytometry.10.RNA interference,plasmid construction and LentivirusesSmall interfering RNAs(si RNAs)that specifically target human LOXL2,ERK1/2,and ATG7 were obtained from Sangon Biotech(Shanghai,China).The LOLX2expression plasmid was obtained from Gene Chem(Shanghai,China)with the corresponding empty vector(EV)as the control.Cells were cultured on 6-well plates to confluency and transfected with si RNAs,plasmid,or negative control using Lipofectamine 3000 reagent(Invitrogen)according to the manufacturer's instructions.Lentivirus carrying the LOXL2 si RNA sequence was obtained from Genechem.Puromycin(10 g/ml)was applied to screen out the stable transfected cells.11.Statistical analysisAll statistical analyses were performed using Graph Pad Prism version 6.0(Graph Pad Software,San Diego,CA,USA)and SPSS version 16.0(SPSS Inc.,Chicago,IL,USA).Data are presented as the mean±SEM or SD.Where applicable,one-way ANOVA,two tailed t-tests,and log-rank tests were applied.Survival distributions were estimated via the Kaplan-Meier method.Unless otherwise stated,P<0.05 was considered statistically significant.Results:1.According to the public databases,the expression of LOXL2 in glioma tissue is significantly higher than that in non-tumor tissues.Moreover,LOXL2 expression was significantly related to glioma grade,IDH mutation status,and glioma subtype.In addition,the expression of LOXL2 is also related to the prognosis of glioma.The higher the expression of LOXL2,the worse the prognosis of patients.2.In glioma cell lines,the expression of LOXL2 is significantly higher than that of normal stellate cells;in addition,the expression level of LXOL2 is also related to the prognosis of chemotherapy.LOXL2 became an independent prognostic factor for gliomas.3.LOXL2 can promote glioma proliferation,migration,invasion and EMT process,and promote TMZ tolerance of glioma cells3.1.Knockout of LOXL2 can significantly inhibit the proliferation,migration and invasion of glioma cells,affect the expression of EMT-related proteins,and enhance the sensitivity of glioma cells to TMZ;3.2.Overexpression of LOXL2 can significantly promote the proliferation,migration,and invasion of glioma cells.The expression of EMT-related proteins has an opposite trend to that of knockout LOXL2,and the sensitivity of glioma cells to TMZ is also reduced.4.LOXL2 can promote the activation of autophagy in glioma cells.Overexpression of LOXL2 can promote autophagy.Conversely,knockout of LOXL2 can inhibit autophagy.In addition,overexpression of LOXL2 and treatment of glioma cells with autophagy inhibitor chloroquine can reverse Regulation of LOXL2 on EMT and TMZ sensitivity5.LOXL2 regulates autophagy by regulating the expression of ATG76.LOXL2 promotes the expression of ATG7 by promoting the phosphorylation of ERK1/2,thereby regulating autophagy.7.LOXL2 silencing increased the sensitivity of glioma cells to TMZ in vivo.Conclusion:As an independent prognostic factor of glioma,LOXL2 has a regulatory effect on glioma proliferation,migration,invasion,EMT and chemotherapy sensitivity.The above effects of LOXL2 are mainly achieved by ERK1/2-ATG7 regulating the autophagy process of glioma cells.LOXL2 may become a potential target for glioma treatment. |
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