【作者】 张青；

【导师】 吴安华；

【作者基本信息】 中国医科大学 ， 外科学（神经外科）， 2021， 博士

【摘要】 背景与目的:胶质瘤是成人颅内最常见的原发恶性肿瘤,预后极差。目前,手术结合放化疗的综合治疗方案被广泛运用于临床胶质瘤的治疗,但治疗效果并不理想,接受手术治疗的胶质瘤呈现高复发的现象,而且预后也极差。其中,多形性胶质母细胞瘤（GBM）患者的平均生存期在诊断后仅约15个月。赖氨酰氧化酶（LOX）家族的蛋白质是分泌型的氨基氧化酶,该家族成员的共同功能是能共价交联细胞外基质中的胶原蛋白和弹性蛋白;该项功能使LOX家族蛋白在维持组织结构的完整性方面具有不可替代的作用。虽然赖氨酰氧化酶样蛋白2（Lysyl-oxidase-like-2,LOXL2）也具有上述功能,但其也有其特有的功能。有研究证实了LOXL2能调节细胞外和细胞内细胞信号通路。LOXL2的异常表达或激活与许多疾病有关,包括纤维化和心脏病。LOXL2在肿瘤中的表达水平和功能取决于肿瘤源发组织类型。尽管已有研究报道在卵巢肿瘤中LOXL2呈低表达状态,但是在结肠和食道肿瘤,口腔、喉和头颈鳞状细胞癌中,LOXL2呈高表达状态且与肿瘤患者的不良预后相关。此外,LOXL2还能促进胃癌和乳腺癌细胞的转移。但是,LOXL2基因在胶质瘤中的作用仍不清楚。替莫唑胺（Temozolomide,TMZ）是临床用于治疗胶质瘤的一线药物。TMZ的最终功效根据胶质瘤内在和获得性耐药性而变化。因此,TMZ耐药性是影响TMZ在胶质瘤治疗疗效方面的主要障碍,对TMZ耐药性的研究对于恢复疗效和减轻患者痛苦至关重要。自噬是真核细胞通过溶酶体介导的降解性生物学过程,它具有高度保守性。研究表明自噬在机体的生存,分化,发育等方面发挥着至关重要的作用。在肿瘤中,自噬却具有双重角色。在肿瘤发生的早期阶段,自噬可以通过抑制活性氧（ROS）,DNA损伤,组织损伤,炎症和基因组不稳定性等肿瘤诱发因素来抑制肿瘤的发生;但在成熟的肿瘤中,自噬能通过增强肿瘤细胞对恶劣环境因素的耐受性来增强肿瘤细胞的存活,迁移,侵袭以及对治疗的抵抗性。在胶质瘤中,自噬不仅会降低胶质瘤细胞对TMZ的敏感性,还会改变其他过程来增加恶性表型,例如上皮到间质（样）转变（EMT）。但是,自噬对胶质瘤的EMT过程以及TMZ敏感性的内在具体调控机制目前尚不清楚。在本研究中,我们首先分析了LOXL2在TCGA（The Cancer Genome Atlas,TCGA）,CGGA（Chinese Glioma Genome Atlas,CGGA）,GSE16011和REMBRANDT（the Repository for Molecular Brain Neoplasia Data,REMBRANDT）中的表达和预后意义,然后研究了其在肿瘤发生中的作用以及其对胶质瘤TMZ的耐药性的调控作用。我们的研究结果表明,LOXL2能促进胶质瘤的发生发展并促进胶质瘤对TMZ的抵抗,其可能构成神经胶质瘤患者的新治疗靶点。研究方法:1.生物信息学分析通过c Bio Portal for Cancer Genomics（http://www.cbioportal.org/）获得来自TCGA的GBM和低级别胶质瘤（LGG）患者的基因表达数据和临床相关数据。用Kaplan-Meier生存曲线和对数秩检验评估比较不同患者的总体生存差异。有关GBM的无进展生存数据来自TCGA GBM的Affymetrix阵列数据。CGGA数据库（http://www.cgga.org.cn）的m RNA表达数据和临床资料自官网下载。来自m RNAseq＿693的数据用于化疗相关的生存分析。使用m RNAseq＿325数据进行其他分析。GSE16011基因表达原始数据和病例对应的临床信息是从GEO（Gene Expression Omnibus,GEO）数据库下载而来。其中基因表达的原始数据使用affy R包的多阵列分析（RMA）方法进行背景矫正和归一化处理。从Glio Vis网站（http://gliovis.bioinfo.cnio.es）下载REMBRANDT的基因表达数据和临床信息。GSE43107患者样品是在III期随机临床试验EORTC26951中收集的,该试验研究了间变性少突胶质神经胶质瘤中辅助前卡巴嗪（procarbazine）,CCNU（洛莫司汀）和长春新碱（PCV）化疗的情况。其中,有45个样本来自HU133plus 2.0阵列,而有95个样本来自外显子阵列（Hu Ex＿1.0＿st阵列）。由于这两个平台之间的显着差异,我们仅挑选了来自外显子阵列的样品。通过GEO数据库获得原始基因表达数据和临床信息。使用Oligo R包处理原始数据,RMA方法用于背景校正和标准化。2.临床病理标本本研究方案经中国医科大学伦理委员会批准通过。所有患者均已签署书面知情同意书。从2016年至2018年在中国医科大学附属第一医院收集了56例胶质瘤患者的临床样本。在同一时期,还从另外6名无任何先前神经系统疾病的患者中抽取了样本,这些患者曾遭受严重的脑外伤并立即接受了手术。所有入选患者均接受了根治性手术切除,而没事先接受化疗或放疗。3.细胞培养和试剂人胶质瘤细胞系U373,U251和U87和人正常星形胶质细胞（Normal human Astrocyte,NHA）购自中国科学院细胞库。T98细胞株是从美国模式培养物保藏所（American Type Culture Collection,ATCC,马纳萨斯,弗吉尼亚州,美国）购买。LN229细胞由姜涛（北京神经外科研究所）提供。所有细胞系均在含有10%胎牛血清（FBS）,1%的青霉素（100 U/m L）和链霉素（100 U/m L）的高糖DMEM（Dulbecco’s Modified Eagle’s Medium,DMEM;Hyclone,Logan,UT,USA）培养基中作为单层细胞进行培养,并在37℃,5%二氧化碳的潮湿环境中孵育。4.q-PCR实验TRIzol（Invitrogen,沃尔瑟姆,马萨诸塞州,美国）被用来分离提取总RNA。使用Prime-Script RT Master Mix（Takara Bio Inc.,Shiga,Japan）合成单链c DNA,然后使用SYBR Green Master Mix进行q PCR检测（PCRLight Cycler（?）480;Roche Diagnostics Ltd.,Basel,Switzerland,Ta Kara）。每个样品进行三次重复测试。每个样品的熔解曲线分析用于评估扩增特异性。使用2^-ΔΔCt方法计算相对基因表达水平。5.迁移和侵袭检测采用划痕实验确定胶质瘤细胞的迁移能力,并使用Image J（美国马里兰州贝塞斯达的美国国立卫生研究院）进行定量。使用基质胶包被的transwell测定法（Corning,8μm;Corning,NY,美国）确定侵袭潜能。将含有2%FBS的DMEM细胞悬液添加到上部孔中,并将含有20%FBS的DMEM作为趋化剂添加到下部孔中。孵育20小时待细胞穿过基质胶涂层进入到小室底面后收集小室,固定、染色细胞并计数。6.蛋白质印迹实验收获细胞或组织碎片并用蛋白质提取剂（Beyotime,北京,中国）裂解。使用双辛可宁酸（BCA）方法定量总蛋白。在每个泳道中,每个样品上样25–50μg,用于十二烷基硫酸钠-聚丙烯酰胺凝胶（SDS-PAGE）电泳,并将电泳分离的蛋白转印到PVDF膜上（0.45μm,Millipore）。用TBST配制的5%脱脂奶粉封闭溶液封闭1小时后,将膜与目标蛋白对应的抗体在4℃孵育16小时。一抗孵育完毕后将膜用TBST清洗三遍并在20℃条件下孵育对应的二抗2小时。使用增强的化学发光试剂（Beyotime）和Chemi Doc?Touch检测系统（Bio-Rad Laboratories,Hercules,CA,美国）并用Image J软件对免疫反应蛋白进行定量。7.细胞克隆实验,细胞增殖和细胞毒性测定克隆实验按如下操作进行:将细胞接种到6孔板中（每孔1000个细胞）,孵育约2周。当单个细胞集落显示大于50个细胞时,用磷酸盐缓冲盐水（PBS）洗涤并用结晶紫染色溶液染色。Cell Titer 96（?）AQueous Non-Radioactive cell proliferation assay kit（Promega,麦迪逊,威斯康星州,美国）被用来评估细胞增殖。配制1,000个细胞/100μL的细胞悬液并以每孔100μL的量接种到96孔板中,每天一次,连续检测5天。对于细胞毒性测定,细胞悬液的密度为5,000细胞/100μL,每孔同样100μL,培养三天。使用酶标仪在490 nm处测量吸光度。计算每个时间点对对照组的光密度值的百分比作为细胞活力。8.透射电子显微镜将收集到的细胞浸泡于2%戊二醛中持续2小时以固定细胞。然后将细胞转移到1%的四氧化锇中。经上述处理后细胞会在梯度乙醇中脱水。然后将细胞包埋切片并用柠檬酸铅染色。最后在透射电镜（H-7650,日本日立）下观察。9.凋亡分析使用Annexin V PE/7-AAD双重染色凋亡检测试剂盒（BD Pharmingen Inc.,圣地亚哥,CA,美国）测定细胞凋亡。简而言之,收获3×10⁵个细胞经冷PBS洗涤后悬浮于1×结合缓冲液中。用5μL Annexin V/PE和10μL 7ADD对10⁵个细胞的等分试样进行染色。通过流式细胞仪分析染色的细胞。10.si RNA,质粒和慢病毒的构建特异性靶向人LOXL2,ERK1/2和ATG7的小干扰RNA（si RNA）从Sangon Biotech（中国上海）获得。LOXL2过表达质粒获自Gene Chem（中国上海）,以相应的空载体（EV）作为对照。将细胞在6孔板上培养至80%-90%汇合,并根据制造商的说明使用Lipofectamine 3000试剂（Invitrogen）作为转染试剂,配以si RNA,质粒或阴性对照转染。带有LOXL2 si RNA序列的慢病毒是从Genechem获得的。嘌呤霉素（10μg/ml）用于筛选出稳定转染慢病毒的细胞。11.统计分析所有统计分析均使用Graph Pad Prism 6.0版（Graph Pad Software,美国加利福尼亚州圣地亚哥）和SPSS 16.0版（SPSS Inc.,美国伊利诺伊州芝加哥）进行。数据表示为平均值±SEM或SD。在适用的情况下,采用单向方差分析,双尾t检验和对数秩检验。生存分布通过Kaplan-Meier方法计算。除非另有说明,否则P<0.05被认为具有统计学意义。结果:1.LOXL2在胶质瘤中表达水平显著高于其在非肿瘤脑组织中的表达水平。并且,LOXL2表达与胶质瘤级别、IDH突变情况以及胶质瘤恶性亚型显著相关。此外,LOXL2表达还与胶质瘤预后相关,LOXL2表达越高,患者预后越差;2.在胶质瘤细胞系中,LOXL2表达显著高于人正常星形细胞（NHA）;此外LXOL2的表达水平还与化疗预后相关。3.LOXL2能够促进胶质瘤的增殖、迁移、侵袭以及EMT过程,并促进胶质瘤细胞的TMZ耐受;3.1沉默LOXL2能显著抑制胶质瘤细胞的增殖,迁移和侵袭,并影响EMT相关蛋白的表达,增强胶质瘤细胞对TMZ的敏感型;3.2过表达LOXL2能显著促进胶质瘤细胞的增殖,迁移和侵袭,EMT相关蛋白的表达与敲除LOXL2呈相反趋势,胶质瘤细胞对TMZ的敏感性也降低;4.LOXL2能够促进胶质瘤细胞自噬的活化,过表达LOXL2能促进自噬,相反敲除LOXL2能抑制自噬,此外在过表达LOXL2同时用自噬抑制剂氯喹处理胶质瘤细胞能逆转LOXL2对EMT以及TMZ敏感性的调控作用;5.LOXL2对自噬的调控是通过调控ATG7的表达实现的;6.LOXL2通过促进ERK1/2的磷酸化来促进ATG7的表达,从而对自噬产生调控作用。7.在体实验验证了LOXL2的作用结论:LOXL2作为胶质瘤的预后因素,对胶质瘤的增殖、迁移、侵袭、EMT以及化疗敏感性都存在调控作用。LOXL2产生上述作用主要通过ERK1/2-ATG7调控胶质瘤细胞自噬过程实现。LOXL2可能成为胶质瘤治疗的潜在靶点。更多还原

【Abstract】 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^-ΔΔCt 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,8μm;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,25–50μg protein per sample was loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）and transferred onto PVDF membranes（0.45μm,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/100μL per well and evaluated daily for 5 d.For cytotoxicity assays,5,000cells/100μL 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 5μL of Annexin V/PE and 10μL 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.更多还原