ZEB2通过CYLD/FASN轴重编程脂质代谢促进胶质母细胞瘤进展

doi:10.12122/j.issn.1673-4254.2026.06.09

摘要/Abstract

摘要：

目的系统评估ZEB2对胶质母细胞瘤代谢网络的调控特征及其潜在分子机制。方法在LN-229细胞及GBM007细胞中构建ZEB2敲低稳转株（shZEB2#1、shZEB2#2）及阴性对照（shNC），建立小鼠颅内移植瘤模型评估肿瘤生长与小鼠生存期。体外采用Nile Red染色与透射电镜观察并定量脂滴，开展脂质组学分析差异脂质类别构成，并以荧光漂白恢复（FRAP）检测膜流动性。机制方面，基于ZEB2敲低RNA测序（RNA-seq）差异基因，结合FASN免疫沉淀-质谱结果及BioGRID数据库中FASN互作蛋白信息（IgG对照）进行交集筛选获得候选分子并以Western blotting验证；采用启动子荧光素酶报告、ChIP-qPCR及启动子突变实验验证ZEB2对去泛素化酶（CYLD）的转录调控；通过 siCYLD、CYLD 回补、免疫共沉淀、蛋白降解通路干预及泛素化检测，评估 CYLD 对 FASN 蛋白稳定性及脂滴表型的调控作用。结果与shNC组相比，shZEB2组小鼠颅内肿瘤生长受抑制，生存期延长（P<0.05）。RNA-seq及通路富集分析提示差异基因富集于脂质代谢相关通路。shZEB2组细胞内脂滴相对面积分数降低（P<0.001）。ZEB2敲低组中饱和脂肪酸相关储存脂质减少，而含多不饱和脂肪酸链的磷脂分子增加。FRAP实验显示ZEB2敲低组膜荧光恢复率升高（P<0.0001），ZEB2敲低下调脂肪酸合成关键酶FASN，过表达FASN可逆转ZEB2敲低导致的脂滴减少。CYLD是潜在的关键中间分子；实验证实ZEB2可直接结合并激活CYLD启动子（P<0.0001）。敲低CYLD显著降低FASN蛋白水平，且过表达CYLD可逆转shZEB2引起的FASN下调及脂滴减少表型。CYLD 与 FASN 存在共定位并可形成蛋白复合物；MG132 可部分恢复 ZEB2 敲低背景下 FASN 蛋白水平，且 CYLD 过表达降低 FASN 泛素化水平，提示 CYLD 至少部分通过抑制泛素化相关降解维持 FASN 蛋白稳定性。结论 ZEB2敲低可抑制GBM颅内肿瘤生长并延长生存，降低脂滴积累并伴随脂质谱重排及膜流动性增加。ZEB2-CYLD-FASN 轴通过连接转录调控、蛋白稳态与脂质合成重编程促进 GBM 进展。

关键词: 胶质母细胞瘤, 锌指E盒结合同源盒2, 脂质代谢, 脂肪酸合成酶, 去泛素化酶

Abstract:

Objective To investigate the role of ZEB2 in lipid metabolic reprogramming of glioblastoma and its mechanism for promoting glioblastoma progression. Methods Mouse models bearing orthotopic intracranial xenografts derived from LN-229 and GBM007 cells with stable ZEB2 knockdown were used to assess tumor growth and mouse survival.. Lipid droplet accumulation, lipid composition, and membrane fluidity in the cells with ZEB2 knockdown were examined using Nile Red staining, transmission electron microscopy, untargeted lipidomics, and fluorescence recovery after photobleaching (FRAP). The candidate mediators were screened by integrating RNA sequencing, fatty acid synthase (FASN) immunoprecipitation-mass spectrometry, and BioGRID interaction data. Promoter luciferase assays, ChIP-qPCR, promoter mutagenesis, co-immunoprecipitation, protein degradation pathway inhibition, and ubiquitination assays were performed to investigate the regulatory role of the ZEB2-CYLD-FASN axis. Results ZEB2 knockdown significantly suppressed intracranial glioblastoma growth and prolonged mouse survival. Glioblastoma cells with ZEB2 silencing showed reduced lipid droplet accumulation, decreased saturated fatty acid-associated storage lipids, increased phospholipid species containing polyunsaturated fatty acyl chains, and enhanced membrane fluidity. Mechanistically, ZEB2 knockdown reduced FASN protein abundance, whereas FASN restoration reversed lipid droplet reduction induced by ZEB2 silencing. Multi-omics screening identified CYLD as a key intermediate. ZEB2 was capable of directly binding to and activating the CYLD promoter. CYLD knockdown decreased FASN protein levels, whereas CYLD restoration recovered FASN abundance and lipid droplet formation. CYLD was co-localized and interacted with FASN. MG132 partially restored FASN abundance under ZEB2 knockdown, and CYLD overexpression reduced FASN ubiquitination. Conclusion ZEB2 promotes glioblastoma lipid metabolic reprogramming and tumor progression by transcriptionally activating CYLD, which maintains FASN protein stability at least in part through ubiquitination-associated regulation.

Key words: glioblastoma, zinc finger E-box binding homeobox 2, lipid metabolism, fatty acid synthase, CYLD

陈镇霖, 柴鹏, 毛洋奇, 刘然新, 宋烨. ZEB2通过CYLD/FASN轴重编程脂质代谢促进胶质母细胞瘤进展[J]. 南方医科大学学报, 2026, 46(6): 1290-1300.

Zhenlin CHEN, Peng CHAI, Yangqi MAO, Ranxin LIU, Ye SONG. ZEB2 promotes glioblastoma progression by reprogramming lipid metabolism through the CYLD/FASN axis[J]. Journal of Southern Medical University, 2026, 46(6): 1290-1300.

图/表 6

图1 ZEB2敲低抑制GBM颅内肿瘤生长并提示脂质代谢相关通路改变

Fig.1 ZEB2 knockdown suppresses intracranial glioblastoma (GBM) growth and induces alterations in lipid-metabolism-related pathways. A: HE staining of orthotopic GBM007 tumors (shNC, shZEB2#1, shZEB2#2). B: Tumor burden quantification (% of the ipsilateral hemisphere). C: Kaplan-Meier survival analysis of the mice bearing LN-229 or GBM007 orthotopic xenografts. D: GO biological process analysis of differentially expressed genes (DEGs) from RNA-seq (LN-229; shZEB2 vs shNC). E: Pathway enrichment bubble plot of the DEGs. n=5 for assessing tumor burden, n=8 for mouse survival, and n=3 for RNA-seq analysis. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

图2 ZEB2敲低减少GBM细胞脂滴积累

Fig.2 ZEB2 knockdown reduces lipid droplet (LD) accumulation in GBM cells. A: Nile Red staining of LDs (red) with DAPI counterstaining (blue) in LN-229 and GBM007 cells (shNC, shZEB2#1, shZEB2#2; scale bar=40 μm); B, C: LD area fraction quantification based on Nile Red staining images (LN-229, B; GBM007, C). D: Transmission electron microscopy (TEM) of LD ultrastructure (arrows) in LN-229 and GBM007 cells. E, F: LD area fraction quantification based on TEM images (LN-229, E; GBM007, F). ***P<0.001, ****P<0.0001 (n=5).

图3 ZEB2敲低重塑脂质谱并增加细胞膜流动性

Fig.3 ZEB2 knockdown remodels lipid profiles and increases membrane fluidity of glioblastoma cells. A: Lipidomics summary of differential lipid classes between shZEB2 and shNC groups (n=3). B: representative fluorescence recovery after photobleaching (FRAP) images (pre-bleach, bleach, and recovery at 5 min) in LN-229 cells (Scale bar=40 μm). C: FRAP recovery (%) quantification ( n =5). ****P<0.0001. TG: Triglycerides; SAFA: Saturated fatty acids; PC: Phosphatidylcholine; PE: Phosphatidylethanolamine; PUFA: Polyunsaturated fatty acids.

图4 ZEB2调控FASN蛋白水平并介导脂滴形成

Fig.4 ZEB2 regulates FASN abundance and LD formation. A: Western blotting of ZEB2 and FASN in LN-229 and GBM007 cells (shNC, shZEB2#1, shZEB2#2). B: Western blotting of ZEB2 and FASN in U87 cells transfected with ZEB2-HA or vector. C: Immuno-fluorescence staining of ZEB2 (green) and FASN (red) with DAPI (blue) in LN-229 cells (shNC, shZEB2#1, shZEB2#2; scale bar=40 μm; n=5). D: Integrated fluorescence quantification of FASN in panel C (n=5). E: Nile Red staining of LDs with DAPI in LN-229 and GBM007 cells (shNC, shFASN, shZEB2+Vector, shZEB2+FASNoe; scale bar=40 μm). F, G: LD area fraction quantification in LN-229 (F) and GBM007 (G) cells (n=5). H: Clinical Proteomic Tumor Analysis Consortium (CPTAC) multi-omics profile of FASN (Copy number variation, methylation, RNA and protein) (n=93). ****P<0.0001.

图5 ZEB2转录激活CYLD并参与FASN调控

Fig.5 ZEB2 activates CYLD transcription and links to FASN regulation. A: Intersection of candidates from RNA-seq DEGs, FASN IP-MS, BioGRID interactors and IgG control. B: Correlation analyses in TCGA-GBM (ZEB2 vs CYLD; FASN vs CYLD). C: qRT-PCR analysis of CYLD and FASN mRNA levels after ZEB2 knockdown. D: Western blotting of ZEB2 and CYLD in LN-229 and GBM007 cells (shNC, shZEB2#1, shZEB2#2). E: Schematic illustration of the predicted ZEB2-binding E-box within the CYLD promoter. F: Dual-luciferase reporter assay for CYLD promoter (WT and MUT). G: ChIP-qPCR assay of ZEB2 occupancy at the CYLD promoter (IgG as control). H: Western blotting of CYLD and FASN after siCYLD in GBM007 and LN-229 cells. I: Rescue design assessed by Western blotting (CYLD-Flag or FASN-Flag as indicated). RNA-seq, n=3; qRT-PCR, luciferase and ChIP-qPCR, n=3. ****P<0.0001. IP-MS: Immunoprecipitation-mass spectrometry; ChIP-qPCR: Chromatin immunoprecipitation followed by qPCR.

图6 CYLD 与 FASN 相互作用并影响 FASN 泛素化水平

Fig.6 CYLD interacts with FASN and regulates FASN ubiquitination. A: Immunofluorescence staining of CYLD (green) and FASN (red) with DAPI counterstaining (blue) in LN-229, T98G and GBM007 cells (Scale bar=20 μm); B: Endogenous Co-IP analysis of the interaction between CYLD and FASN in GBM007 and T98G cells. C: Schematic illustration of full-length Flag-FASN and truncated Flag-FASN constructs, including WT, A (1-436 aa), B (437-992 aa), C (993-1151 aa), D (1152-1704 aa) and E (1705-2391 aa). D: Co-IP analysis of CYLD-HA binding to full-length or truncated Flag-FASN constructs. E: structural schematic of FASN. F: Western blotting of FASN and ZEB2 in LN-229 shNC and shZEB2 cells treated with DMSO, 3-MA or MG132. G: Ubiquitination assay of FASN after ZEB2 or CYLD overexpression, including total ubiquitination and K48/K63-linked ubiquitination signals. Co-IP: Co-immunoprecipitation; IP: Immunoprecipitation; IB: Immunoblotting; WCL: Whole-cell lysate; Ub: Ubiquitin; 3-MA: 3-methyladenine.

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