血管生成拟态(VM)作为血管样通道,为肿瘤生长提供重要物质,是胶质母细胞瘤(GBM)耐药性的主要因素。人抗原R(HuR)-一种mRNA结合蛋白-在GBM中高度表达,与肿瘤进展密切相关,并被认为是潜在的药物靶标。尽管一些小分子化合物已被鉴定为破坏HuR与靶mRNA的结合,它们仍处于临床前研究阶段,这表明需要进一步验证和开发HuR抑制剂。在我们的研究中,我们旨在筛选潜在的HuR抑制剂,并研究其在GBM中的疗效和分子机制。我们采用荧光偏振方法从天然化合物库中鉴定HuR抑制剂,证实了胡桃醌有效抑制HuR与AREVegf-a结合的功效。通过电泳迁移率分析,在蛋白质水平上进一步验证胡桃醌与HuR的结合,表面等离子体共振,和分子对接。此外,胡桃醌在体外和体内表现出对神经胶质瘤生长和VM形成的抑制作用。此外,观察到胡桃醌通过抑制VEGF-A/VEGFR2/AKT/SNAIL信号通路逆转上皮-间质转化。最后,我们通过HuR敲低建立了胡桃醌靶向U251细胞HuR的能力,mRNA稳定性,和细胞热转移测定。因此,这项研究确定胡桃醌是一种新型的HuR抑制剂,通过靶向HuR,有可能成为GBM抗VM治疗的先导化合物。缩写:AKT,蛋白激酶B;ARE,富含腺嘌呤和尿苷的元素;CETSA,细胞热转移测定;DMEM,Dulbecco's改良Eagle's培养基;ELISA,酶联免疫吸附测定;EMSA,电泳迁移率变动分析;EMT,上皮间质转化;FP,荧光偏振;GBM,胶质母细胞瘤;HTS,高通量筛选;HuR,人抗原R;IF,免疫荧光;PAS,高碘酸-希夫;PI3K,磷酸肌醇-3激酶;qRT-PCR,定量实时PCR;RRM,RNA识别基序;SPR,表面等离子体共振。TMZ,替莫唑胺;VM,血管生成拟态;VEGF-A,血管内皮生长因子-A;VEGFR2,血管内皮生长因子受体-2。
Vasculogenic mimicry (VM) serves as a vascular-like channel that provides important substances for tumor growth and is a primary factor in glioblastoma (GBM) drug resistance. Human Antigen R (HuR)-an mRNA-binding protein-is highly expressed in GBM, closely related to tumor progression, and deemed a potential drug target. Although some small-molecule compounds have been identified to disrupt HuR binding to target mRNA, they remain in the preclinical research stage, suggesting the need for further validation and development of HuR inhibitors. In our study, we aim to screen for potential HuR inhibitors and investigate their efficacy and molecular mechanisms in GBM. We employed the fluorescence polarization method to identify HuR inhibitors from a natural compound library, confirming the efficacy of juglone in effectively inhibiting the binding of HuR to AREVegf-a. Further validation of the binding of juglone to HuR at the protein level was conducted through electrophoretic mobility shift analysis, surface plasmon resonance, and molecular docking. Furthermore, juglone demonstrated inhibitory effects on glioma growth and VM formation in vitro and in vivo. Moreover, it was observed that juglone reversed epithelial-mesenchymal transition by inhibiting the VEGF-A/VEGFR2/AKT/SNAIL signaling pathway. Finally, we established the capability of juglone to target HuR in U251 cells through HuR knockdown, mRNA stability, and cell thermal shift assays. Therefore, this study identifies juglone as a novel HuR inhibitor, potentially offering promise as a lead compound for anti-VM therapy in GBM by targeting HuR. Abbreviations: AKT, protein kinase B; ARE, adenine-and uridine-rich elements; CETSA, cellular thermal shift assay; DMEM, Dulbecco\'s modified Eagle\'s medium; ELISA, enzyme linked immune sorbent assay; EMSA, electrophoretic mobility shift assay; EMT, epithelial mesenchymal transition; FP, fluorescence polarization; GBM, glioblastoma; HTS, high-throughput screening; HuR, human antigen R; IF, Immunofluorescence; PAS, periodic acid-Schiff; PI3K, phosphoinositide-3 kinase; qRT-PCR, quantitative real-time PCR; RRMs, RNA recognition motifs; SPR, surface plasmon resonance. TMZ, temozolomide; VM, vasculogenic mimicry; VEGF-A, Vascular endothelial growth factor-A; VEGFR2, Vascular endothelial growth factor receptor-2.