Inhibition of COX-2, mPGES-1 and CYP4A by isoliquiritigenin blocks the angiogenic Akt signaling in glioma through ceRNA effect of miR-194-5p and lncRNA NEAT1
Inhibition of COX-2, mPGES-1 and CYP4A by isoliquiritigenin blocks the angiogenic Akt signaling in glioma through ceRNA effect of miR-194-5p and lncRNA NEAT1
复制标题
异甘草素对 COX-2、mPGES-1 和 CYP4A 的抑制作用通过 miR-194-5p 和 lncRNA NEAT1 的 ceRNA 效应阻断神经胶质瘤中的血管生成 Akt 信号传导
DOI：
10.1186/s13046-019-1361-2
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发表时间：
2019-08-22
影响因子：
11.3
通讯作者：
Yang，Jing
中科院分区：
文献类型：
--
作者：
Wang，Chenlong;Chen，Yaxin;Yang，Jing
研究背景花生四烯酸（AA）代谢酶包括环氧合酶-2（考克斯-2）、微粒体前列腺素E合成酶-1（mPGES-1）和细胞色素P450（CYP）4A 11在胶质瘤血管生成中起重要作用。因此，迫切需要确定潜在的机制，并制定战略，以克服themes.MethodsA同源性模型的人CYP 4A 11使用SYBYL-X 2.0构建。使用SYBYL套件的Surflex-Dock对考克斯-2、mPGES-1和CYP 4A 11进行基于结构的虚拟筛选。在斑马鱼胚胎和兔角膜血管生成模型中进一步评估候选物的抗血管生成活性。激光多普勒分析用于测量肿瘤灌注。免疫荧光法检测CD 31和α-SMA的表达。Western blot检测HIF-1、Akt和p-Akt的表达。使用qPCR测定FGF-2、G-CSF、PDGF、TGF-β、Tie-2、VEGF、lncRNA NEAT 1和miR-194- 5 p的基因表达。采用ELISA法检测FGF-2、TGF-β和VEGF的表达。结果利用考克斯-2、mPGES-1和CYP 4A 11三维模型对中药数据库中近36 043个化合物进行筛选，鉴定出17个黄酮类化合物。在斑马鱼筛选中，异甘草素（ISL）表现出最有效的抗血管生成活性，EC 50值为5.9 μM。相反，ISL在斑马鱼和兔角膜模型中的抗血管生成作用部分被20-羟基二十碳四烯酸（20-HETE）或前列腺素E2（PGE 2）逆转。ISL使胶质瘤血管系统正常化，并提高了替莫唑胺治疗在大鼠C6胶质瘤模型中的疗效。ISL抑制考克斯-2、mPGES-1和CYP 4A可降低C6和U87胶质瘤细胞中FGF-2、TGF-β和VEGF的产生，并伴有p-Akt下调，而Akt过表达可逆转上述作用。此外，ISL在U87胶质瘤细胞中下调lncRNA NEAT 1，但上调miR-194- 5 p。重要的是，lncRNA NEAT 1过表达逆转了ISL介导的miR-194- 5 p表达增加，从而减弱了FGF-2、TGF-β和VEGF的产生。结论类黄酮ISL重编程考克斯-2、mPGES-1和CYP 4A介导的胶质瘤AA代谢通过miR-194- 5 p和lncRNA NEAT 1的ceRNA效应抑制血管生成Akt-FGF-2/TGF-β/VEGF信号传导，有望成为治疗人脑胶质瘤的新策略。
BackgroundArachidonic acid (AA) metabolic enzymes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1) and cytochrome P450 (CYP) 4A11 play important roles in glioma angiogenesis. Thus, there is an urgent need to identify the underlying mechanisms and develop strategies to overcome them.MethodsA homology model of human CYP4A11 was constructed using SYBYL-X 2.0. Structure-based virtual screening against COX-2, mPGES-1 and CYP4A11was performed using the Surflex-Dock of the SYBYL suite. The candidates were further evaluated their antiangiogenic activities in a zebrafish embryo and rabbit corneal angiogenesis model. Laser doppler analysis was used to measure tumor perfusion. The expression of CD31 and α-SMA was measured by immunofluorescence. Western blot was used to measure the expression of HIF-1, Akt and p-Akt. The gene expression of FGF-2, G-CSF, PDGF, TGF-β, Tie-2, VEGF, lncRNA NEAT1 and miR-194-5p were determined using qPCR. The production of FGF-2, TGF-β and VEGF were analyzed using ELISA. Bioinformatic analysis and luciferase reporter assays confirmed the interaction between lncRNA NEAT1 and miR-194-5p.ResultsThe nearly 36,043 compounds from the Traditional Chinese Medicine (TCM) database were screened against COX-2, mPGES-1 and CYP4A11 3D models, and the 17 top flavonoids were identified. In zebrafish screening, isoliquiritigenin (ISL) exhibited the most potent antiangiogenic activities with the EC50values of 5.9 μM. Conversely, the antiangiogenic effects of ISL in the zebrafish and rabbit corneal models were partly reversed by 20-hydroxyeicosatetraenoic acid (20-HETE) or prostaglandin E2 (PGE2). ISL normalized glioma vasculature and improved the efficacy of temozolomide therapy in the rat C6 glioma model. Inhibition of COX-2, mPGES-1 and CYP4A by ISL decreased FGF-2, TGF-β and VEGF production in the C6 and U87 glioma cells with p-Akt downregulation, which was reversed by Akt overexpression. Furthermore, ISL downregulated lncRNA NEAT1 but upregulated miR-194-5p in the U87 glioma cell. Importantly, lncRNA NEAT1 overexpression reversed ISL-mediated increase in miR-194-5p expression, and thereby attenuated FGF-2, TGF-β and VEGF production.ConclusionsReprogramming COX-2, mPGES-1 and CYP4A mediated-AA metabolism in glioma by flavonoid ISL inhibits the angiogenic Akt- FGF-2/TGF-β/VEGF signaling through ceRNA effect of miR-194-5p and lncRNA NEAT1, and may serve as a novel therapeutic strategy for human glioma.