Lang H-L, Hu G-W, Chen Y, Liu Y, Tu W, Lu Y-M, Wu L, Xu G-H
Department of Anesthesiology, The Second Affiliated Hospital of Nanchang University, Nanchang, China.
Eur Rev Med Pharmacol Sci. 2017 Mar;21(5):959-972.
Angiogenesis is a key event in the progression of gliomas, and emerging evidence suggests that exosomes are signaling extracellular organelles that modulate the tumor microenvironment and promote angiogenesis and tumor progression. This study aimed to explore the mechanism by which glioma-derived exosomes affect angiogenesis.
qRT-PCR was used to determine the expression level of linc-POU3F3 in glioma tissue as well as glioma cell lines. Ultrafiltration combined with a purification method was used to isolate exosomes derived from A172 cells (A172-Exo) and linc-POU3F3 shRNA-treated A172 cells (shA172-Exo). Transmission electron microscopy, Western blot and tunable resistive pulse sensing (TRPS) were used to identify exosomes. In vitro migration, proliferation, and tube formation experiments, as well as in vivo CAM assays, were used to analyze the pro-angiogenesis ability of exosomes. qRT-PCR and Western blot were used to identify expression levels of angiogenesis-related genes and proteins in human brain microvascular endothelial cells (HBMECs) after being cultured with exosomes.
The levels of linc-POU3F3 were upregulated in glioma tissue and significantly correlated with the advanced tumor stage. A172 cells exhibited the highest expression level. A172-Exo was similar to shA172-Exo (50-100 nm in diameter) and expressed Alix, Tsg101 and CD9, while the expression level of linc-POU3F3 in A172-Exo was significantly higher than that in shA172-Exo. HBMECs rapidly internalized A172-Exo and shA172-Exo, and the linc-POU3F3 expression level in HBMECs treated with A172-Exo was significantly higher than the level in HBMECs treated with shA172-Exo. A172-Exo exhibited better function in promoting HBMECs migration, proliferation, tubular-like structure formation in vitro and arteriole formation in vivo. The gene and protein expression level of bFGF, bFGFR, VEGFA, and Angio in HBMECs treated with A172-Exo was much higher than that of HBMECs treated with shA172-Exo.
These results indicated that gliomas can induce angiogenesis by secreting exosomes enriched in linc-POU3F3. Exosomes and lncRNA-POU3F3 may, therefore, function as a putative therapeutic target in glioma.
血管生成是胶质瘤进展中的关键事件，新出现的证据表明，外泌体是调节肿瘤微环境、促进血管生成和肿瘤进展的信号细胞外细胞器。本研究旨在探讨胶质瘤来源的外泌体影响血管生成的机制。
采用qRT-PCR法检测胶质瘤组织及胶质瘤细胞系中linc-POU3F3的表达水平。采用超滤结合纯化方法分离A172细胞来源的外泌体（A172-Exo）和经linc-POU3F3 shRNA处理的A172细胞来源的外泌体（shA172-Exo）。采用透射电子显微镜、蛋白质印迹法和可调电阻脉冲传感技术（TRPS）鉴定外泌体。通过体外迁移、增殖、管腔形成实验以及体内鸡胚绒毛尿囊膜实验分析外泌体的促血管生成能力。采用qRT-PCR和蛋白质印迹法鉴定外泌体培养后人脑微血管内皮细胞（HBMECs）中血管生成相关基因和蛋白的表达水平。
linc-POU3F3在胶质瘤组织中表达上调，且与肿瘤晚期显著相关。A172细胞表达水平最高。A172-Exo与shA172-Exo相似（直径50-100nm），表达Alix、Tsg101和CD9，而A172-Exo中linc-POU3F3的表达水平显著高于shA172-Exo。HBMECs能快速摄取A172-Exo和shA172-Exo，用A172-Exo处理的HBMECs中linc-POU3F3的表达水平显著高于用shA172-Exo处理的HBMECs。A172-Exo在促进HBMECs体外迁移、增殖、管状样结构形成及体内小动脉形成方面表现出更好的功能。用A172-Exo处理的HBMECs中bFGF、bFGFR、VEGFA和Angio的基因和蛋白表达水平远高于用shA172-Exo处理的HBMECs。
这些结果表明，胶质瘤可通过分泌富含linc-POU3F3的外泌体诱导血管生成。因此，外泌体和lncRNA-POU3F3可能作为胶质瘤的潜在治疗靶点。
