State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin, China.
Methods Mol Biol. 2021;2248:1-18. doi: 10.1007/978-1-0716-1130-2_1.
Vascular endothelial growth factor (VEGF) plays a pivotal role in promoting neovascularization. Tumor necrosis factor superfamily 15 (TNFSF15) is an antiangiogenic cytokine prominently produced by endothelial cells in a normal vasculature. In this study, Western blot, quantitative polymerase chain reaction (qPCR), and dual luciferase reporter gene assay were used to validate the mechanisms of TNFSF15-mediated suppression of VEGF production in endothelial cells. We report that TNFSF15 inhibits VEGF production via microRNA-29b (miR-29b) targeting the 3'-UTR of VEGF transcript in mouse endothelial cell line bEnd.3. Neutralizing antibody against TNFSF15, 4-3H, inhibits the level of miR-29b and reinvigorates VEGF. In addition, TNFSF15 activates the JNK signaling pathway as well as the transcription factor GATA3, resulting in enhanced miR-29b production. SP600125, an inhibitor of JNK, eradicates TNFSF15-induced GATA3 expression. Moreover, GATA3 siRNA suppressed TNFSF15-induced miR-29b expression. Together, this study provides evidence and method of activation of the JNK-GATA3 signaling pathway by TNFSF15 that suppresses VEGF gene expression, which gives rise to upregulation of miR-29b.
血管内皮生长因子（VEGF）在促进新血管生成中起着关键作用。肿瘤坏死因子超家族 15（TNFSF15）是一种抗血管生成细胞因子，主要由正常血管内皮细胞产生。在这项研究中，我们使用 Western blot、定量聚合酶链反应（qPCR）和双荧光素酶报告基因检测来验证 TNFSF15 介导的内皮细胞中 VEGF 产生抑制的机制。我们报告称，TNFSF15 通过靶向 VEGF 转录本 3'-UTR 的 microRNA-29b（miR-29b）抑制 VEGF 的产生。抗 TNFSF15 的中和抗体 4-3H 抑制 miR-29b 的水平并重新激活 VEGF。此外，TNFSF15 激活 JNK 信号通路以及转录因子 GATA3，导致 miR-29b 的产生增加。JNK 的抑制剂 SP600125 消除了 TNFSF15 诱导的 GATA3 表达。此外，GATA3 siRNA 抑制了 TNFSF15 诱导的 miR-29b 表达。总之，这项研究提供了证据和方法，证明了 TNFSF15 通过激活 JNK-GATA3 信号通路抑制 VEGF 基因表达，从而导致 miR-29b 的上调。
