Department of Pancreatic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, China.
Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, China.
Oxid Med Cell Longev. 2022 Oct 15;2022:8445093. doi: 10.1155/2022/8445093. eCollection 2022.
It has been reported that signaling from the nerve growth factor (NGF) pathway associated with peripheral nerves is able to contribute to perineural invasion (PNI) of pancreatic cancer (PC). Nevertheless, the underlying mechanism by which NGF leads to PNI remained poorly understood.
Western blotting was employed to determine NGF level in PC and paracarcinoma tissues and in PC cell lines as well as pancreatic ductal epithelial cells. MiaPaCa-2 and CFPAC-1 cells were treated with 100 ng/ml of NGF or the NGF inhibitor Tanezumab for 24 h, CCK-8 and Transwell assays were employed to test cell proliferation, invasion, and migration, respectively. TrkA expression was knocked down in MiaPaCa-2 and dorsal root ganglion (DRG) cells treated with NGF to determine its effect on the Warburg effect. To reveal that the NGF-TrkA signaling pathway was closely associated with PC PNI, neuroinvasion model was established by using MiaPaCa-2 cells via coculturing DRG cells in Matrigel. Further, exosomes were extracted from PC cells and identified by examining the levels of specific markers for exosomes. Then RT-qPCR was applied to test miR-21-5p level in tumor derived exosomal (TDE-miR-21-5p). RIP assay was performed to validate NGF and miR-21 binding ability in MiaPaCa-2 cells. Rescue experiments were performed by using coprocessing of Tanezumab and miR-21-5p mimic on MiaPaCa-2 cells, followed by coculture with DRG cells. Subsequently, we used a model of neuroinvasion in nude mice to assess the effect of NGF on tumor nerve invasion as well as on nociceptive transmission.
NGF level was preeminently higher in PC tissues and cell lines than in paracarcinoma tissues and normal pancreatic epithelial cell lines. NGF promoted MiaPaCa-2 and CFPAC-1 cell invasion and migration, while Tanezumab treatment showed the opposite results. Besides, NGF binding to TrkA receptors encouraged the intracellular Warburg effect in PC and DRG cells. TrkA blocking-up could restrain NGF induced PC cell migration and neural invasion. Mechanistically, NGF could upregulate TDE-miR-21-5p levels, and DRG cells took up TDE to activate the Warburg effect and stimulate nociceptor gene expression. miR-21-5p inhibitor could abolish the facilitative effect of NGF on PNI in MiaPaCa-2 cells. tumorigenesis experiments, Tanezumab markedly alleviated nerve invasion of PC cells as well as relieved nociceptive conduction in animal models.
These findings displayed that NGF/TrkA encouraged the neuroinvasive potential of PC cells by activating the Warburg effect in DRG cells through upregulation of TDE-miR-21-5p expression.
有报道称，与周围神经相关的神经生长因子（NGF）信号通路能够促进胰腺癌（PC）的神经周围侵犯（PNI）。然而，NGF 导致 PNI 的潜在机制仍知之甚少。
采用 Western blot 法检测 PC 及癌旁组织和 PC 细胞系及胰腺导管上皮细胞中的 NGF 水平。用 100ng/ml 的 NGF 或 NGF 抑制剂 Tanezumab 处理 MiaPaCa-2 和 CFPAC-1 细胞 24 小时，分别采用 CCK-8 法和 Transwell 法检测细胞增殖、侵袭和迁移。用 NGF 处理 MiaPaCa-2 和背根神经节（DRG）细胞，敲低 TrkA 表达，以确定其对瓦伯格效应的影响。为了揭示 NGF-TrkA 信号通路与 PC PNI 密切相关，通过在 Matrigel 中与 DRG 细胞共培养，建立了 MiaPaCa-2 细胞的神经侵袭模型。然后，从 PC 细胞中提取外泌体，并通过检查外泌体特异性标志物来鉴定。然后采用 RT-qPCR 检测肿瘤来源的外泌体（TDE-miR-21-5p）中的 miR-21-5p 水平。采用 RIP 测定法验证 MiaPaCa-2 细胞中 NGF 和 miR-21 的结合能力。然后，在 MiaPaCa-2 细胞中共同处理 Tanezumab 和 miR-21-5p 模拟物，再与 DRG 细胞共培养，进行挽救实验。随后，我们使用裸鼠神经侵袭模型评估 NGF 对肿瘤神经侵袭和伤害感受传递的影响。
PC 组织和细胞系中的 NGF 水平明显高于癌旁组织和正常胰腺上皮细胞系。NGF 促进 MiaPaCa-2 和 CFPAC-1 细胞侵袭和迁移，而 Tanezumab 处理则显示出相反的结果。此外，NGF 与 TrkA 受体结合促进了 PC 和 DRG 细胞内的瓦伯格效应。TrkA 阻断可抑制 NGF 诱导的 PC 细胞迁移和神经侵袭。机制上，NGF 可以上调 TDE-miR-21-5p 水平，DRG 细胞摄取 TDE 以激活瓦伯格效应并刺激伤害感受器基因表达。miR-21-5p 抑制剂可消除 NGF 对 MiaPaCa-2 细胞 PNI 的促进作用。在肿瘤发生实验中，Tanezumab 显著减轻了 PC 细胞的神经侵袭，并在动物模型中缓解了伤害感受传导。
这些发现表明，NGF/TrkA 通过上调 TDE-miR-21-5p 的表达，在 DRG 细胞中激活瓦伯格效应，从而增强 PC 细胞的神经侵袭潜力。
