RIP1调控TNF-α-NF-κB-VEGF-C信号通路促胆囊癌微淋巴管形成的作用及其机制

RIP1 is a key molecule in TNF- alpha downstream signaling pathways and decides which way, TNF- alpha induced cell survival or death pathway, to be activated. Our previous preliminary research has shown that TNF-alpha promotes the expression of VEGF-C through the activation of NF- kappa B in gallbladder cancer cells, and then promotes lymphangiogenesis in gallbladder cancer. The expression of RIP1 is found to be high in the tissue of gallbladder cancer. Silencing RIP1 with siRNA reduces the formation of lymphangiogenesis in the transplanted tumor of gallbladder carcinoma. Therefore, we speculate that RIP1 plays an important role in the activation of TNF- alpha induced cell survival pathway in gallbladder cancer cells, that is, RIP1 regulates TNF-alpha-NF-kappa B-VEGF-C signaling pathway in promoting lymphangiogenesis in gallbladder cancer. In order to confirm this hypothesis and to elucidate its mechanism, we will aim to address whether RIP1 can regulate TNF-alpha-NF-kappa B-VEGF-C signaling pathway in promoting lymphangiogenesis in gallbladder cancer through in vitro cell experiment, in vivo animal experiment and clinical gallbladder carcinoma specimen detection experiment. On this basis, we reveal the mechanism that RIP1 regulates TNF-alpha-NF-kappa B-VEGF-C signaling pathway through three aspects: the existence form of RIP1-complex,different sites of ubiquitination of RIP1 and the integrity of RIP1 protein. These research results will provide a new theoretical basis for foundation study and the design of targeted drugs for preventing lymphatic metastasis in gallbladder cancer.

RIP1是TNF-α下游信号通路的关键分子，参与决定TNF-α诱导的细胞生存或死亡通路。本课题组前期研究证实：TNF-α作用胆囊癌细胞后，通过激活NF-κB促进VEGF-C表达，进而促进胆囊癌微淋巴管形成；RIP1在胆囊癌组织高表达；沉默RIP1降低胆囊癌移植瘤微淋巴管形成。推测在胆囊癌细胞中，RIP1使TNF-α诱导了细胞生存通路，即通过TNF-α-NF-κB-VEGF-C信号通路促胆囊癌微淋巴管形成。为了证实此推测并阐明其机制，本研究拟通过体外细胞、动物实验及临床胆囊癌标本检测，证明RIP1通过调控TNF-α-NF-κB-VEGF-C信号通路促胆囊癌淋巴管形成；在此基础上，从RIP1复合体存在形式、RIP1不同位点泛素化和RIP1蛋白的完整性这三个方面揭示RIP1诱导TNF-α-NF-κB-VEGF-C信号通路的机制。研究结果将为抗胆囊癌淋巴转移基础研究和设计靶向药物提供新的理论依据。

本项目探究RIP1通过调控TNF-α-NF-κB-VEGF-C信号通路促胆囊癌淋巴管形成的作用与机制：（1）证实TNF-α增强胆囊癌细胞RIP1蛋白表达；RIP1是TNF-α激活胆囊癌细胞NF-κB活性的必需因子，并通过NF-κB信号通路调控TNF-α诱导的胆囊癌VEGF-C的表达，从而影响了胆囊癌微淋巴管生成和淋巴转移。以上结果证实：RIP1通过调控TNF-α-NF-κB-VEGF-C信号通路促进胆囊癌微淋巴管形成。（2）证实cIAP2在胆囊癌组织中高表达并与淋巴转移有关；cIAP2促进TNF-α诱导的NF-κB核转移及NF-κB活性，并促进TNF-α诱导的VEGF-C表达。证实胆囊癌细胞内cIAP2可结合RIP1，并作为E3泛素连接酶泛素化修饰RIP1蛋白63位赖氨酸。以上结果证实：胆囊癌细胞中TNF-α通过募集RIP1、cIAP2形成“信号复合体I（TNFR1/TRADD/RIP1/cIAP2等）”，并激活NF-κB开启生存通路；而RIP1 调控胆囊癌细胞TNF-α-TNFR1-VEGF-C信号通路的机制是 RIP1 63位赖氨酸被cIAP2泛素化增强所致。（3）证实cIAPs抑制剂“Smac模拟物”可引导TNF-α促进胆囊癌细胞凋亡，其机制与RIP1降解、RIP1 K48多聚泛素化及形成RIP1复合体I减少、RIP1复合体II（TRADD/RIP1/FADD等）增加有关。该项目研究结果明确了RIP1是TNF-α促胆囊癌微淋巴管形成的关键分子及其调控机制，进一步完善了炎症因子TNF-α促胆囊癌微淋巴管形成的机制；在此基础上，发现cIAPs抑制剂“Smac模拟物”可引导TNF-α促进胆囊癌细胞凋亡并阐明其机制。为抗胆囊癌淋巴转移的基础研究和设计靶向药物提供新的理论证据。完成研究目标，发表SCI论文8篇，培养研究生7名。