NLRP3炎症小体/IL-1β信号通路在口腔鳞癌中的作用研究

The inflammatory cytokine IL-1β has been reported to play an important role in carcinogenesis and tumor development. The secretion of IL-1β is dependent on two steps: the production of pro-IL-1β mediated by the activation of NF-κB signaling pathway and the cleavage of pro-IL-1β into mature IL-1β by the activated inflammasome. In our previous study, we detected higher expression of TLRs in the oral squamous cell carcinoma (OSCC) cells, and activation of these TLRs led to the activation of the NF-κB signaling pathway. Moreover, in our preliminary work, higher expression of NLRP3 inflammasome and its activation product IL-1β was found in OSCC cells and tissues, compared to that in the normal oral epithelial cells and tissues. Thus, these studies hint that the NLRP3 inflammasome/IL-1β pathway probably play an imortant role in the tumorigenesis and progression of OSCC. Therefore, in this project, we will get further to investigate the role of the activated NLRP3 inflammasome/IL-1β pathway in OSCC cell growth/epithelial-mesenchymal transition, the effect of it on the stromal cells and its related mechanisms; to investigate the expression of NLRP3 inflammasome and IL-1β in OSCC tissues and their relationship with the clinical characteristics; and finally to identify the function of NLRP3 inflammasome/IL-1β pathway in OSCC with in vivo experiments by using the Nlrp3-/- mice. The present study will provide theoretical and experimental evidence to the elucidation of OSCC pathological process from the new perspective of the NLRP3 inflammasome/IL-1β signaling pathway, so as to provide new and effective targets for the OSCC prevention and therapy.

炎性因子IL-1β在肿瘤的发生发展中发挥重要作用，其分泌有赖于NF-κB信号通路激活产生IL-1β前体和炎症小体对IL-1β前体的活化。我们的前期研究显示口腔鳞癌细胞中TLRs表达升高并介导NF-κB信号通路活化，预初实验又发现口腔鳞癌细胞高表达NLRP3炎症小体及其活化产物IL-1β，这些结果表明NLRP3炎症小体/IL-1β信号通路很可能在口腔鳞癌发生发展中发挥重要作用。为此，本项目拟探讨NLRP3炎症小体/IL-1β信号通路的激活对口腔鳞癌细胞生长及上皮-间质转化和基质细胞的影响及相关机制；分析口腔鳞癌组织中NLRP3炎症小体和IL-1β的表达及其临床相关性；最后，通过Nlrp3-/-小鼠建立口腔鳞癌小鼠模型，在体内验证该信号通路在口腔鳞癌中的作用。本项目试图从NLRP3炎症小体/IL-1β信号通路这一新视角为揭示口腔鳞癌病理过程提供理论和实验依据，进而为口腔鳞癌的防治提供新的思路。

炎症参与肿瘤的发生发展，其调控机制复杂。本项目通过对临床组织标本、细胞系和动物模型的系统研究，（1）阐明了NLRP3炎症小体/IL-1β信号通路与口腔鳞癌（OSCC）的临床相关性：患者OSCC组织中NLRP3表达明显高于癌旁组织，其表达与肿瘤大小、淋巴结侵袭以及成熟IL-1β的表达相关，且与患者的不良预后相关；（2）阐明了NLRP3炎症小体/IL-1β信号通路影响OSCC进展的机制：通过OSCC细胞培养体系，发现敲减NLRP3表达可抑制OSCC细胞的增殖、克隆形成以及侵袭转移能力，使与上皮细胞-间充质转化（EMT）呈正相关的N-钙粘蛋表达降低，而与EMT呈负相关的E-钙粘蛋白与波形蛋白表达上调；敲减NLRP3表达可抑制肿瘤相关成纤维细胞中IL-1受体、IL-6和COX-2的表达；5-氟尿嘧啶（5-FU）处理可显著提高OSCC细胞内NLRP3炎症小体与IL-1β的表达，敲减NLRP3基因表达可明显增强5-FU的细胞凋亡诱导作用；5-FU可抑制OSCC细胞超氧化物歧化酶（SOD2）和过氧化氢酶（catalase）的表达，促使细胞内活性氧（ROS）水平升高，从而激活NLRP3炎症小体使IL-1β前体被剪切为成熟有功能的IL-1β；（3）为进一步验证NLRP3炎症小体/IL-1β信号通路在OSCC中的作用，通过建立裸鼠荷瘤模型和致癌剂4-硝基喹啉-1-氧化物（4-NQO）诱导的小鼠舌鳞癌模型，发现敲减NLRP3可抑制OSCC的体内成瘤能力；NLRP3炎症小体功能缺失小鼠舌鳞癌的发生率和进展速度低于野生型对照小鼠，且NLRP3炎症小体功能缺失小鼠对5-FU的敏感性显著高于野生型对照小鼠；（4）初步阐述了OSCC中NLRP3炎症小体/IL-1β信号通路异常激活的机制，发现miR-22能够靶向调控NLRP3的表达，OSCC组织和细胞中miR-22异常下降，过表达miR-22可明显抑制OSCC细胞的增殖、克隆形成、迁移侵袭和体内成瘤能力。因此，本项目的研究结果表明OSCC中异常表达和激活的NLRP3炎症小体/IL-1β信号通路促进OSCC的发生、发展及5-FU化疗抵抗，靶向NLRP3炎症小体/IL-1β信号通路可抑制OSCC的生长和提高OSCC对5-FU的敏感性。本项目在丰富OSCC发病机理研究的同时，也为OSCC的治疗提供了新的方向。