IL-18影响舌鳞状细胞癌侵袭转移作用的机制研究

Cancer invasion and metastasis promotes tumor progression by allowing tumor to develop to a higher level of malignant tumor, invading the blood or lymphatic vessels and migrating to other tissues or organs. In the process, the most significant change is the decline of the intercellular adhesion. Interleukin-18 (IL-18) is a proinflammatory cytokine, and studies have revealed that IL-18 enhances the expression of ICAM-1. In addition, through enhancing the production of IL-18, VEGF is showed to increasing the migration of human gastric cancer cells. However, the underlying mechanism between IL18 and migration/metastasis is not yet fully uncovered. Tongue squamous cell carcinoma (TSCC) is the most common cancer of head and neck. In this project, we try to release the role of IL-18 in the EMT process using TSCC cell line. A IL-18 over-expression cell line in established, and the IL-18 knockout cells are also generated by Crispr-Cas9 technique. Applying TGFβ1, the key inducer of EMT, the changes in gene expression in IL-18 over-expression and knockout cells are examined. Furthermore, the factors interacting between IL-18 and EMT process are identified by co-immunoprecipitation. To confirm the functions of this factor between IL-18 and EMT process, gene knock in and knockout experiment is also performed. Tumor stem cells are isolated from TSCC cells by ALDEFLUORTM Kit to further confirm the function of this identified factor and the roles of IL-18 on cancer development. The above results are also evaluated in the in vivo experiments. Utilizing siRNA injecting into tumors, the interaction between IL-18 and EMT process are further confirmed. Injecting the IL-18 over-expression and knockout cells into nude mice through tail vein, respectively, the effects of IL-18 on the in vivo migration and metastasis are exploited. This study will provide strong and convincing evidence for comprehensively evaluating the roles of IL-18 TSCC treatment, especially on EMT process.

癌症侵袭与转移过程中最显著的变化是细胞间粘附作用下降。IL-18影响细胞粘附，发挥抑癌和促癌的双重作用。本项目拟研究IL-18在舌鳞状细胞癌侵袭转移进程中的作用。申请人构建IL-18过表达细胞系，采用Crispr-Cas9技术制备IL-18敲除细胞系；应用TGFβ1因子诱导EMT的发生，检测干扰IL-18表达水平后EMT相关基因的表达变化；运用免疫共沉淀等技术，寻找与IL-18直接或间接作用的侵袭转移相关分子，阐明IL-18与EMT基因相互作用的机制；选用流式细胞术分离舌鳞癌干细胞，进一步验证IL-18对侵袭转移进程的影响。将IL-18遗传修饰细胞植入裸鼠肋部，对瘤体组织注射目的siRNA，体内验证与IL-18相互作用的EMT分子；尾静脉注射入裸鼠体内，测定干扰IL-18表达水平对肿瘤转移的影响。本项目将探讨IL-18影响EMT进程的作用机制，从更深层面揭示IL-18在癌症中的双重作用。

上皮间质转化是（EMT）指上皮细胞通过特定程序转化为具有间质表型细胞的生物学过程。对于上皮源性的恶性肿瘤细胞来说，EMT是其局部浸润突破基底膜的重要过程，并通过渗入血管或淋巴管内形成远端转移。IL-18在多种肿瘤中扮演着抗瘤活性，并能提高机体宿主防御，但同时也能促进多种肿瘤细胞的转移。因此，在前期研究基础上，本项目拟探究IL-18对舌鳞癌细胞侵袭转移的影响。.在本项目研究中，我们收集了临床患者口腔鳞癌组织标本，发现IL-18可作为一种肿瘤恶性程度的指标，表达水平越低则代表疾病进展较差，此外，IL-18可以通过TNKS2分子降低β-cantenin信号通路活性抑制口腔鳞癌EMT行为的发生。我们构建了过表达IL-18的舌鳞癌细胞系，发现IL-18能有效抑制舌鳞癌细胞的增殖并促进其凋亡和G1期阻滞，另外，过表达的IL-18可以增强舌鳞癌细胞迁移和侵袭能力。除此之外，我们还构建了构建裸鼠移植瘤模型，证实了IL-18具有体内抗瘤作用，并且能活化β-cantenin信号促进舌鳞癌细胞上皮间充质转化。由此可见，舌鳞癌细胞EMT行为的发生与β-cantenin信号通路有着密切的联系。.综上所述，本项目通过成功构建过表达IL-18的舌鳞癌细胞系，分别从体内和体外角度验证了IL-18抑制舌鳞癌细胞增殖的效果，但同时也促进了肿瘤细胞EMT侵袭转移的能力。针对舌鳞癌IL-18是一种可靠的细胞因子，但值得注意的是，靶向提升IL-18表达水平后，会通过活化β-cantenin信号促进舌鳞癌细胞的迁移和侵袭能力。因此，如何有效的发挥IL-18的抗瘤效果，并降低其促肿瘤细胞转移能力，是下一阶段研究的重点，也为后期抑制肿瘤细胞EMT行为提供理论依据。