IL-8/IL-8R轴对胃癌上皮间质转化的调控作用以及消痰散结方干预机制研究

Emerging evidence suggests that epithelial-mesenchymal transition (EMT) plays a pivotal role in tumor metastasis. In previous studies of various cancers, it was reported that the abnormality of cell’s microenvironment, notably the aberrant of interleukin-8 (IL-8)/IL-8 receptor axis, has a great impact on EMT, however, additional studies are urgently needed and data concerning the role of IL-8 in EMT of gastric cancer is rare. The therapy based on the phlegm theory of traditional Chinese medicine has been confirmed to inhibit invasion and metastasis of gastric cancer. However, the potential mechanisms remain unclear. The phlegm theory of traditional Chinese medicine could be well applied in the understanding of cell’s microenvironment and it regards the pollution of phlegm as the essential mechanism for the invasion and metastasis of gastric cancer, of note, which includes the irregular of inflammation factor in the tumor cell’s microenvironment. Based on the theory, our previous study indicated that IL-8 promotes tumor invasion and Xiaotan Sanjie decotion could down-regulate the expression of IL-8 and E-cadherin, a mark of EMT, and inhibit IL-8-induced invasion. In this study, we aimed at investigating the role of IL-8/IL-8R axis in EMT of gastric cancer and the efficacy, as well as the underlying mechanism of Xiaotan Sanjie decotion, which include the research of cell’s morphologic variation, key protein and signaling pathway of EMT such as E-cadherin, N-cadherin, vimentin, fibronectin, Snail and Slug respectively. In order to reach the above purpose, we are going to establish a Brachyury-induced EMT model (in vitro) and use blocking antibodies specific for the IL-8 receptors, in addition, we also plan to perform study in vivo, which targets at observation the role of IL-8 in EMT of gastric cancer and the efficacy, as well as the underlying mechanism of Xiaotan Sanjie decotion by in situ transplantation gastric cancer model and tail vein gastric cancer metastasis model in nude mice.

上皮间质转化（EMT）是肿瘤侵袭迁移的关键，白介素8及其受体轴（IL-8/IL-8R轴）通过自/旁分泌在EMT中发挥了关键作用，我们前期发现IL-8促进胃癌侵袭迁移，但IL-8/IL-8R轴在胃癌EMT中的机制尚不清楚。临床证实胃癌从痰论治采用消痰散结法抑制胃癌侵袭转移有效可行，前期研究证实消痰散结方下调胃癌IL-8及EMT特征蛋白E-Cad的表达，且抑制IL-8诱导的胃癌细胞迁移侵袭，据此我们提出假设：IL-8/IL-8R轴在胃癌EMT中具有关键作用，也是消痰散结方通过调控EMT抑制胃癌迁移侵袭的作用靶点。我们将以细胞形态、EMT特征性蛋白、迁移侵袭能力为观察指标，体外建立Brachyury诱导EMT模型，运用IL-8受体阻断剂；体内通过两种转移模型，慢病毒转染，探索IL-8/IL-8R轴在胃癌EMT中的作用和消痰散结方调控EMT的IL-8/IL-8R轴机制，为胃癌从痰论治提供理论支持。

本研究采用慢病毒感染建立Brachyury过表达胃癌细胞株，构建胃癌上皮间质转化（EMT）模型，通过Western Blot、qPCR、ELISA、Transwell等方法研究在IL-8直接作用、自分泌、旁分泌三种状态下胃癌EMT相关蛋白和mRNA表达和EMT相关迁移侵袭能力的变化，再使用消痰散结方和IL-8受体阻断剂Reparixin来观察三种状态下两者的交互关系，探索消痰散结方抑制胃癌转移的可能IL-8轴机制。研究发现Brachyury过表达胃癌细胞株（MGC803、SGC7901、BGC823细胞）可以发生EMT，表现为特征性的EMT标志（E-Cadherin、N-Cadherin、Vimentin、Fibronectin、Slug、Snail）蛋白与基因表达的改变和细胞迁移侵袭能力的增加；IL-8可以促进模型的EMT表现，EMT特征性蛋白与mRNA表达增加或减弱，细胞迁移和侵袭能力增加，且该种改变可以被IL-8特异性受体阻断剂Reparixin所抑制；消痰散结方可以抑制模型的EMT特征，包括蛋白和mRNA表达的改变和细胞迁移侵袭能力的降低，且该种效应能部分被Reparixin所阻断。从软琼脂克隆形成实验也证实IL-8过表达胃癌细胞较IL-8沉默胃癌细胞和空白细胞生长更为迅速，细胞团为多，消痰散结方可以抑制IL-8过表达细胞的生长与成团。同时IL-8可以促进胃癌侵袭转移关键步骤之一肿瘤血管生成，且该种效应能被消痰散结方对抑制。项目研究结果提示IL-8和受体结合是消痰散结方抑制侵袭和迁移的关键环节之一。