基于Wnt信号通路调控胃癌细胞EMT的化痰通瘀解毒方抗胃癌侵袭转移作用机制研究

Studies have shown that epithelial mesenchymal transition plays a key role in the process of tumor cell invasion and distant spread. Wnt signaling pathways can induce EMT process, and it is closely related to invasion and metastasis of gastric cancer. TCM holds that combined action of phlegm, blood stasis and toxin is the key factor of recurrence and metastasis of gastric cancer. Resolving phlegm, dissipating blood stasis and detoxicating are the fundamental treatment principal. Our preceding study shows Huatantongyujiedu decoction can significantly reduce the density of transplanted tumor tissue microvascular of mices, inhibit VEGF expression of tumor tissue, excessive activation of Wnt/β-catenin signaling pathway of gastric cancer cells and excessive expression of COX-2, MMP-9 and VEGF of its downstream target molecules. Based on our preceding studies, this project proposes classical and non-classical way to regulate EMT by wnt signaling pathway. RT-PCR, Western blot, flow cytometry, immunofluorescence and immunohistochemistry are used. To study the relevance of the EMT process of different differentiation of gastric cancer cells and Wnt signaling pathways, and the intervention targets of Huatantongyujiedu decoction by in vivo and in vitro methods, and further reveal the mechanism of Huatantongyujiedu decoction against the invasion and metastasis of gastric cancer.

研究表明，上皮–间质转化（EMT）在肿瘤细胞向周围侵袭和远处播散过程中发挥着关键作用，wnt信号通路可诱导EMT进程，与胃癌侵袭转移密切相关。中医认为痰、瘀、毒相互胶结是胃癌复发、转移的重要因素，化痰、行瘀、解毒是其根本治疗大法。本项目组既往研究显示，化痰通瘀解毒方能够显著降低小鼠移植瘤组织微血管密度、抑制瘤组织VEGF表达，并能抑制胃癌细胞Wnt/β-catenin信号通路的过度激活及其下游靶分子COX-2、MMP-9和VEGF过表达。本项目拟在既往研究基础上，从wnt信号通路经典途径与非经典途径调控EMT入手，运用RT-PCR、Western blot、流式细胞术、免疫荧光和免疫组织化学等技术，从体内、体外两方面研究不同分化程度胃癌细胞EMT进程与Wnt信号通路的相关性，以及化痰通瘀解毒方的干预靶点，进一步揭示化痰通瘀解毒方抗胃癌侵袭转移作用机制。

侵袭转移是影响胃癌预后的重要因素，EMT是肿瘤侵袭转移的关键环节，Wnt信号通路参与了胃癌细胞EMT进程，已成为肿瘤治疗的重要靶点。本课题组既往研究显示：化痰通瘀解毒方能抑制胃癌细胞Wnt/β-catenin 信号通路活性及肿瘤侵袭转移，下调VEGF、MMP-9和E-Cadherin等与侵袭转移和EMT相关的蛋白表达，提示该方抑制胃癌侵袭转移作用可能与其调控Wnt/β-catenin 信号通路抑制胃癌细胞EMT有关。本项目分别构建激活或沉默Wnt/β-catenin和Wnt5a /Ca2+ /NFAT信号通路的人胃癌MGC-803细胞模型，以及裸鼠移植瘤模型，观察胃癌细胞侵袭转移能力、EMT标记蛋白和Wnt经典信号通路和非经典信号通路相关蛋白表达变化，初步阐明Wnt信号通路对人胃癌细胞EMT和侵袭转移的调控机制，以及化痰通瘀解毒方的干预效应。研究结果显示：化痰通瘀解毒方抑制胃癌细胞侵袭转移作用与其抑制EMT作用密切相关；该复方抑制胃癌细胞EMT及侵袭转移作用与其抑制经典信号通路Wnt/β-catenin和非经典信号通路Wnt5/ Ca2+/ NFAT活性均密切相关；该复方调控胃癌细胞Wnt通路的机制在于：其既可抑制Wnt/β-catenin信号通路GSK-3β磷酸化，稳定GSK-3β、Axin和APC蛋白表达，促进β-catenin磷酸化，减少胞浆β-catenin蓄积及其核移位、降低核因子TCF4转录活性，又可下调Wnt5/ Ca2+/ NFAT信号通路CaN蛋白表达、促进NFAT磷酸化而降解，减少NFAT胞浆蓄积及其转移入核、降低其转录活性。本研究成果为化痰通瘀解毒方广泛应用于胃癌的防治奠定了坚实的实验基础。