AMPK介导的14-3-3σ/Bax交互作用在川芎嗪抗胃癌作用中的机制研究

Gastric cancer is a disease which belongs to traditional chinese medicine such as”zheng jia” or “accumulation”. The use of blood stasis to treat gastric cancer, corresponding with the dialectical principles of diagnosis and treatment of traditional chinese medicine. Previous experiments have confirmed that tetramethylpyrazine(TMP) activates AMPK to promote apoptosis of gastric cancer cells, but its signal pathway and downstream target is still not clear. The project intends to grasp the key link of "interation of 14-3-3σ and Bax" by modern molecular biology technology, surround the "AMPK activation → p53-induced 14-3-3σ expression → 14-3-3σ-Bax complex formation → Bax localization in mitochondrial membrane → mitochondrial membrane permeability changes → apoptosis". The corresponding gene knockout gastric cancer cell lines will be constructed by CRISPR-Cas9 gene editing technique. We will study the interaction and regulatory factors of 14-3-3σ and Bax in the process of apoptosis of gastric cancer cells induced by TMP, and effects of mitochondrial subcellular localization of 14-3-3σ-Bax complex on mitochondrial function and mitochondrial apoptotic pathway. Finally these results will be verified by in vivo experiments. We hope we can provide experimental evidence for the clinical application of traditional Chinese medicine for gastric cancer.

胃癌属于中医“癥瘕”、“积聚”等范畴，运用活血化瘀药治疗胃癌，符合中医辩证论治的诊疗原则。本课题组前期实验已证实川芎嗪激活AMPK促进胃癌细胞凋亡，但其涉及的信号通路及下游靶点并不明确。本课题拟借助现代分子生物学手段，抓住 “14-3-3σ和Bax交互作用”之关键环节，围绕“AMPK激活→p53诱导14-3-3σ表达→14-3-3σ-Bax复合物形成→Bax定位于线粒体膜→线粒体膜通透性改变→细胞凋亡”这一主线，拟采用CRISPR-Cas9基因编辑技术构建相应的基因敲除胃癌细胞系，研究川芎嗪促进胃癌细胞凋亡过程中14-3-3σ与Bax的相互作用及调控因素，以及14-3-3σ-Bax复合体的线粒体膜亚细胞定位对线粒体功能、线粒体凋亡通路的影响。最后还原到整体动物水平进行验证。以期为传统活血化瘀药类中药治疗胃癌的临床应用提供实验依据。

本课题组在前期实验中已发现TMP能抑制胃癌SGC7901细胞的增殖，促进细 胞凋亡，此作用与AMPK激活有关，但具体机制并不明确。鉴于主要是AMPKα1亚型在肿瘤的生长、增殖中发挥作用，故拟用CRASPR-Cas9基因组编辑技术，获得精准靶向AMPKα1的稳定胃癌细胞株，并在二个胃癌细胞系中进行机制探讨。研究结果: 1. 成功利用CRISPR/Cas9技术靶向构建 AMPKα1基因敲除人胃癌SGC-7901/MKN45 细胞系。通过MTT法、软琼脂克隆形成实验、划痕实验、Transwell 和流式证实TMP是通过激活AMPK来发挥其对胃癌细胞的抗肿瘤作用。2. 成功应用CRISPR/Cas9技术靶向构建能够稳定敲除14-3-3σ基因的CRISPR/Cas9慢病毒系统，通过qPCR、WB、MTT、软琼脂克隆形成实验、划痕实验、Transwell和流式实验，确认14-3-3σ为AMPKα1/p53通路靶基因。3. 成功构建GV287-14-3-3σ、GV287-Bax的重组慢病毒表达载体，通过免疫共沉淀技术确认存在 14-3-3 σ与 Bax 的相互作用;通过双标记免疫荧光法证实 14-3-3σ-Bax 共同由胞浆迁移至线粒体膜。4.证实14-3-3σ-Bax复合物通过影响线粒体膜电位、ROS 系统活性、mPTP 开放及 CytC 释放，最终导致细胞死亡。5.成功构建人胃癌细胞异体移植瘤模型，并在整体层面验证结论。该研究的完成，填补了相关的理论空白，并为后续的研究提供基础。