沉默ARK5基因逆转乏氧诱导胃癌多药耐药的机制研究

The multidrug resistance（MDR）is the basic cause of failure of chemotherapy in patients with gastric cancer,and hypoxia is one of the important cause of MDR.The resistance could be overcome by regulating the key molecules in multidrug resistance induced by hypoxia.Our research group has found in patients with invalid neoadjuvant chemotherapy the ARK5 and it's phosphorylated activation state ( p-ARK5 ) was strongly positive, and it was reported recently ARK5 was an important intracellular " energy monitor ", which regulated energy metabolism in hypoxic conditions and made the cell survive. Combined with the previous work, we put forward the following hypothesis: ARK5 play an important role in MDR induced by hypoxia through the mechanism that under the hypoxic condition HIF-1α regulated the ARK5 transcription, and activated Akt/ARK5 signaling pathway, p-ARK5 translocated from endochylema to chondrosome by the conduction fo 14-3-3 zeta, finally achieved energy regulation.This project aims to systematicly elucidate the mechanism of ARK5 and its signal transduction pathway in hypoxia induced gastric carcinoma MDR by building low differentiation human gastric cancer drug-resistant cell line,using the adenovirus vector, plasmid transfection, RNA interference, Co-IP, double labeling immunofluorescence technique, in vivo and in vitro, respectively under normoxic and hypoxic conditions. And further to provide the theoretical foundation for the molecular targeted therapy aimed at ARK5.

多药耐药(MDR)导致胃癌化疗失败，乏氧是MDR产生的重要原因，发现并调控乏氧诱导胃癌MDR的关键分子可以克服胃癌耐药性。本课题组发现新辅助化疗无效患者ARK5及其磷酸化活化状态（p-ARK5）呈强阳性，最新研究报告ARK5是细胞内 "能量监控分子"，在乏氧条件下重新调节能量代谢，使细胞幸免。结合前期工作，我们假设：ARK5在乏氧所致的MDR中发挥重要作用，其机制是乏氧通过HIF-1α调控ARK5表达，激活Akt/ARK5信号通路，p-ARK5在14-3-3ζ"分子向导"作用下由胞浆迁移至线粒体，实现能量调控作用。本课题拟构建低分化人胃癌耐药细胞株，通过体内及体外实验，分别在常氧及乏氧条件下，采用腺病毒载体构建、质粒转染、RNA干扰、Co-IP、双标记免疫荧光法等技术，系统地阐明ARK5及其信号传导通路在乏氧诱导胃癌细胞MDR中的机制，为开发ARK5分子靶向治疗药物奠定理论基础。

本项目在前期工作中证实：在接受新辅助化疗后，肿瘤无明显缩小或降期的患者胃癌组织中检测到ARK5及其磷酸化激活（p-ARK5）均高表达；最新研究报告作为“能量监控分子”的ARK5，能在乏氧条件下重新调节能量代谢，使细胞幸免；又鉴于多药耐药（MDR）是导致胃癌化疗失败的主要原因，而乏氧又是MDR产生的重要原因；故拟对ARK5在乏氧诱导的胃癌多药耐药中具体作用机制进行深入地探讨。研究结果表明：1、免疫组化、RT-qPCR及Western blot均显示胃癌组织较慢性胃炎中ARK5、HIF-1α、14-3-3ζ mRNA及蛋白高表达且与恶性程度正相关；2、构建ARK5 RNAi重组慢病毒载体，发现沉默ARK5能逆转胃癌细胞的耐药；3、构建GV205-HIF-1α慢病毒重组载体和HIF-1α-RNAi重组慢病毒，发现HIF-1α通过Akt/ARK5通路磷酸化激活来调控ARK5基因转录、表达，证实HIF-1α是Akt/ARK5通路的上游信号调控分子；6、构建GV205-14-3-3ζ慢病毒重组载体和14-3-3ζ-RNAi重组慢病毒，验证人胃癌耐药细胞在乏氧环境状态下，14-3-3ζ协助ARK5迁移至线粒体，以及完成此亚细胞定位在胃癌细胞MDR中的作用；7、通过裸鼠成瘤实验，验证ARK5在胃癌MDR发生、发展中的关键作用；8、通过基因分析寻找ARK5的下游靶基因，为后续实验提供理论依据。