扶正泻肝方通过TREM2/DUSP6/ERK介导糖代谢影响肝癌侵袭转移的作用机制研究

Invasion and metastasis of hepatocellular carcinoma (HCC) are the important reasons for the failure of HCC treatment, which is closely related to abnormal carbohydrate metabolism. Many Chinese formulas can adjust body metabolism and alleviate the symptoms of HCC, but the application is limited because of the unclear mechanisms. In our previous study, we found that Fuzheng Xiegan (FZXG) formula from Jinghua Ye had a good effect on the treatment of HCC. FZXG could also inhibit invasion and metastasis in vivo and in vitro experiments. Furthermore, in the different HCC cells, FZXG could down-regulate the expression of TREM2, which was proved as an oncogene of HCC in our published paper. Using bioinformatics analysis and in vitro experiments, we found that the activation of ERK pathway and the uptake of glucose could be inhibited by FZXG treatment and TREM2 interference. We also found that there were some interactions between TREM2 and DUSP6, a negative feedback enzyme of ERK. Therefore, we propose the hypothesis that FZXG may inhibit the invasion and metastasis in HCC via TREM2/DUSP6/ERK pathway mediating carbohydrate metabolism. The study aims to investigate the effects of FZXG on TREM2/DUSP6/ERK pathway, carbohydrate metabolism, invasion and metastasis of HCC, and reveal the mechanism of FZXG on the treatment for HCC using gene interference or overexpression, pull-down assay, ubiquitylation analysis and so on. This study will provide the experimental basis for FZXG in HCC treatment.

肝癌的侵袭转移是临床肝癌治疗失败的重要原因，而肿瘤侵袭转移与糖代谢异常密切相关。许多中药方剂能够调节机体代谢、改善肝癌症状，但由于作用机制不清，使得推广应用受限。前期研究发现，上海市名中医叶景华经验方扶正泻肝方对肝癌治疗具有良好的临床疗效，体内外实验发现该方能够抑制肝癌侵袭转移；TREM2在肝癌中发挥癌基因作用，扶正泻肝方能够抑制其表达；同时，扶正泻肝方和TREM2干扰均可能抑制葡萄糖的摄取和ERK通路的激活，而负反馈调节ERK通路的DUSP6可能和TREM2互作。由此提出假说，扶正泻肝方通过下调TREM2表达来调节DUSP6/ERK通路，影响糖代谢，进而抑制肝癌侵袭转移。本项目拟通过基因干扰和过表达、Pull-down实验、泛素化分析等技术，考察扶正泻肝方在体内外对TREM2/DUSP6/ERK通路、糖代谢，以及肝癌侵袭转移的影响，揭示该方作用机制，为其治疗肝癌的临床应用提供科学依据。

肝癌的发病率逐年升高，且仍缺乏有效治疗药物。中药复方扶正泻肝方，在长期临床实践中治疗肝癌安全有效，为了揭示该方作用机制，本项目首先从体外实验和体内实验两个方面考察扶正泻肝方对肝癌的影响；接着，通过质谱分析鉴定扶正泻肝方有效成分，网络药理学预测相关作用靶点，生物信息学寻找关键基因和通路并进行验证；最后，体内外考察TREM2对肝癌的影响，探索TREM2下游作用机制并进行验证。在体外实验中，扶正泻肝方作用于肝癌细胞后，发现细胞周期阻滞、细胞凋亡增加、侵袭和迁移被抑制；体内动物实验发现，扶正泻肝方显著抑制裸鼠原位移植瘤的生长。接着，质谱分析鉴定出复方的31种有效成分，网络药理学预测出扶正泻肝方作用的相关靶点和通路主要集中于侵袭和转移功能上，随即对预测靶点进行了验证，结果表明，扶正泻肝方显著下调侵袭转移相关靶点VEGF、FGF2、HSP90AA1、MDM2、CDK6的表达；进一步通过转录组测序找出肝癌关键基因TREM2，验证后发现，扶正泻肝方显著下调TREM2表达；同时，基因富集分析发现扶正泻肝方抑制ERK通路的激活。最后，考察TREM2与肝癌的关系，结果表明，TREM2在肝癌细胞株和组织中的表达显著上调；TREM2基因干扰后，肝癌细胞增殖能力减弱、凋亡数量增加、细胞周期阻滞、裸鼠原位移植瘤生长减慢，TREM2干扰与扶正泻肝方作用效果相同；紧接着，探究TREM2下游的作用机制发现，TREM2高表达与ERK信号通路的激活呈正相关，并发现TREM2的互作蛋白DUSP6，随即验证结果表明，扶正泻肝方下调TREM2的同时，上调DUSP6，抑制ERK通路的激活。本项目确证了扶正泻肝方能够抑制肝癌的发展，初步探索了扶正泻肝方通过TREM2/ DUSP6/ERK通路影响肝癌发展的作用机制。通过本项目的研究，为扶正泻肝方治疗肝癌的临床应用提供一定的科学依据。