解毒消癥饮对肝癌细胞中HIF-1/miR-210正反馈回路介导的能量代谢重编程的影响研究

Under hypoxic microenvironment , the tumor cells promote metastasis eventually by choosing to open energy reprogramming and up-regulating the expression of angiogenesis factor. In this process, HIF-1/miR-210 positive regulation loop is involved in tumor energy metabolic reprogramming through enhancing glucose uptake, activating glycolysis pathway and enhancing lactic acid accumulation. Our previous studies have found that under hypoxia condition Jiedu Xiaozheng Yin inhibited angiogenesis by up-regulating the VEGF protein level through the HIF-1/PHD/VEGF signaling pathway, transcriptional coactivator P300/CBP in hepatoma cells. And the recent research also found that Jiedu Xiaozheng Yin inhibited the glucose consumption and extracellular acidification rate. In this study, we will use the SPECT/PET/CT, Three-dimensional imaging system and Cell Energy Metabolism Workstation in our laboratory to research if the Jiedu Xiaozheng Yin regulated energy metabolic reprogramming by HIF-1/miR-210 positive regulation loop under hypoxia and further affect the angiogenesis, invasion and metastasis in vivo and in vitro.

在缺氧微环境下，肿瘤细胞应激性上调HIF-1/miR-210后开启能量代谢重编程，使糖代谢流从线粒体氧化磷酸化转变为无氧糖酵解模式，形成新的生长代谢平衡，促进肿瘤侵袭转移。在这一过程中，HIF-1/miR-210正调控回路单元通过增强葡萄糖摄取、活跃糖酵解途径、促进乳酸堆积等全程参与了肿瘤细胞能量代谢重编程过程。课题组前期研究发现，低氧条件下解毒消癥饮调控肝癌细胞中HIF-1/PHD/VEGF信号通路并通过转录辅因子P300/CBP影响VEGF的表达从而抑制血管新生及侵袭转移，解毒消癥饮亦显著抑制葡萄糖的消耗及胞外酸化率。本研究以HIF-1/miR-210正调控回路为切入点，利用小动物荧光发光、小动物PET-CT成像系统及细胞能量代谢工作站等仪器结合分子生物学、细胞生物学及组织化学等技术从体内外不同层面探讨解毒消癥饮对肝癌细胞能量代谢重编程的调控作用，以期阐明解毒消癥饮的抗肿瘤作用机制。

本项目利用3株不同肝癌细胞HepG2、Hep3B、Huh7建立低氧模型后，于常氧及低氧不同条件下予解毒消癥饮（JXY）干预后发现JXY时间依赖性的抑制低氧条件下上述肝癌细胞HIF-1α及其miR-210的表达，抑制此反馈回路后进一步抑制了肝癌细胞的增殖和迁移能力，诱导了肝癌细胞的凋亡，通过抑制糖酵解相关酶蛋白的水平（GLUT-1、HK2、PFK1和MCT-4））降低了糖酵解通路对葡萄糖的利用度。进而选择较敏感细胞株Huh7过表达HIF-1α后建立低氧刺激模型，JXY干预后抑制HIF-1α进而抑制miR-210的水平，降低葡萄糖的消耗量、耗氧量及ATP、乳酸和胞外酸化率的产生，分子层面检测发现，JXY通过抑制糖酵解相关酶蛋白（GLUT1、LDHA、MCT4、PFK1）的表达进而抑制糖酵解的发生，但未显著提高CoA和ROS的水平，且JXY诱导了细胞凋亡的发生。体外建立小鼠原位肝癌移植瘤模型后，JXY干预后抑制了原位肿瘤的生长，未见转移瘤出现，但对体重及肝重、脾重、肝功能未见影响，对葡萄糖代谢酶蛋白HIF-1α、HK2、GLUT1、PFK1、PDK具有显著抑制作用。