人锌铁调控蛋白hZIP1通过泛素-蛋白酶体系统调控低氧诱导因子HIF-1α的表达影响肾癌细胞糖代谢重编程的机制研究

Human ZRT, IRT-like protein-1 (hZIP1) not only directly involved in the cellular zinc transportation, but also affects the oncogenesis and progression of malignant tumors through complex mechanism. In addition, in the study of the pathogenesis of renal cell carcinoma (RCC), the most prominent genes are related to the expression of hypoxia inducible factor-1α(HIF-1α). However, the relationship between hZIP1 and HIF-1α is unclear yet. Meanwhile, abnormal metabolism of glucose is closely related to oncogenesis and progression of malignant tumors. Our previous work showed that the expression defect of hZIP1 in RCC cells induced the upregulation of HIF-1α; this process is likely to be mediated by zinc through Ubiquitin-Proteasome System (UPS), then affect the metabolic reprogramming of glucose through HIF-1α and its downstream genes (such as PDK1), finally promoting cellular proliferation, migration and invasion of RCC. Therefore, we put forward the hypothesis about "hZIP1—Zinc—HIF-1α—metabolic reprogramming of glucose”, trying to clarify the mechanism of hZIP1’s influence on cellular metabolic state by regulating zinc level, and to discuss the effect and mechanism of zinc regulating the key factor HIF-1α through UPS. Finally get a further understanding of the molecular mechanism of the oncogenesis and progression of RCC, and provide probable target to RCC therapy.

人锌铁调控蛋白hZIP1参与细胞的锌离子转运, 同时还通过复杂的机制影响恶性肿瘤的发生和发展。在肾癌发病机制的研究中，最突出的相关基因均与低氧诱导因子HIF-1α的表达相关，但hZIP1与HIF-1α的关系尚不清楚。同时，糖代谢异常与肿瘤的发生发展密不可分。我们的前期工作表明，肾癌细胞中hZIP1的表达缺陷诱导了HIF-1α的上调；这一过程极有可能由锌离子通过泛素-蛋白酶体系统（UPS）所介导，最终通过HIF-1α及其下游基因PDK1影响整个细胞的糖代谢重编程，促进肾癌细胞的增殖和侵袭迁移。因此，我们提出了“hZIP1-锌离子-HIF-1α-糖代谢重编程”调控网络的猜想，期望从肿瘤细胞糖代谢重编程的角度入手，阐明hZIP1通过调节锌离子水平影响细胞糖代谢状态的机制，同时探讨锌离子通过UPS调控HIF-1α表达水平的机制，以深入对肾癌发生发展分子机制的认识，为肾癌的临床治疗提供可能的靶点。

hZIP1参与细胞的锌离子转运，同时还影响多种恶性肿瘤的发生发展。HIF-1α与肾癌发病机制显著相关，但hZIP1与HIF-1α的关系尚不明晰。通过转录组学、蛋白组学和代谢组学联合分析揭示hZIP1影响的物质代谢和信号通路，我们发现hZIP1显著改变肾癌细胞的嘌呤和嘧啶代谢、氨基酸和衍生物代谢、乳糖代谢和游离脂肪酸代谢，并促进肾癌细胞的铁死亡，调节PI3K-AKT、cAMP和PPAR信号通路。通过免疫印迹实验、RT-qPCR和IHC实验检测肾癌组织和癌旁正常组织，我们发现HIF-1α的表达在肾癌组织中显著下调，FBXO30则显著上调，而PDK1无显著变化。通过生物学功能实验和动物实验，我们发现HIF-1α显著促进肾癌细胞恶性生物学行为和裸鼠体内肿瘤形成，FBXO30则呈现肿瘤抑制作用。通过核蛋白提取实验，我们发现过表达hZIP1导致NF-kB1入核减少并显著下调HIF-1α的表达。通过蛋白质半衰期实验、MG132抑制实验、免疫荧光实验、Co-IP实验和补锌实验，我们发现hZIP1通过募集锌离子上调FBXO30，并促进其与HIF-1α的直接结合并介导HIF-1α经过泛素-蛋白酶体系统降解。因此我们得出结论，hZIP1通过抑制肾癌细胞代谢与干扰相关信号通路发挥肿瘤抑制作用。hZIP1减少NF-kB1入核并下调HIF-1α，通过锌离子上调FBXO30以促进其介导HIF-1α泛素化降解，从而抑制HIF-1α的致癌作用。本研究揭示hZIP1下调HIF-1α从而发挥抑癌作用的具体机制，不仅为肾癌的发生发展提供全新见解，还为肾癌临床治疗提供了潜在生物标志物和治疗靶点。

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