高尔基体相关蛋白GP73增强脯氨酸羟化酶PHD3泛素化降解稳定HIF-1α促肝癌生长转移机制和干预研究

Central to the secretary pathway, the Golgi apparatus modifies and systematizes cell products and transfers them to their final destinations within the cell. It is assumption that the balance of protein trafficking between different subcellular compartments, mainly modulated by Golgi apparatus, can activate either pro-survival or pro-apoptosis mechanisms depending on the specific cell microenvironments, which offers a potential therapeutic opportunity for cancer.However, the exact mechanisms of Golgi apparatus involving the growth and metastasis of cancer are still unclear. Based on the gene expression profiling of hepatocellular carcinomas (HCC) complicated with lung metastasis, we found a higher expression level of Golgi associated protein GP73 was correlated with early metastatic recurrence of HCCs, and the similar results were obtained by real-time PCR and immunohistochemical staining of two independent sets.Downregulation of GP73 could significantly inhibit the proliferation, migration，invasion, and angiogenesis of HCC cells with high endogenous GP73 levels, rather than those with lower intrinsic GP73 levels.The results of mass spectrometry indicated one of GP73 interacted proteins was prolyl hydroxylase 3 (PHD3), a key regulator of hypoxia-inducible factor(HIF), and the endogenous and exogenous interaction between GP73 and PHD3 was demonstrated by CO-IP assays. HIF-1 is a transcriptional regulator playing a key role in many aspects of oxygen homeostasis. The heterodimeric HIF complex is regulated by proteolysis of its α-subunits, following oxygendependent hydroxylation of specific prolyl residues. So we detected the expression of HIF-1α in HCC cells when GP73 was down- or up-regulation, and found HIF-1α decreased when GP73 was knocked down, and increased when GP73 was overexpression. Culturing HCC cells in hypoxia, the amplitude of decrease or increase was even bigger. Whether the regulatory role of GP73 on the HIF-1α is dependent on the PHD3 will be demonstrated with specific plasmid targeting PHD3. Furthermore,we will design and construct lentiviral vector containing GP73 sigal peptide mutation or transmembrane domain deletion to explore the role of secretion and membrane anchor in regulating the interact between GP73 and PHD3. Based on the results, we will transfect EGFP-GP73 plasmid and Dysred-PHD3 plasmid into HCC cells to observe their space distribution and correlations in hypoxia. HA-tagged fragments of GP73 were coexpressed with PHD3 in 293T cells and immunoprecipitatedwith anti-PHD3 antibody. The presence of associated GP73 protein was assessed by immunoblotting using anti-HA antibody. Concerning the key orle of HIF-1α on the angiogenesis, the profile of growth factor in supernatant of GP73 high or low expression HCC cells will be detected, and the role of GP73 on HCC angiogenesis will be demonstrated in vivo test. The prognostic value of GP73 on overall survival and the effects of Sorafenib will be analyzed retrospectivelly.

高尔基体的改变参与了肿瘤的发生发展,其相关蛋白GP73是肝癌诊断和预后重要标志物，但生物学功能尚不明确。前期研究显示GP73高表达肝癌患者切除术后复发高预后差。恶性度高肝癌细胞中GP73高表达，慢病毒介导敲低其表达可抑制细胞增殖、运动、侵袭，回补对shRNA有抗性GP73 cDNA能恢复其恶性表型。质谱结果和内、外源性Co-IP均提示羟基化HIF-1α促进其泛素化降解的关键酶PHD3是与GP73相互作用的蛋白。常氧、缺氧条件下敲低和过表达GP73可下调、上调HIF-1α表达。进一步将构建GP73信号肽突变、不定位高尔基膜突变及GP73、PHD3双过表达细胞，通过缺氧、动态激光共聚焦、染色质免疫沉淀等方法研究GP73外分泌和膜定位对PHD3空间分布的调节并促进其泛素化降解,及HIF-1α对GP73转录激活作用(正反馈)，明确GP73促肿瘤血管生成作用及对Sorafenib治疗疗效的预测。

肝细胞肝癌因其迅速进展至转移，是肝胆系统常见恶性肿瘤中最致命的一种。转移包含了在血液中迁移、侵袭和存活等多步过程。直接微环境（细胞之间或细胞与细胞基质之间相互作用）和扩展的肿瘤微环境（例如氧有效度的波动）对肿瘤转移都有深远的影响。在慢性肝病和不完整的畸形新生血管网基础上，肝癌细胞无限制增殖，尽管肝细胞肝癌是一种富含血管的肿瘤，仍会出现严重缺氧的区域。HIF是缺氧诱导的肿瘤进展中一个关键介质。HIF是一个转录因子异质二聚体，缺氧情况下被激活，调控一系列适应缺氧功能的基因，包括糖代谢、血管生成和上皮-间质细胞转换。在缺氧诱导的肿瘤进展中，多个通路包括VHL、REDD1等促进了HIF失调性激活。GOLM1是一个位于高尔基体的73-kD跨膜蛋白，它的过表达和肝细胞肝癌患者的转移和预后差强烈相关。然而，作为常见的一种损伤应激——缺氧，是否是GOLM1过表达的潜在诱发因素，以及GOLM1在缺氧诱导肝细胞肝癌恶变中的作用仍未明确。. 在我们之前的研究中发现它可通过促进EGFR／RTK循环加快肝细胞肝癌转移。本篇文章中，我们试图探究GOLM1是否参与了肝细胞肝癌癌基于缺氧产生的恶性进展。首先，我们发现了缺氧应激通过HIF1α 直接作用GOLM1启动子上调GOLM1的mRNA和蛋白水平。其次，在缺氧诱导的肝细胞肝癌转移过程中，GOLM1通过稳定HIF1α 蛋白，参与了HIF1α -twist和VEGFA信号通路。除了定位在反面高尔基体网,意外发现部分GOLM1定位于线粒体，可上调ROS的产生，抑制HIF1α 的降解。检测GOLM1和HIF1α 的结合水平比单独检测GOLM1或HIF1水平更有评估预后的价值。最后我们发现GOLM1在缺氧诱导的索拉非尼耐药过程中起到关键作用。结论：GOLM1和HIF1α 形成一个互相作用的正性通路促进肝细胞肝癌的转移和对索拉非尼耐药。