乏氧应激上调GOLM1促进肝细胞癌恶性潜能的作用及其机制研究

It is well acknowledged that hypoxic stress and malignant phenotype such as invasion, metastasis as well as therapy resistance are closely related in hepatocellular carcinoma(HCC).Previously,we had demonstrated that expression of GOLM1 was closely correlated with hypoxia-associated malignant phenotype in HCC by means of a high-throughput genomic filtering technology. In-depth study, we found that GOLM1 was significantly up-regulated in hypoxic environment which in turn regulated hypoxia-related malignant phenotype such as invasion, metastasis, angiogenesis and threapy resistance of HCC cells. Based on our previous study about the protein interactions, we speculated that HIF1A may have an important role as a connected molecular between GOLM1 and hypoixa-related invasion, metastasis, angiogenesis and treatment resistance. So we suggest that there is a new molecular mechanism as an axis of GOLM1-HIF1A-hypoxia-malignant potential to take effect and we are going to carry out some researches on this functional axis as below: 1. Analyze the connection between the GOLM1,HIF1A both structurally and functionally in HCC for the confirmation of this axis. 2. Determinate the expression of these molecular in clinical specimens to investigate the correlation between this axis and recurrence ，metastasis and poor prognosis of HCC patients. 3. Disturb this functional axis by some in vitro and in vivo assays for further validation that it can effectively predict the recurrence and serve as a potential therapeutic target in HCC.

乏氧应激与肝癌侵袭、转移及治疗抵抗等恶性潜能密切相关。我们前期通过高通量基因组学筛选技术发现，GOLM1与肝癌乏氧相关恶性潜能密切联系；深入研究证实，GOLM1在肝癌乏氧应激环境中显著上调，并促进肝癌乏氧相关侵袭、转移及治疗抵抗恶性潜能；基于蛋白-蛋白相互作用分析及前期研究提示，我们又推测HIF1A是GOLM1调控肝癌乏氧应激相关恶性潜能的关键中间衔接分子，由此提出"GOLM1-HIF1A-乏氧应激-恶性潜能"功能轴这一新的分子机制。本课题拟在前期研究基础上，进一步围绕此功能轴展开以下研究：①分析肝癌细胞中GOLM1、HIF1A在表达和功能上的联系，对此功能轴进行验证；②在肝癌临床样本中检测GOLM1、HIF1A的表达，探讨此功能轴与肝癌患者转移复发及预后的关系；③通过体内、体外实验，干预此功能轴，明确其对肝癌侵袭转移及治疗抵抗的影响；为肝癌转移复发提供有效的预测模型和干预靶点。

乏氧是包括肝癌在内实体肿瘤由于过度生长和新生血管畸形所产生的一个重要微环境，与肿瘤侵袭、转移及治疗抵抗等恶性潜能高度相关。肿瘤内乏氧被认为是肿瘤预后差的一个重要因素。然而目前为止，对乏氧应激导致肿瘤，特别是肝癌细胞侵袭、转移及治疗抵抗等恶性潜能中涉及的关键分子及具体机制仍不清楚。通过构建乏氧细胞模型，我们在多株肝癌细胞系中证实，乏氧应激通过HIF-1显著上调肝癌细胞内GOLM1的表达水平；肝癌细胞中上调的GOLM1能显著促进肝癌细胞生长、侵袭和转移等恶性潜能；GOLM1在肝癌患者，特别是转移的肝癌患者中显著升高，并与患者血管侵犯和不良预后密切相关；进一步基于“蛋白-蛋白”相互作用筛选与GOLM1潜在相互作用的分子发现：受体酪氨酸激酶家族的成员EGFR和GOLM1存在潜在相互作用；我们进一步通过免疫共沉淀和激光共聚焦共定位分析技术证实，GOLM1和EGFR在细胞内存在相互作用，且两者之间的相互作用在EGFR的配体EGF作用下显著增强，抑制GOLM1的表达能显著影响EGFR在配体诱导的下游相关信号通路如：AKT,ERK,mTOR的活化；进一步研究发现，在配体EGF的作用下，EGFR内吞至细胞内，与高尔基体蛋白GOLM1动态作用，进而促进内吞的EGFR循环至细胞膜上是其调控EGFR持续活化的重要机制；最后，我们还进一步深入分析了GOLM1反馈调控HIF-1可能性及相关机制。我们发现GOLM1部分定位于细胞内的线粒体，通过影响细胞内线粒体ROS生成，通过阻止HIF1蛋白的脯氨酰羟基化抑制降解，从而稳定HIF1，形成了“GOLM1-HIF”正反馈的恶性回路。我们的研究首次揭示了以往被主流研究忽视的亚细胞器的重要结构高尔基体在肿瘤（特别是肝癌）转移中起重要作用，为肝癌转移研究的打开了新方向，后续研究针对“GOLM1-HIF”回路设计药物干预肝癌转移意义重大。