缺氧环境诱导AEG-1基因促卵巢癌侵袭的作用机制

Astrocyte elevated gene 1 (AEG-1), as a novel oncogene, has emerged in recent years as a potentially crucial mediator of tumor malignancy. Recent studies have demonstrated that AEG-1 is associated with tumor angiogenesis, invasion and metastasis induced by HIF-1α. However, the precise role of AEG-1 on angiogenesis, invasion and metastasis induced by HIF is still unknown in ovarian cancer. Through immunohistochemical and western blot analysis, we previously revealed that AEG-1 was over-expressed in tissues from ovarian cancer patients with chemo-resistant, peritoneal dissemination and lymph node metastasis. By silenceing AEG-1 in ovarian cancer cell, we also found that AEG-1 over-expression is correlated with NF-κB expression levels. Our present studies will be in-depth investigations of above findings. By hypoxic experiments in vitro and in vivo, we will further investigate whether and how AEG-1 and NF-κB will regulate hypoxic ovarian cancer invasion induced by HIF. Using co-immunoprecipitation,immunofluorescence, luciferase reporter system technology,we furhter explore the HIF transcription factors involved in regulating the process of AEG-1 gene promoter region; determine how the interaction between HIF and NF-κB; detect whether VEGF as an essential factor of AEG-1 to NF-κB activation. These findings will provide new insights into mechanisms how AEG-1 pathway promoting ovarian cancer invasion induced by hypoxia, and will prove its potential therapeutic target value for ovarian cancer patients.

AEG-1作为新发现的原癌基因与缺氧诱导因子(HIF)-1α诱导的肿瘤血管生成、侵袭及转移密切相关，但其作用机制尚不清楚。我课题组前期研究发现AEG-1过表达普遍存在于伴有大网和淋巴结转移及对铂类不敏感的卵巢癌组织中；同时发现通过稳定抑制卵巢癌细胞AEG-1表达，VEGF及NF-κB的表达量也随之显著降低。根据前期研究基础提出假设：缺氧条件下HIF通过AEG-1与NF-κB之间相互作用促卵巢癌侵袭过程。本研究拟在不同缺氧环境下, 以卵巢癌组织、细胞及裸鼠移植瘤为研究对象，采用免疫共沉淀、免疫荧光、荧光素酶报告系统等技术，明确HIF参与AEG-1基因启动子区调节的转录因子；明确AEG-1与NF-κB之间如何相互作用以及VEGF是否为AEG-1活化NF-κB必要因子。本研究有助于进一步证实AEG-1在卵巢癌侵袭中的关键作用及其机制，为AEG-1特异性靶向治疗提供理论依据。

AEG-1 作为新发现的原癌基因与缺氧诱导因子(HIF)-1α诱导的肿瘤血管生成、侵袭及转移密切相关，但其作用机制尚不清楚。前期研究发现AEG-1 过表达普遍存在于伴有大网和淋巴结转移及对铂类不敏感的卵巢癌组织中；同时发现通过稳定抑制卵巢癌细胞AEG-1 表达，VEGF 及NF-κB 的表达量也随之显著降低。课题根据前期研究基础提出假设：缺氧条件下HIF 通过AEG-1 与NF-κB 之间相互作用促卵巢癌侵袭过程，拟在不同缺氧环境下, 以卵巢癌组织、细胞及裸鼠移植瘤为研究对象，采用免疫共沉淀、免疫荧光、荧光素酶报告系统等技术，明确HIF 参与AEG-1 基因启动子区调节的转录因子；明确AEG-1与NF-κB 之间如何相互作用以及VEGF 是否为AEG-1 活化NF-κB 必要因子。.课题三年的后续研究，检测并评估卵巢癌患者肿瘤组织中AEG-1, NF-κB, 及 VEGF的表达均较非卵巢癌患者表达升高；AEG-1、VEGF与HIF-1α的表达具有相关性；且高表达AEG-1及HIF-1α与患者的组织学类型、转移、FIGO评分及肿瘤大小相关，提示了更差的预后。在体外细胞学实验中，通过RT-PCR和westernblot方法检测了设定不同缺氧时段后(0, 2, 4, 6, 8, 10, 12, and 24h)，AEG-1, NF-κB, VEGF及HIF-1α的mRNA和蛋白水平的表达，发现随着缺氧时间的延长以上四因子的表达逐渐升高，在缺氧8h后达到顶点；然而经过敲除AEG-1后，即使经缺氧处理NF-κB, VEGF表达升高不明显并且不具有统计学意义。在动物学实验中，敲除了AEG-1基因的癌细胞株成瘤能力明显下降，并且成瘤后对照未敲除AEG-1基因的癌细胞株的成瘤组织，其组织中VEGF的表达明显降低。通过以上体内外实验，研究表明AEG-1是卵巢癌在缺氧环境中肿瘤生长、侵袭的重要调控因子，并对肿瘤微血管环境的形成有着关键作用。本研究有助于进一步证实在卵巢癌侵袭中的关键作用及其机制，为AEG-1 特异性靶向治疗提供理论依据。