neurotrophin-3激活HIF-1α促进肿瘤血管生成的分子机制研究

Angiogenesis is intimately engaged in all processes of tumor development, progression and prognosis. Various studies demonstrated that cancer cells can release various soluble factors or protien to affect angiogenesis in a paracrine or autocrine manner. Furthermore, the biological characteristics of angiogenesis and tumor cells can induce positive feedback loop between them. However, the underlying mechanism is still largely unknown. Our previous data indicated that co-culturing with gastric cancer cells can increase angiogenesis. Most interestingly neurotrophin-3 (NT-3), which plays an important role in growth, differentiation and stability in neuron, is the main regulator. However, acting as tumor-secreted protein, its effect on gastric cancer, especially in tumor angiogenesis is still unclear. Based on the results, our study intend to detect the expression of NT-3 in gastric cancer tissues and cells, uncovering their correlation, to explore the effect on angiogenesis in gastric cancer and to clarify the mechanism of NT-3 inducing tumor angiogenesis through the activating of HIF-1α mediated by PI3K/PTEN/AKT pathway and the interaction between NT-3 and HIF-1α through a series of molecular biological method. It provides a novel insight into the mechanism of tumor-promoting angiogenesis and may contribute to the development of therapeutic applications.

新生血管形成与肿瘤发生、发展及预后密切相关。肿瘤细胞分泌多种细胞因子或蛋白，以旁分泌或自分泌的方式影响血管生成，新生血管-肿瘤细胞的生物学特征使二者形成正反馈环路，但其关键分子及作用机制尚不明确。我们前期研究证实，与胃癌细胞共培养可增加血管形成能力，并成功筛选出可发挥促进神经元生长、诱导分化及维持稳定作用——神经营养因子3（NT-3）为促进肿瘤血管生成的关键因子。但是NT-3作为肿瘤分泌蛋白在胃癌，尤其是胃癌新生血管中作用尚不清楚。因此，本研究拟采用分子生物学方法①检测胃癌组织及细胞中NT-3表达，明确其与胃癌相关性；②通过增加或降低细胞中NT-3表达，在体内外探讨NT-3对胃癌新生血管生成作用；③ 阐明NT-3诱导PI3K/PTEN/AKT信号通路激活HIF-1α促进胃癌血管生成分子机制，及NT-3与HIF-1α作用关系，为揭示肿瘤细胞促进血管生成及抗肿瘤治疗提供新的理论依剧。

胃癌作为消化系统最常见的恶性肿瘤，在亚洲国家位居所有肿瘤致死率的第二位，胃癌的发生发展、预后不良与组织中血管生成密切相关。研究表明，血管生成发生于肿瘤组织形成的早期阶段，是启动肿瘤生长、侵袭、转移的关键步骤。然而，促进血管生成的关键分子及作用机制尚不十分明确。前期试验中我们采用共培养系统发现胃癌细胞可促进体外血管生成，通过收集细胞培养液上清，利用细胞因子芯片技术，筛选出神经营养因子-3（NT-3）作为促进血管生成的关键因子。NT-3是神经营养因子家族成员，近来研究发现其在多种肿瘤中发挥重要的调节作用。本研究采用多种分子生物学方法①发现NT-3在胃癌组织及细胞中表达显著升高，且NT-3高表达水平与肿瘤分化程度、TNM分期、淋巴转移及患者不良预后密切相关，而与患者的年龄及性别无关。另外，NT-3表达对胃癌具有一定的诊断价值；②MTT试验显示利用内源性过表达或外源性刺激NT-3均可促进胃癌细胞增殖；③双荧光素酶报告基因表明miR-200c/b通过与NT-3的3’UTR结合调节NT-3表达，进一步分析显示在胃癌组织中NT-3与miR-200c/b表达呈显著负相关。另外，本研究还发现过表达NT-3后细胞中HIF-1α表达水平明显增加，且PI3K/AKT信号通过被激活。利用siRNA技术下调过表达NT-3细胞中HIF-1α水平，发现胃癌细胞增殖能力显著下降，且HIF-1α下游分子VEGF及MMP表达亦显著降低。因此，本研究结果显示NT-3在胃癌的发生发展中发挥癌基因功能，且可被miR-200c/b调节并通过诱导HIF-1α激活发挥作用，这为阐明胃癌发生发展、血管生成提供新的理论依据，为治疗肿瘤提供新的分子靶点。