RI与Angiogenin相互作用调控PI3K/AKT/mTOR信号通路和ANG的核转位在膀胱癌发生发展中的机制研究

Recent research showed that Angiogenin (ANG) could effectively promote angiogenesis and tumor cell proliferation. ANG plays an important role in the development of cancer. ANG might stimulate ribosomal RNA transcription and the ribosome biogenesis through nuclear translocation and PI3K/Akt/mTOR signaling pathway. The experiment demonstrated that Ribonuclease inhibitor (RI) could inhibit ANG activity by binding to it in vitro. However, the underlying mechanism remains largely unknown to date. Our previous works suggest that RI might have tumor suppressor gene function. The experiments also showed that RI and ANG have a colocalization and interaction between them with Laser Scanning Confocal Microscope and Co-Immunoprecipitation. Moreover we found that RI and AKT phosphorylation express have a negative correlation. Therefore, we suppose that RI could block nuclear translocation of ANG and regulate PI3K/AKT signaling pathways and related signaling pathways cross-talk to inhibit tumor growth and metastasis. Using bladder cancer as a model, we would elucidate the relationships among the expressions of RI, ANG and PI3K/AKT pathways molecules and tumor growth as well as metastasis in cancer cells and clinical specimens; We are going to get direct evidence that RI could combine with ANG and to confirm their interaction in vivo; In addition, we will explore whether RI expression might adjust ANG activity，function and effects in vitro and in vivo. The purposes of our study are to further reveal the molecular mechanism of RI in inhibiting tumor growth and metastasis and to provide new molecule target for bladder cancer therapeutics.

新近研究表明血管生成素(ANG)能有效促进血管生成和肿瘤细胞增殖，在肿瘤发生发展中起重要作用。ANG通过核转位和激活PI3K/AKT/mTOR信号通路刺激rRNA转录和核糖体生成。体外实验证明核糖核酸酶抑制因子(RI)能与ANG结合而抑制其活性。然而，机制尚不清楚。前期工作发现RI具抑癌基因功能；RI与ANG在细胞内共定位；Co-IP初步证实其相互作用；RI与AKT磷酸化表达负相关。因此，推测RI可能结合ANG封锁其核转位和调控PI3K/AKT/mTOR信号通路及其相关通路cross-talk抑制肿瘤生长及转移。拟以膀胱癌为模型，明确RI与 ANG和PI3K/AKT通路的分子在细胞及临床标本中的表达与肿瘤细胞生长及转移的关系；获取RI与ANG在体内结合的直接证据及证实相互作用；在细胞和动物模型上探讨RI调节ANG活性、功能及其作用。旨在阐明RI抑制肿瘤的机制并为膀胱癌治疗提供新的分子靶点。

项目的背景 .新近研究表明血管生成素(ANG)能有效促进血管生成和肿瘤细胞增殖，在肿瘤发生发展中起重要作用。ANG通过核转位和激活PI3K/AKT/mTOR信号通路刺激rRNA转录和核糖体生成。体外实验证明核糖核酸酶抑制因子(RI)能与ANG结合而抑制其活性。然而，机制尚不清楚。前期工作发现RI具抑癌基因功能；RI与ANG在细胞内共定位；Co-IP初步证实其相互作用；RI与AKT磷酸化表达负相关。因此，推测RI可能结合ANG封锁其核转位和调控PI3K/AKT/mTOR信号通路及其相关通路cross-talk抑制肿瘤生长及转移。旨在阐明RI抑制肿瘤的机制并为膀胱癌治疗提供新的分子靶点。.主要研究内容 .观察RI和ANG在细胞中的定位；采用GST pull-down、Co-IP及FRET和定点突变实验及生物信息学等证明和确认RI与ANG在体内外的结合和相互作用。获取RI与ANG相互作用的可靠证据；在前期工作的基础上，发现RI可能与ANG相互作用参与调节PI3K/AKT/mTOR信号通路和抑制ANG核转位，进一步在细胞、临床标本和动物水平上阐明RI表达能够调节ANG活性、功能及其PI3K/AKT/mTOR信号通路和相关通路间的交互应答（cross-talk）并影响肿瘤的血管生成、生长及转移。从一个全新的角度深入探索RI的功能，阐明RI抑制肿瘤生长转移的分子机制。.重要结果、关键数据.1.证明了RI与ANG在体内的直接结合及相互作用，从而解除其RNase的功能并阻止了ANG的核转位和促rRNA转录活性；获取了RI对ANG促rRNA基因的启动子转录活性影响可靠证据。.2．通过本项目的系统研究,发现了RI与ANG的相互作用途径，从调控ANG介导的PI3K/AKT/mTOR信号通路及其相关通路间的交互应答(cross-talk)和封锁了ANG核转位和核酸酶活性中心，抑制了ANG促rRNA转录活性，导致阻断其促肿瘤血管生成和细胞增殖功能的角度揭示了RI抑制肿瘤生长和转移新颖机制。.3．RI可望成为一个有希望的抗肿瘤蛋白新药和血管生成抑制剂，在肿瘤防治方面具有重要的理论和实践意义。.4.在国际杂志上发表SCI论文5篇, 培养研究生5名。.科学意义.该项目的科学意义旨在通过其系统研究, 以RI可能与ANG相互作用参与调节PI3K/AKT/mTOR信号通路和抑制ANG核转位入手，从一个