药用资源植物麻疯树核糖体失活蛋白家族抗肿瘤活性及其作用机制的研究

Jatropha curcas, a member of Euphorbiaceae, is a traditional medicinal resource plant in tropical and subtropical regions. The ribosome inactivating proteins (RIPs) found its endosperm and cotyledons have been proved to have unique antitumor activities. Based on previous investigation, the project plans to systematically predict and identify the members of RIPs family in Jatropha curcas genome. The expression profiles of candidates would be invested by the transcriptome data, the promoter sequences analyzation, followed by fluorescence quantitative PCR, Western blot and reporter gene expression verification. About 4-8 RIP candidates with typical structural differences would be prepared by direct purification from plant materials or via prokaryotic expression systems. Their anti-tumor activities would be studied and 2-4 RIPs candidates of significant differences in activities would be used to further action mechanism survey, including their position and distribution in tumor cell surface and internal structures. The expression changes of tumor cells at transcriptional level would also be analyzed by doing RNA seq to uncover the molecular mechanism of jatropha RIPs inhibiting tumor cell proliferation in vitro. The implementation of the project is expected to provide scientific information and basis for the modern utilization of this medicinal plant widely distributed in the world and the exploration of new antitumor drugs.

大戟科植物麻疯树是热带和亚热带地区的传统药用资源植物，其胚乳和子叶中的核糖体失活蛋白（RIPs）已被证明具有独特的抗肿瘤活性。本项目拟在已有的研究基础上，对麻疯树RIPs基因家族进行系统的预测和鉴定工作，对转录组数据、上游启动子区序列进行分析，采用荧光定量PCR、Western杂交结合报告基因的方式，摸清麻疯树RIPs的表达模式（时空和环境互作模式），采用直接纯化手段或原核表达系统制备4-8个具有典型结构差异的蛋白样品，比较它们的抗肿瘤活性，并通过调查2-4个具有显著活性差异的样品在肿瘤细胞表面和内部的定位和分布，以及处理后肿瘤细胞在转录组水平的基因表达变化，初步阐明其体外抑制肿瘤细胞增殖的分子机制，为这一在世界广泛分布的药用植物资源的现代化开发和在抗肿瘤药物开发领域的应用提供科学依据。

项目以传统药用资源植物麻疯树为对象，对具有抗肿瘤活性的麻疯树核糖体失活蛋白（JcRIPs）家族进行预测和鉴定工作，挖掘出12个成员，根据等电点分为酸性和碱性两组，除JcRIP10外，均为I型RIP。JcRIPs的三级结构具有较高的相似性，差异主要存在于结构域Ⅲ。构成Ricin活性位点的 10个氨基酸中有7个在JcRIPs中十分保守。分子对接结果显示， 7个成员与底物分子腺嘌呤之间的互作方式存在较高的相似性， 4个成员（JcRIP2.1、JcRIP2.2、JcRIP4和 JcRIP7.2）与底物的结合能较低，推测可能具有较高的N-糖苷酶活性。大部分成员的表达值都极低，仅少数（JcRIP2和JcRIP4）在种子中有高表达。JcRIP 基因启动子区包含多个光、生长发育、胁迫和激素响应元件，胁迫处理后都有成员呈表达上调趋势，暗示JcRIPs在应答环境胁迫中的可能作用。采用原核表达系统制备了全部12个成员的重组蛋白并比较了它们的抗肿瘤活性，发现除curcin和curcin C以外的另外两个高抗肿瘤活性成员（JcRIP3和JcRIP12）。转录组数据分析表明curcin C对MAPK信号通路会产生明显影响，但curcin C对骨肉瘤的抗肿瘤活性机制是多路径、多个基因相互协调作用的结果，进一步的实验证明Curcin C可能通过诱导活性氧的积累激活JNK信号途径抑制ERK信号通路，从而导致人骨肉瘤U2OS细胞在S、G2/M期阻滞、出现凋亡和自噬，使得肿瘤细胞的增殖受阻。通过内吞抑制剂、基因沉默等技术发现Curcin和Curcin C的内化效率和进入途径存在差异，而导致两者活性差异的主要原因是curcin无法通过LRP1介导的网格蛋白依赖性内吞途径进入细胞。综上，本项目获得的信息为这一在世界广泛分布的药用植物资源的现代化开发和在抗肿瘤药物开发领域的应用提供了科学依据。