长链非编码RNAZFAS1结合HuR激活Wnt-β-catenin通路促进结肠癌转移的机制研究

We previously discovered that up-regulated LncRNAZFAS1 in colon cancer tissues and patients’ plasma could potentiate the progression of epithelial to mesenchymal transition (EMT) , which was significantly associated with regulation of ZEB1 expression. It was confirmed that there were β-catenin/TCF4 binding sites in the promoter area of ZEB1,while activation of Wnt-β-catenin signaling pathway could increase the β-catenin nuclei translocation and enhance the transcriptional activity of ZEB1.Furthermore, pilot RNA immunoprecipitation (RIP) assay validated none of the endogenous direct binding between lncRNA ZFAS1and β-catenin, but bioinformatics prediction algorithms indicated there were putative binding sites of HuR in LncRNAZFAS1. HuR had been shown to increase the β-catenin mRNA nuclei translocation. On the basis of above findings,we propose that LncRNA ZFAS1 might interact with the mRNA stabilizing protein HuR to promote the metastatic progression of colon cancer via activation of Wnt-β-catenin signaling pathway. In this project, a series of in vitro and in vivo assays will performed to demonstrate the signaling axis by which LncRNA ZFAS1 interact with HuR and then activate the key Wnt-β-catenin signaling pathway of colon cancer based on the preceding results.The project would extend our knowledge of the metastasis cascade of colon cancer, and might provide new insights into how to block these signals and their transduction processes.It would also be facilitating individualized treatment programs.

前期发现ZFAS1在结肠癌组织及血浆中表达增高并促进结肠癌细胞上皮-间质转化，与调控ZEB1表达有关。ZEB1启动子区域存在β-catenin/TCF4结合位点，Wnt-β-catenin通路激活后胞质β-catenin入核增加并与TCF/LEF家族成员结合形成复合物增强ZEB1转录。预实验显示ZFAS1与β-catenin无法直接结合，生物信息学显示ZFAS1与HuR存在结合位点，且HuR促进β-catenin mRNA入核。据此推测ZFAS1可能与HuR结合增加β-catenin入核，进而激活Wnt通路、增强ZEB1转录，加速结肠癌细胞上皮-间质转化进程，影响结肠癌的转移。本项目将验证ZFAS1对结肠癌转移的影响，探讨其通过HuR影响Wnt通路并调控下游信号的分子机制，加深对结肠癌转移级联反应机制的理解，并可能为有效地干预结肠癌转移的信号转导环节提供新线索，有利于制定个体化治疗策略。

本项目拟综合运用分子生物学、生物信息学、细胞学、动物模型和医学统计学等方法，围绕lncRNA ZFAS1的表达水平、功能以及在其结肠癌细胞发挥生物学功能的具体分子生物学机制进行研究，阐明lncRNA ZFAS1可能通过结合蛋白HuR 影响Wnt-β-catenin 信号通路从而调控结肠癌细胞的上皮-间质转化及结肠癌转移的分子机制，可以丰富结肠癌相关分子生物学理论，为结肠癌预后评估及治疗提供新的策略和靶点，同时对理解肿瘤精准治疗有重要意义。包括以下方面：1）lncRNA ZFAS1在结肠癌中的表达变化及其功能的系统研究；2）lncRNA ZFAS1对Wnt-β-catenin的表达水平的影响的研究；3）lncRNA ZFAS1与其结合蛋白HuR相互结合的研究；4）HuR与Wnt-β-catenin的相互作用在结肠癌中的分子机制研究。本研究运用 RNA-pulldown、RNA 结合蛋白免疫沉淀及实时定量 PCR 等技术，发现RNA结合蛋白HuR与ZNFAS1存在结合，同时HuR与β-catenin存在结合，且ZNFAS1能够在转录和蛋白水平调控HuR；shRNA干扰和基因过表达转染技术，双向调节结肠癌细胞HuR表达水平，利用分子生物学技术如：qRT-PCR、Cell Counting Kit-8 kit (CCK-8) 、Western blotting和流式细胞术验证HuR 转染前后的表达水平并观察细胞增殖、凋亡、迁移、侵袭、转移潜能的变化以及对Wnt-β-catenin靶基因表达水平的影响；同时调控ZFAS1和HuR的表达，利用分子生物学技术如： CCK-8 、Western blotting回复实验验证ZFAS1通过调控HuR介导Wnt-β-catenin通路；TOP/FOP活性进一步确认ZFAS1对Wnt-β-catenin通路的调控依赖于HuR；在动物模型整体水平上比较 lncRNA ZFAS1过表达载体、HuR 抑制载体等分别或联合应用与 Wnt-β-catenin 通路抑制剂(XAV-939)动物成瘤的影响，评估其应用于结肠癌预防的潜能。并通过大样本临床病例分析 ZFAS1 及其调控分子的相关性及意义，采用多因素回归模型等统计学方法建立方程，构建出预测结肠癌转移的多分子联检模型。