lincRNA-UFC1在肝癌发生发展中的作用及其机制探讨

We have demonstrated that overexpression of lincRNA-UFC1 correlated with tumor recurrence and poor prognosis in patients with hepatocellular carcinoma (HCC). Our data have been proved that miR-34a could bind to lincRNA-UFC1 and decrease its expression level through reducing its half-life. Interaction of lincRNA-UFC1 and HuR leads to stabilization expression of β-catenin, which further promotes the HCC cell epithelial-to-mesenchymal transition (EMT). Based on our preliminary data, we first present that lincRNA-UFC1 is one of the key genes in HCC recurrence. To test the hypothesis, firstly, we will explore the physiological and functional roles of lincRNA-UFC1 in HCC by mouse xenograpt and transgene zebrafish model; secondly, we will deeply understand the mechanism of lincRNA-UFC1 in HCC using RIA-seq，RNA pull down assay and mass spectrometry; last but not least, the effect of lincRNA-UFC1 and its related effectors will be identified by clinical multicenter sample in HCC. From this work, the functional role of lincRNA-UFC1 in HCC progression will be explored and this will be helpful to discover lincRNA-UFC1 as a potential therapeutic target in HCC.

我们预实验证明，lincRNA-UFC1是肝癌复发和预后不良的分子标志物；与miR-34a直接结合，半衰期被缩短从而表达水平降低；与HuR直接结合，进而稳定β-catenin mRNA的表达，促进肝癌细胞的EMT过程。据此我们首次提出：lincRNA-UFC1是肝癌复发转移的关键基因之一。本项目拟通过肿瘤体内外实验及转基因动物实验探讨lincRNA-UFC1在肝癌中的生物学功能；其次利用RIA-Seq、RNA pull down、质谱等技术探讨lincRNA-UFC1促进肝癌复发的分子机制；最后通过临床多中心大样本检测lincRNA-UFC1及其相关蛋白质、miRNA在预警肝癌复发转移及判断预后方面的作用。本项目完成可望揭示lincRNA-UFC1在肝癌发生发展中的作用机制，对肝癌复发转移、预后判断具有重要的理论及临床价值，为肝癌治疗的新靶点提供充分的理论依据。

lincRNA-UFC1是新发现的lincRNA，我们探讨其在肝癌中的生物学功能并探讨相应分子机制。我们首先利用芯片筛选出7对癌及癌旁组织中差异表达的lncRNA，随后利用qRT-PCR、原位杂交等方法检测肝癌组织中lincRNA-UFC1的表达，分析lincRNA-UFC1表达与肝癌患者预后的关系。接着，分别构建lincRNA-UFC1稳定过表达及敲除的肝癌细胞株，利用MTT、体外侵袭、迁移实验、裸鼠皮下成瘤等实验明确lincRNA-UFC1在肝癌中的生物学功能，最后利用双荧光素酶报告实验、RIP、RNA pull down、western blot等实验探讨lincRNA-UFC1促进肝癌增殖、侵袭及转移的分子机制。结果表明lincRNA-UFC1在肝癌中高表达，其高表达与肝癌患者不良预后相关，体内外实验证明lincRNA-UFC1促进肝癌细胞增殖、侵袭及转移。LincRNA-UFC1与HuR直接结合，从而促进HuR靶基因β-catenin的表达，肝癌组织中lincRNA-UFC1与β-catenin表达呈正相关关系；而miR-34a与lincRNA-UFC1结合降低lincRNA-UFC1的表达。总之，lincRNA-UFC1作为miR-34a的靶基因，通过与HuR直接结合促进β-catenin的表达，进而促进肝癌细胞的增殖、侵袭及转移。.同时我们发现，受linRNA-UFC1调控的miR-139-5p，其在肝癌中表达下调。随后我们利用体内外实验探讨miR-139-5p在肝癌中的生物学功能，并用双荧光素酶报告实验、ChIP、western blot等实验阐明miR-19-5p抑制肝癌增殖的分子机制。结果表明过表达miR-139-5p能抑制肝癌细胞的增殖、侵袭、迁移及糖酵解。MiR-139-5p通过与ETS1（ETS1能与HK1及PFKFB3的启动子结合进而促进HK1及PFKFB3的表达）的3’-UTR结合抑制其表达，从而下调HK1及PFKFB3的表达。过表达ETS1能逆转miR-139-5p对肝癌细胞增殖、侵袭、迁移的抑制作用。而ETS1能够通过转录后水平调控Drosha的表达，进而抑制miR-139-5p的表达。上述结果表明miR-139-5p与ETS1形成负反馈环路调控肝癌糖酵解、增殖及转移，这为肝癌治疗提供新治疗靶点。