长链非编码RNA CUDR在肝干细胞分化和恶变中的作用机制

Hepatic stem cells are a class of infinite proliferative capacity, self-renewal and differentiation into hepatic cells and bile duct cells. Differentiation of liver stem cells is affected by many factors and involved in a variety of regulatory factors and complex signaling pathways network. Hepatocarcinogenesis is closely associated with blocked differentiation of liver stem cells. Epigenetic modifications and chromatin reprogramming play an important role in the abnormal differentiation of liver stem cells，and the non-coding RNA is one of the novel ways as epigenetic modifications. The non-coding RNA CUDR was highly expressed in human liver cancer cells, but its functional mechanism is unknown. In this study, we will separate and induce the liver-derived and non-liver-derived liver stem cells, then to study whether CUDR overexpression or knockdown effects the development of liver stem cells and its malignant transformation, while taking advantage of CUDR knockout and transgenic mice, and high-throughput the transcriptome / proteomic detection means ,we will further analyse whether CUDR actions the key signaling molecules (e.g., CTCF), signaling pathway network, chromatin reprogramming, epigenetic modification and indicate its molecular mechanism during the development and hepatocarcinogenesis from liver stem cells in vivo. We will try to reveal CUDR's key functions in the regulation of hepatic stem cell fate, and provide a comprehensive understanding of the hepatic stem cellular differentiation control mechanisms in a novel perspective which will contribute to promte a new direction for the study pathogenesis and cure of malignant liver cancer.

肝干细胞是一类具有无限的增殖能力、自我更新和能向肝细胞和胆管细胞分化的细胞。肝干细胞的分化受多种因素影响，涉及多种调控因子和复杂信号通路网络。肝癌的发生与肝干细胞的分化受阻有关，表观遗传修饰和染色质重编程在肝干细胞的异常分化中起了重要，非编码RNA 是新近发现的表观遗传修饰方式之一。非编码RNA CUDR在人类肝癌细胞中高表达，但其作用机制不详。本研究通过分离和诱导肝源性和非肝源性肝干细胞，然后研究CUDR过表达或缺失对肝干细胞的发育及其恶性转变的作用，同时利用CUDR敲除、转基因小鼠和高通量的转录组学／蛋白质组学等检测手段进一步分析CUDR对肝干细胞体内发育和癌变中关键分子（如CTCF）、信号通路网络、染色质重编程、表观遗传修饰的影响及其分子机制，从而揭示CUDR在调控肝干细胞命运上的重要功能，并为全面认识肝干细胞的分化调控机制提供新视角，也为研究肝癌发病机制及根治恶性肝癌提示新的方向。

肝癌的发生与肝干细胞的异常分化有关。表观遗传修饰在肝干细胞的异常分化中起重要作用。本研究发现肿瘤中上调表达的长链非编码RNA CUDR一方面能通过调控组蛋白3第27赖氨酸的三甲基化修饰诱导胚胎干细胞向肝样干细胞分化；另一方面，过量的CUDR又能通过抑制非编码RNA HULC启动子的甲基化和促进β-catenin的启动子－增强子间DNA环的形成引发了胚胎干细胞的恶性转化。本研究表明异常的组蛋白甲基化转移酶SET1A 和CUDR协同作用加速了胚胎干细胞诱导的肝干细胞的恶变进程。显然,CUDR增加了RB1的磷酸化修饰和增强了pRB1和甲基化转移酶SET1A间的相互作用。CUDR作为海绵垫连接了SET1A 和 pRB，并且激活了pRB1-SET1A。该复合物结合到组蛋白3第四赖氨酸位点，特异性地增加了H3K4me3修饰。因此，更多的H3K4me3结合到了TRF2的启动子区域，导致了 TRF2 的过量表达。最终过量的TRF2与端粒结合，延长了端粒的寿命。本研究发现CUDR协同IL6促发了人类胚胎干细胞诱导的肝样细胞的恶性转化。进一步研究显示CUDR协同IL6引发METTL3与组蛋白甲基化转移酶SUV39h1 mRNA 3’UTR结合，促进了SUV39h1稳定表达。过量的SUV39h1又能导致更多的H3K9me3修饰，接着又依赖H3K9me3增强了肝样细胞中的NF-κB表达和磷酸化修饰，并且pNF-κB促进了Stat3的表达和磷酸化修饰。又发现磷酸化的Stat3 与miRs and lncRNAs的启动子结合，从而改变了肝样细胞中的关键micoRNAs和lncRNAs（如MEG3）的表达。我们证明过表达的CUDR在过量的CyclinD1或缺失PTEN的背景下，通过改变TERT和C-myc表达而加速肝癌干细胞的恶性生长。同时又揭示双突变P53(N340Q/L344R)在PKM2介导下上调Pim1。我们的结果还显示：HOTAIR通过下调SETD2、HULC协同MALAT1通过端粒结合蛋白2、IKKα/β/γ通过异染色质蛋白1-HOTAIR 改变端粒分别加速了肝癌干细胞的生长。另外，还发现在肝癌细胞中miR675通过激活EGR1上调非编码RNA　H19表达。本研究首次证明了CUDR及其相关基因在人类肝癌干细胞的恶性进展中起重要作用，这些结果为研究肝癌发病机制及根治肝癌提示新的方向。