lncRNA介导的染色质三维空间架构对肝癌干细胞的调控机制研究

Several evidences suggest that the three-dimensional chromatin structure plays a critical role in transcriptional regulation and other biological processes. However, the regulatory role of three-dimensional chromatin structure and the underlying mechanism still remain elusive in Cancer Stem Cells (CSCs), which are considered as the root of cancer recurrence and metastasis. Our preliminary work had successfully sorted Side Population (SP cells) form HepG2 which were rich in hepatocellular carcinoma cancer stem cells (HCCSCs), with obvious properties of stem cells. Meanwhile, we developed a new HiC-RNA method to screen the lncRNAs which participate in the chromatin interactions in the whole genome and illustrated lncRNA-mediated three-dimensional chromatin structure had an important function in the process of cell senescence. Therefore, we proposed a hypothesis that lncRNA-mediated three-dimensional chromatin structure is an important factor in regulation of HCCSCs. In this study, we will use SP cells as model system, to construct the interaction map of HCCSCs through Hi-C (High-throughput / resolution chromosome conformation capture) and screen the target lncRNA involved in the three-dimensional structure of chromatin, utilizing HiC-RNA, RNA-Seq, ChIRP-Seq (Chromatin isolation by RNA purification), FISH and other methods. Furthermore, we will use knock-down assay to analyze the mechanism of regulatory function of lncRNA-mediated three-dimensional chromatin structure in HCCSCs. Taken together, this application will deepen our understanding towards regulation mechanism of CSCs from a new perspective, and may also provide new strategy into clinical intervention therapy for hepatocellular carcinoma.

基因组三维空间架构在转录等生物学过程起重要调控作用,但其对癌症复发转移“根源”的癌症干细胞的调控作用和机制不明。我们前期成功分选出富含高纯度肝癌干细胞的HepG2侧群细胞并开发了在全基因组范围研究参与染色质互作的lncRNA的新方法（HiC-RNA），且发现lncRNA参与形成染色质三维空间架构并在细胞衰老中起关键调控作用。由此提出假说：lncRNA介导的染色质三维空间架构是调控肝癌干细胞的重要因子。为验证此假说，本项目将以HepG2侧群细胞为研究模型，采用Hi-C技术构建肝癌干细胞染色质互作图谱，应用HiC-RNA结合RNA-Seq、CHIRP-Seq、FISH等方法筛选、研究参与介导染色质三维架构的lncRNA，并通过敲降实验解析其介导的染色质三维空间架构对肝癌干细胞的调控机制。本项目将从三维基因组结构这一新视点加深我们对肝癌干细胞调控机制的认识，为肝细胞癌临床干预治疗提供新思路。

研究表明具有自我更新和分化潜能的肝癌干细胞是肝细胞癌复发、侵袭、转移及耐药的根源。目前，针对肝癌干细胞的靶向治疗主要从四个方面入手：鉴定特异的表面标志物；调控异常激活的信号通路；改变肝癌干细胞的微环境；解析肝癌干细胞干性维持的表观调控机制。而肝癌干细胞与正常肝干细胞的相似性极大地限制了现在已经发现的细胞表面标记物、相关信号通路在靶向治疗中的应用。虽然对肝癌干细胞的表观调控机制已有诸多报道，但染色质高级结构在其干性维持方面的功能机制尚不明确。.鉴于我们前期从肝癌细胞系和新鲜肝癌组织样本中分离出的SP细胞（Side Population，侧群细胞）具有相同的肝癌干细胞特性；同时考虑到肝细胞癌肿瘤样本的异质性及个体差异，我们采用一种简单确定的模型，即从肝癌细胞株中分离SP细胞，以此为研究对象，通过增殖实验、Transwell迁移侵袭实验、细胞球形成实验等对其进行干性检测；利用RNA-seq系统鉴定SP细胞特有的基因表达模式，通过对SP细胞和NSP（Non-SP）细胞的HiC、ATAC-seq以及公共的ChIP-seq数据资源的综合分析，探究SP细胞中染色质区室Compartment A/B的变化与特有的基因表达模式之间的关系，寻找SP细胞中特异的关键转录因子和靶基因。在本项目支持下，我们构建了肝癌干细胞染色质相互作用图谱，系统解析了表观遗传变化尤其是染色质高级结构在肝癌干细胞干性维持中的作用机制，有效地鉴定出一组靶向基因治疗的药物靶点，并提出Nr5a2可能通过转录调控提高CYP1B1的表达进而调控肝癌干细胞的干性维持。这一研究有助于加深我们对肝癌干细胞干性维持机制的认识，为肝癌的靶向治疗提供新的诊疗方案。