HOTAIR在胶质瘤超级增强子功能调控中的作用及分子机制

Super enhancers (SE) are large clusters of transcriptional enhancers. A catalog of SEs in a broad range of human cell types had been reported, and these SEs associate with genes that control and define the biology of these cells. Interestingly, cancer cells generate super enhancers at oncogenes and other genes important in tumor pathogenesis. ChIA-PET data had revealed that super-enhancer-driven genes generally occur within chromosome higher ordr structures that are formed by the looping of two interacting CTCF sites co-occupied by cohesin. These looped structures are important for proper expression of local genes. ..HOTAIR expression was closely associated with glioma grade and poor prognosis. In previous study, we performed RNA-seq experiment to uncover the transcriptome changes associated with HOTAIR, and ChIRP-seq experiment to characterize the binding profile of HOTAIR in the genome of U87 cell. Interestingly, about 1/5 glioblastoma super enhancers are associated with HOTAIR. Among them, 9 super enhancers were bound with enriched HOTAIR. These data give out a strong evidence that HOTAIR may play an important role in regulating the functions of super enhancers in glioblastoma...Here we will carry out a series of experiments to study the function and mechanism of HOTAIR in regulating the super enhancers in gliboblastoma. First, we will use RNAi technique to knock down the expression of HOTAIR in glioblastoma model cell lines, and performe chromatin conformation capture-seq experiment to clarify the role of HOTAIR in control the higher order chromatin structure of its associated super enhancers. Then we will use CRISPR-cas9 technique to generate HOTAIR mutant model cell which lost the EZH2 binding motif, to analyze whether the HOTAIR associated PRC2 function is independable in modulating the interaction of super enhancers with their targets. ..Another research interest of this project is whether HOTAIR play a role in formaing the acquired super enhancers in gliboblastoma. We will use ChIP-seq or ChIP-PCR to study the super enhancer catalog and the synergetic effects of HOTAIR and associated factors. We will use 3C-PCR, FISH experiment to confirm the interaction of super enhancers and its target gene promoters, and use knock out experiment to verify the association of super enhancers and its key regulated genes. Finally we will study the role of HOTAIR associated important super enhancers in cancer cell progress, and perfome RNA-seq experiment to analyze the functional network of these super enhancers.

超级增强子已经被发现在包括胶质瘤在内的多种生理病理过程中起到至关重要作用，但肿瘤细胞中超级增强子形成和效应的机制也所知甚浅。前期研究发现HOTAIR这一表观遗传学调控的明星分子在胶质瘤中与超级增强子有密切关系，本课题拟以胶质瘤中HOTAIR对MYC和NOTCH2这两个超级增强子关联基因的调控机制为切入点，从染色质高级空间结构的角度，采用分子生物学、细胞生物学和免疫细胞化学等手段，研究HOTAIR在超级增强子与靶启动子特异性长程相互作用中的调控机制；从HOTAIR和相关转录因子协同作用的角度，探究肿瘤获得性超级增强子形成的机制。本项目可揭示HOTAIR在超级增强子形成和功能调节层面对肿瘤关键基因转录调控的多重复杂网络，发现超级增强子在胶质瘤中作用的直接证据和并部分揭示其分子机制，为将HOTAIR及其相关转录调控机器应用成为胶质瘤新的临床标记物提供理论基础，并可能提供新的临床干预靶点。

LncRNA HOTAIR与胶质瘤的恶性进展密切相关，它可能在增强子，特别是超级增强子形成特定三维基因组结构的过程中发挥重要的调控作用，进而调控关键基因表达影响肿瘤的表型。以往报道的HOTAIR主要通过募集PRC2和LSD1等组蛋白修饰因子抑制抑癌基因表达，从而促进肿瘤恶性进展；本研究通过细胞分子生物学手段研究发现，lncRNA HOTAIR主要通过正调控靶基因的表达促进胶质瘤细胞增殖和转移，其负调控的基因对肿瘤患者生存期没有明显影响。通过对HiChIP-seq、ChIRP-seq、ChIP-seq和RNA-Seq等数据进行生物信息学整合分析，描绘了HOTAIR介导的增强子、超级增强子以及靶基因启动子之间相互作用的图谱，鉴定出了HOTAIR直接调控的、促进胶质瘤恶性进展的关键靶基因和信号通路；HOTIAR介导超级增强子调控GPC6、CACNA1C、FLJ22447、ADAM12、COL1A1、TSHZ2、LOXL1、GDF5等多个参与信号转导和细胞间通讯的重要基因表达，其中TSHZ2基因可作为胶质瘤患者生存的独立预测因子，是潜在的肿瘤治疗新靶点。研究还发现，定位于HOTAIR基因座上的增强子元件可以同染色质长程相互作用调控靶基因的表达，减低了肿瘤细胞对化疗药物替莫唑胺的敏感性。胶质瘤中高表达的lncRNA HOTAIRM1可作为增强子RNA，通过组织染色质相互作用促进肿瘤细胞的增殖和迁移。这些研究成果从染色质高级结构的角度，部分揭示了胶质瘤发生发展的机制，可能为胶质瘤的诊断和精准临床干预提供理论支持。