特异阻断HOTAIR功能结构域与PRC2结合的小分子化合物筛选及其抑制肿瘤侵袭和转移的研究

Non-coding RNA (ncRNA) are functional RNAs that do not encode proteins. NcRNAs include microRNAs (miRNAs), long non-coding RNAs (lncRNAs), tRNAs, snoRNAs, and siRNAs. Different from miRNAs, lncRNAs generally comprise non-protein-coding RNAs that consist of more than 200 nucleotides. Recent reports have indicated that lncRNAs can serve as prognostic markers in various cancer types.We recently discovered that HOTAIR is a cell cycle-related lncRNA in human glioma, and its expression is closely associated with glioma staging and poor prognosis.We also demonstrated that the binding between 5’ functional domain of HOTAIR and EZH2 (component of PRC2 complex) is the key factor for HOTAIR induced uncontrollable growth of glioma cells. We mainly focus on “exploring small molecule inhibitor compounds blocking the formation of HOTAIR 5’ domain-EZH2 network and inhibiting malignant phenotypes of glioma cells” in this study. According to structural modeling and molecular docking, candidate compounds will be obtained. Firstly, H3K27-NLK virus reporting system will be introduced to compare the function of both wild type and mutant type of HOTAIR 5’ domain in recruiting PRC2 complex and to screen the best candidate compound in vitro. Secondly, signaling pathway investigation, gene restoration experiments, gene-protein sequencing analysis techniques will be performed to study the regulation network in Wnt signal downstream pathways of this compound. Finally, tumor invasion and metastasis inhibitory effects of this candidate compound will be examined in an orthotopic mice model.This study will be instructive for finding lncRNA targeted small molecule inhibitors, and has the potential to guide lncRNA based translational medicine research.

申请人前期研究发现：HOTAIR这个lncRNA与胶质瘤的分级、分型和预后相关，其5’功能结构域与PRC2复合物组分EZH2的结合是发挥HOTAIR功能、促进肿瘤无控性增殖的关键因素。本课题集中在“阻断HOTAIR功能结构域与PRC2结合的小分子化合物筛选与逆转肿瘤恶性表型”的方向。在结构模拟和分子对接的基础上获得候选化合物群，应用H3K27-NLK病毒报道系统和免疫沉淀、信号通路分析等方法，在细胞水平比较研究HOTAIR的5’功能结构域及其突变体对PRC2复合物的核募集调控作用、得到最优候选化合物；借助于信号通路分析、基因恢复实验、基因-蛋白质测序分析技术研究该化合物对Wnt下游信号通路的调控网络；最后利用裸鼠原位移植瘤模型研究候选小分子药物对肿瘤侵袭和转移的在体抑制作用。本研究对寻找lncRNA靶向小分子抑制剂的先导化合物具有指导意义，并具有临床转化潜在价值。

长链非编码RNA——HOTAIR被报导参与肿瘤细胞的表观遗传调控，5' 端结合PRC2复合体，3' 端募集LSD1复合体，沉默肿瘤抑制因子的转录。课题组前期工作发现HOTAIR是胶质瘤独立预后因子，与胶质瘤组织中细胞周期蛋白的表达呈正相关，且主要依赖5' 端与EZH2的结合。本项目以HOTAIR-PRC2功能结构特点，以HOTAIR-EZH2结合位点为靶点，通过结构模拟，分子对接和化合物库高通量筛选技术，筛选靶向HOTAIR-EZH2的候选小分子；进一步实验筛选候选小分子化合物，阐明小分子化合物的靶点和作用机制；建立HOTAIR-PRC2调控网络，发现小分子化合物的靶基因和靶向信号通路；探索小分子化合物与其它抑制剂的联合用药方案。本研究确定了两个小分子化合物——AC1NOD4Q（ADQ）和AC1Q3QWB（AQB）能够特异性阻断HOTAIR-EZH2结合，从而影响HOTAIR对PRC2复合体的募集，分别逆转肿瘤抑制因子NLK和APC2的表观沉默，抑制Wnt/β-Catenin信号通路，抑制胶质母细胞瘤和乳腺癌的增殖和转移。另外，低剂量AQB可以增强EZH2抑制剂DZNep的药效，联合用药不仅降低用药剂量，还增强了药物治疗效果。本研究可揭示HOTAIR-EZH2作为新的抗癌靶点，为胶质母细胞瘤和乳腺癌的新治疗途径提供临床前实验依据。