LncRNA-Neu对人胚胎干细胞神经分化的调控作用和机制研究

Problems in neural development are the basis of neural tuber defects (NTD). In vitro, ES cells can differentiate into neural epithelial cells, therefore they are an excellent model to study the molecular mechanisms underlining neural development, and also to highlight the pathologic research in NTD. Our recent study found that the expression of lncRNA-Neu was up-regulated during the neural differentiation from human ES cells based on the RNA-Seq data. Further study found that inhibiting lncRNA-Neu could significantly impair the ability of human ES neural differentiation. Our preliminary data showed that lncRNA-Neu might affect the histone epigenetic modifications during neural differentiation and be involved in the transcriptional regulation of key molecules in the early stage of neural differentiation. Therefore, this project tried to systematically research the functions of lncRNA-Neu during neural differentiation and use the Tet-On inducible transgenic system to elucidate the lncRNA-Neu “functional window” during the differentiation. Furthermore, we would explore the molecular mechanisms of lncRNA-Neu and speculated that lncRNA-Neu might regulate the neural differentiation through forming the RNA- epigenetic enzymes complex with techniques such as RNA pull-down, RIP and ChIP-Seq. Finally, signaling pathways chip and reporter gene expression system will be uesd to further explore and validate the target genes and signaling pathways of lncRNA-Neu. Our study will not only reveal the mechanisms and functional roles of lncRNA-Neu during neural differentiation of human ES cells, but also provide a target for the optimization of in vitro neural differentiation system and therapies for NTD.

人胚胎干细胞（hES）神经分化体系是研究神经发育的理想模型，基于神经分化过程中的高通量数据分析，我们发现lncRNA-Neu在hES神经分化过程中上调表达；初步研究显示，抑制lncRNA-Neu能显著影响hES的神经分化能力，并发现lncRNA-Neu可能在神经分化过程中影响组蛋白修饰状态而调控神经分化早期关键转录因子的表达。在此基础上，本研究将系统地研究lncRNA-Neu在神经分化中的功能，确定lncRNA-Neu起作用的时间窗；并运用RNA pull-down、RIP、ChIP-Seq等技术探索lncRNA-Neu是否与特定表观遗传酶复合物结合而调节hES的神经分化及其作用机制，进一步运用信号转导通路芯片和报告基因系统探索调控lncRNA-Neu的信号转导通路。该研究将揭示lncRNA-Neu在hES神经分化中的功能和机制，为优化神经分化体系和神经发育疾病发病机制研究提供基础。

lncRNAs广泛存在于神经系统中，其功能尚不明确。本项目基于干细胞神经分化系统，研究一条跨越SOX1的lncRNA SOX1-OT（即LncRNA-Neu），发现无论是在背侧皮层分化，还是在腹侧MGE分化中，抑制SOX1-OT既不影响人ESC分化成为神经上皮细胞，也不影响其分化成为特定区域的神经祖细胞，但却不能进一步分化成为神经元，显示其表达对于神经元发生是至关重要的。本项目发现SOX1-OT能够通过其5’端与HDAC10结合，缺失SOX1-OT后，HDAC10在SOX1基因组区域结合增加而导致SOX1基因组区域乙酰化水平的降低，显示SOX1-OT作为HDAC10的decoy，充当了内源HDAC10的抑制剂而维持了SOX1的表达，进而促进了神经元的分化。作为SoxB1家族中的一员，SOX1在神经细胞的命运决定和神经元的分化中发挥重要的作用。本项目研究表明，过表达SOX1能够促进ASCL1的表达，且SOX1能够直接结合到ASCL1的启动子区，表明SOX1是通过直接激活ASCL1而促进神经元的分化。该研究将有助于我们更好的理解长链非编码RNA如何与表观修饰分子共同调控下游基因的表达，并进一步揭示人脑神经元分化过程中的调控机制。