干细胞分化中的长非编码RNA功能鉴定及调控机制研究

More than third quarters of human genome is transcribed and could generate thousands of long noncoding RNAs (lncRNAs) with the size over 200 nucleotides. LncRNA is capable to regulate gene expression and actively participates in various biological processes, but very little is known regarding the functional link between lncRNAs and human early development. In addition, the studies of human early development and germ-layer specification are largely delayed due to the very limited access to human embryo materials, thus human pluripotent stem cells are considered as a promising alternative. Previously we have profiled the transcriptome of human endoderm lineage and identified dozens of lncRNAs which are specifically expressed in human endoderm cells. Some of those lncRNAs are located close to endoderm-related genes, some are located close to the gene which seems not functional in endoderm development, and the rest are located in gene-desert. Following our preliminary data, this proposal will combine the cutting-edge techniques of stem cell differentiation and CRISPR/Cas9-based genome editing, to investigate the roles of those three classes of lncRNAs in human endoderm differentiation and cell fate determination. The completion of this proposal will not only reveal several novel functional lncRNAs in human endoderm differentiation, but also provide invaluable insights for lncRNA-centered regulatory network in cell fate determination.

人类基因组超过3/4可以转录，产生数万个长度大于200碱基的长非编码RNA。长非编码RNA能够调节基因表达，已知参与多个生物学过程，但在人早期发育和干细胞分化方向的研究还很缺乏。另外，人类早期胚胎发育的研究极大地受制于难以获得合适的人胚胎样品，故人多能性干细胞被认为提供了一个极好的补充。我们最近获得了人内胚层谱系的动态转录组数据，发现了数十个内胚层特异表达的长非编码RNA。这些长非编码RNA在基因组上有些位于内胚层相关编码基因附近的，有些其临近基因和内胚层分化尚无关联性报道，还有一些附近不存在已知的编码基因。因此，本项目拟联合基因组编辑技术和干细胞分化技术来系统研究这几类长非编码RNA在人内胚层细胞分化中的作用，并寻找其行使功能的分子机制。我们相信，完成该项目不仅可以揭示出更多的新的有生物学功能的长非编码RNA，同时也将为长非编码RNA为中心的调控网络提供新的视角和思考。

人类基因组可以转录产生数万个长度大于200碱基的长非编码RNA。这些长非编码RNA能够调节基因表达，但在人早期发育和干细胞分化是否有生物学功能还知之甚少。在本项目中，我们利用人胚胎干细胞往内胚层的定向分化体系，分析了人内胚层谱系的动态转录组数据，发现了数十个内胚层特异表达的长非编码RNA。我们按这些长非编码RNA在基因组位置附近是否有编码基因进行分类研究。通过shRNA敲低以及基因组启动子敲除实验，我们证明了多个长非编码RNA对内胚层分化是必要的。进一步的分子机制显示，GATA6-AS1可以通过与核内的SMAD2/3相互作用调节GATA6的转录，lncM可以通过与胞质的IMP互作影响下游WNT信号分子稳定性。利用内胚层胰岛分化体系，我们也进一步探索了长非编码RNA在胰岛发育分化中的作用。通过本项目，我们揭示了多个新的参与内胚层分化的长非编码RNA，同时研究了其与转录因子、信号分子之间的相互作用，对长非编码RNA为关键节点的调控网络提供了更多的线索和支撑。