Dlk1-Dio3印记域内miRNA簇在小鼠胚胎干细胞神经分化中的作用与机制

Located on chromosome 12qF1 in mice, the imprinted Dlk1-Dio3 region has a miRNA (microRNA) cluster. Silencing of the imprinted miRNA cluster is responsible for the variability of the development potential of pluripotent stem cells. Also, the down-regulation of this cluster is closely associated with schizophrenia. Basing on recent research and bioinformatic analysis, the miRNA cluster may play important roles in the nerual differentiation, but the underlying molecular mechanisms and functional roles are largely unknown. In our previous study, we have successfully induced the Sox1-GFP ES cells differentiation toward the neural lineage cells and discovered that seven members of the miRNA cluster showed significant expression changes during this differentiation process. On this basis, in this project, we will first demonstrate the functions of the seven miRNAs and filter out the key players among them in the regualtion of neural differentiation by loss- and gain-of-function experiments. We will then use the Tet-On inducible transgenic system to elucidate the miRNAs "functional window" during the differentiation. Finally, microarray chip, signaling pathways chip and reporter gene expression system will be uesd to further explore and validate the the target genes and signaling pathways of the discovered key miRNAs. Our study will not only reveal the mechanisms and functional roles of the imprinted miRNA cluster located within the Dlk1-Dio3 region in neural differentiation of mouse ES cells, but also provide a target for the optimization of in vitro neural differentiation system and gene therapies.

最近研究发现，Dlk1-Dio3印记域内miRNA簇是决定多能干细胞发育分化潜能的关键基因，且其表达降低与神经系统疾病紧密关联，表明该miRNA簇在神经发育分化中可能扮演重要角色，但其具体功能和机制尚不清楚。前期工作中我们建立了小鼠ES细胞神经分化体系，发现该簇中有7个miRNAs的表达伴随着分化进程有显著变化。在此基础上本项目将运用基因操作手段改变这7个miRNAs表达水平，筛选出参与调控神经分化的关键miRNAs；利用可诱导表达系统，明确这些miRNAs在神经分化过程中发挥作用的具体时间段。进一步运用表达谱和信号通路芯片及报告基因等技术，探索和验证miRNAs在神经分化中的靶基因和信号通路机制。本项目的完成将深入阐明Dlk1-Dio3域内miRNA簇在ES细胞神经分化中的机制，为优化体外神经分化体系和相关疾病的治疗提供靶标。

Dlk1-Dio3印记域内miRNA簇在神经发育分化中可能扮演重要角色，但其具体功能和机制尚不清楚。我们在前期工作中筛选出两个miRNA作为进一步的研究对象。其中一个是随着分化过程逐渐下降的miR-377，一个是随着分化过程逐渐上升的miR-495。miRNA-377的下游靶基因Sirt2在ES细胞分化中起着重要作用。基因操作的手段使该基因表达减少将会大大抑制神经分化。进一步发现SIRT2减少使得GSK3β磷酸化位点Ser9修饰减少，从而激活了GSK3β达到抑制神经分化的作用。另外，我们发现miRNA簇另一成员miRNA-495参与调控神经分化。运用基因操作手段等，发现过表达miRNA-495会促进外胚层神经基因的表达并抑制其中、内胚层表达，而抑制miRNA-495会抑制外胚层神经基因的表达并促进中、内胚层表达。进一步运用报告基因等技术，在下游基因筛选中我们发现了miRNA-495是通过DNA甲基化酶Dnmt3a，而不是Dnmt3b来发挥作用。本项目中所研究的miRNA簇的两个成员虽然在同一体系中表达趋势相反，却通过调控不同的靶基因达到同一个目的：促进神经分化。我们的研究揭示了Dlk1-Dio3域内miRNA簇部分成员在ES细胞神经分化中的机制。