LncRNA在小脑发育和功能活动中作用的研究

Recent progress of long non-coding RNAs (lncRNAs) indicates their important roles in nervous system. Cerebellum is the second largest organ in the central nervous system and plays critical roles in many basic life activities. However, the research on cerebellar lncRNA is very limited. To obtain systematic information about cerebellum-highly expressed lncRNA (CeH-lncRNA), we performed microarray analysis using mouse cerebellum as well as tissues isolated from other brain regions. Our results showed that the number of CeH-lncRNA is much higher than most of the other tested brain regions. Moreover, we identified two CeH-lncRNAs whose shRNAs significantly changed the expression levels of several well-known cerebellum-associated mRNA genes, e.g. Cbln3. These data together suggested the two CeH-lncRNAs may play some essential roles in cerebellum development and function. In this proposal, we will modify the expression level of CeH-lncRNA in cultured neuronal cells to further investigate their working molecular mechanisms in regulating their targeting cerebellum-associated mRNA genes in vitro. Meanwhile, we will generate CRISPR knockout mice for the CeH-lncRNAs to study their roles in cerebellum development and activity in vivo. Our work will provide previously undiscovered information about the roles of lncRNAs in cerebellum development/function, and help to expand fundamental knowledge to the better understanding of the molecular mechanisms in the central nervous system.

长链非编码RNA（lncRNA）的研究进展正将lncRNA与神经系统越来越紧密地结合在一起。小脑作为中枢神经系统中第二大器官，在多种基础生命活动中发挥着重要的调节作用。然而，小脑是目前神经系统lncRNA研究尚缺乏的脑区。申请人课题组的前期研究结果显示小脑中超高表达的lncRNA（CeH-lncRNA）的数目远远高于其他受检的多数脑区，并且发现了两个对小脑发育重要相关基因（如小脑肽3）有显著调控作用的CeH-lncRNA。本项目以此为切入点，拟从分子和细胞水平对CeH-lncRNA调控下游靶基因的分子机制进行深入探讨，同时建立目的CeH-lncRNA基因的CRISPR敲除小鼠，进而对CeH-lncRNA在小脑发育和功能活动中的作用从整体动物水平进行研究。本项目将提供新的lncRNA在小脑发育和功能活动中发挥作用的实验证据，深化神经系统复杂调控分子机制的理论基础。

“LncRNA在小脑发育和功能活动中作用的研究”这一项目是基于神经系统长链非编码RNA（long noncoding RNA, lncRNA）的作用和功能亟需深入研究的基础上提出并开展的。在研究中，我们以丰富的内容开展了项目书中计划的研究内容。工作主要从前后相互依托的2个方面展开：（1）鉴定包含小脑在内的多个脑区表达的lncRNA；（2）对所鉴定出的小脑高表达lncRNA（CeH-lncRNA）的功能与机制进行深入研究。取得以下主要进展：（1）在鉴定小脑及其他受检脑区高表达lncRNA的过程中，发现了一组由在中枢神经系统高表达且保守性较高的非编码RNA基因组成的新型基因簇结构CNIBs，并通过CRISPR-Cas9斑马鱼敲除模型对其中保守性最高的CNIB1在个体发育和视觉神经调控中的作用进行了研究。（2）探索了两条CeH-lncRNA分子在小脑功能调控中的作用及其分子机制。通过病毒介导的小鼠小脑特异性敲减，我们证明了这两条CeH-lncRNA分子在小脑结构、突触可塑性以及小脑相关运动功能中发挥着重要作用，并解答了其调控的分子机理。本项目的结果进一步丰富了lncRNA相关的基础理论知识，论证了中枢神经系统lncRNA在人类疾病和小脑功能调控中的重要作用，为深入理解人类基因组提供了新的实验依据，具有重要的科学意义和潜在的应用前景。