MicroRNA Function in the Development of Cerebral Cortex

描述（申请人提供）：发育中的大脑皮层神经祖细胞增生分化异常，可引起大脑畸形，导致癫痫、智力低下等脑功能失调。新出现的证据表明，与蛋白质编码基因类似，非编码microrna （mirna）在皮层发育中起着关键作用，并与人类神经系统疾病的病因有关。我们之前的研究已经通过产生Dicer条件敲除小鼠证明了miRNA在皮层发育中的功能，其中miRNA的生物发生在胚胎皮层中被特异性阻断。我们已经使用蛋白质组学方法和RNA测序技术鉴定了潜在的miRNA靶基因。我们还研究了特定mirna在皮质发育中的作用。mirna通常位于编码基因的内含子区，与宿主基因一起转录。然而，对于内含子miRNAs如何与靶基因和宿主基因相互作用，以及在皮质发育过程中miRNAs、靶基因和宿主基因的表达如何被精确、适当地调控，这些方面的知识还存在空白。在这个项目中，我们将验证一个假设，即mirna的调控环，它们的靶基因和宿主基因协同工作，以确保发育中的皮层中神经祖细胞的正常发育。我们的初步研究已经确定了一个在发育中的小鼠皮层中表达的内含子mirna家族。我们已经开发了新的工具来改变miRNA的表达水平，并在体外和体内皮层发育过程中检测miRNA沉默对靶基因的特异性。基于这些发现，我们提出的研究主要集中在三个具体目标：目标1是确定该miRNA家族在体内和培养系统中控制神经祖细胞增殖和分化中的作用。目的2是通过鉴定特异性靶基因来阐明miRNA调控的分子机制。目的3是揭示miRNA靶基因在控制皮层神经祖细胞准确数量和神经正常发生过程中对该miRNA及其宿主基因表达的反馈调控。我们的建议将解决一个基本问题，即mirna的调控环，它们的靶基因和宿主基因如何控制适当的皮质发育。我们项目的成功应该使我们能够产生新的工具来操纵mirna及其靶基因在体外和体内的表达。由于皮质神经祖细胞的正常发育对正常的脑功能至关重要，因此我们的研究结果将为开发一种利用mirna治疗人脑畸形和神经系统疾病的新诊断和治疗手段提供重要的见解。

DESCRIPTION (provided by applicant): Abnormal proliferation and differentiation of neural progenitors in the developing cerebral cortex can cause brain malformation and result in dysregulation of brain function such as epilepsy and mental retardation. Emerging evidence has shown that similar to protein coding genes, noncoding microRNAs (miRNAs) play critical roles in cortical development and are associated with the etiology of human neurological disorders. Our previous studies have demonstrated miRNA functions in cortical development by generating Dicer conditional knockout mice, in which miRNA biogenesis is specifically blocked in the embryonic cortex. We have identified potential miRNA target genes using proteomic approaches and RNA sequencing technique. We have also examined the role of specific miRNAs in cortical development. miRNAs are often located in the intronic regions of coding genes and transcribed together with host genes. However, the knowledge gap is how intronic miRNAs interact with target genes and host genes, and how expressions of miRNAs, target genes and host genes are precisely and properly regulated during cortical development. In this project, we will test a hypothesis that a regulatory loop of miRNAs, their target genes and host genes works cohesively to ensure proper development of neural progenitors in the developing cortex. Our preliminary study has identified a family of intronic miRNAs that is expressed in the developing mouse cortex. We have developed new tools to alter miRNA expression levels and to examine the specificity of miRNA silencing effects on target genes in vitro and in vivo during cortical development. Based on these findings, our proposed research focuses on three specific aims: Aim 1 is to determine the role of this miRNA family in controlling neural progenitor proliferation and differentiation in vivo and in a culture system. Aim 2 is to elucidate molecular mechanisms of the miRNA regulation by identifying specific target genes. Aim 3 is to reveal the feedback regulation of the miRNA target gene on expression of this miRNA and its host gene in the process of controlling accurate numbers of cortical neural progenitors and proper neurogenesis. Our proposal will address a fundamental question of how a regulatory loop of miRNAs, their target genes and host genes controls proper cortical development. The success of our project should allow us to generate new tools to manipulate expressions of miRNAs and their target genes in vitro and in vivo. Because proper development of cortical neural progenitors is essential for normal brain function, our proposal should provide significant insights into developing a new diagnostic and therapeutic means using miRNAs for human brain malformations and neurological disorders.