Department of Internal Medicine, University of Iowa, Iowa City, Iowa, United States of America.
PLoS One. 2012;7(6):e38562. doi: 10.1371/journal.pone.0038562. Epub 2012 Jun 11.
MicroRNA (miRNA) function is required for normal animal development, in particular in differentiation pathways from stem cell and precursor populations. In neurogenesis, it is becoming increasingly appreciated that miRNAs act at many stages to ensure proper progression. In this study we examined the role of miR-34a in neural progenitor cells (NPC) derived from murine embryonic cortex. We found that over-expression of miR-34a in NPC significantly reduced the neuron yield upon in vitro induction of differentiation. MiR-34a has several predicted targets in the Notch pathway, which operates to balance progenitor self-renewal and differentiation during cortical neurogenesis. We tested several Notch pathway players for regulation by miR-34a in undifferentiated NPC, and found that mRNA and protein levels of Numbl, a negative regulator of Notch signaling, as well as two downstream pro-neural genes usually blocked by Notch signaling, NeuroD1 and Mash1, were diminished, while Notch1 and Cbf1 transcripts were enhanced by miR-34a over-expression. Using a luciferase reporter assay, we verified the Numbl 3'-UTR as a direct miR-34a target. Correspondingly, knock-down of endogenous miR-34a resulted in increased Numbl, NeuroD1 and Mash1, and reduced Notch1 transcript levels. Together these results implicate Numbl as a physiologically relevant target of miR-34a in NPC, allowing for enhanced Notch signaling and inhibition of neuronal differentiation. This work extends our understanding of miR-34a-mediated control of cell differentiation from cancer to mammalian nervous system development.
微小 RNA（miRNA）的功能对于正常动物的发育是必需的，特别是在干细胞和前体细胞分化途径中。在神经发生中，miRNA 在许多阶段发挥作用以确保适当的进展，这一点越来越受到人们的重视。在这项研究中，我们研究了 miR-34a 在源自鼠胚胎皮层的神经祖细胞（NPC）中的作用。我们发现，在 NPC 中过表达 miR-34a 会显著减少体外诱导分化时神经元的产量。miR-34a 在 Notch 通路中有几个预测的靶点，该通路在皮层神经发生中起作用，以平衡祖细胞的自我更新和分化。我们在未分化的 NPC 中测试了几个 Notch 通路成员是否受 miR-34a 的调节，发现 Notch 信号的负调节剂 Numbl 的 mRNA 和蛋白水平，以及通常被 Notch 信号阻断的两个下游促神经基因 NeuroD1 和 Mash1 的水平降低，而 Notch1 和 Cbf1 的转录物则被 miR-34a 的过表达增强。通过荧光素酶报告基因检测，我们验证了 Numbl 3'-UTR 是 miR-34a 的直接靶标。相应地，内源性 miR-34a 的敲低导致 Numbl、NeuroD1 和 Mash1 的增加，以及 Notch1 转录物水平的降低。这些结果表明，Numbl 是 NPC 中 miR-34a 的一个生理相关靶标，允许增强 Notch 信号并抑制神经元分化。这项工作扩展了我们对 miR-34a 介导的细胞分化控制的理解，从癌症到哺乳动物神经系统发育。
