MiR-34a represses Numbl in murine neural progenitor cells and antagonizes neuronal differentiation.
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.
Department of Internal Medicine, University of Iowa, Iowa City, Iowa, United States of America.
Sarah K
SK
Fineberg
Poppy
P
Datta
Colleen S
CS
Stein
Beverly L
BL
Davidson
D000818:Animals; D015153:Blotting, Western; D001973:Bromodeoxyuridine; D002454:Cell Differentiation / Q000502:physiology*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental / Q000235:genetics / Q000502:physiology*; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008156:Luciferases; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D058953:Neural Stem Cells / Q000502:physiology*; D055495:Neurogenesis / Q000235:genetics / Q000502:physiology*; D051880:Receptors, Notch / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D018709:Statistics, Nonparametric; D014161:Transduction, Genetic
D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; C551848:MIRN34a microRNA, mouse; D035683:MicroRNAs; D009419:Nerve Tissue Proteins; C106292:Numbl protein, mouse; D051880:Receptors, Notch; D008156:Luciferases; D001973:Bromodeoxyuridine