miR-132 regulates the differentiation of dopamine neurons by directly targeting Nurr1 expression.
Although it is well established that embryonic stem (ES) cells have the potential to differentiate into dopamine neurons, the molecular basis of this process, particularly the role of microRNAs (miRNAs), remains largely unknown. Here we report that miR-132 plays a key role in the differentiation of dopamine neurons by directly regulating the expression of Nurr1 (also known as nuclear receptor subfamily 4 group A member 2; Nr4a2). We constructed a mouse ES cell line CGR8, which stably expresses GFP under the tyrosine hydroxylase (TH) promoter, so the TH-positive neurons could be easily sorted using fluorescence-activated cell sorting (FACS). Then, we performed a miRNA array analysis on the purified TH-positive neurons and found that 45 of 585 miRNAs had more than a fivefold change in expression level during dopamine neuron differentiation. Among the 45 miRNAs, we were particularly interested in miR-132 because this miRNA has been reported to be highly expressed in neurons and to have a potential role in neurodegenerative diseases. We found that the direct downregulation of endogenous miR-132 induced by miR-132 antisense oligonucleotide (miR-132-ASO) promoted the differentiation of TH-positive neurons, whereas ectopic expression of miR-132 in ES cells reduced the number of differentiated TH-positive neurons but did not change the total number of differentiated neurons. Furthermore, we identified that miR-132-ASO could substantially reverse the miR-132-mediated suppression of TH-positive neuron differentiation. Moreover, through a bioinformatics assay we identified the Nurr1 gene as a potential molecular target of miR-132. Using a luciferase-reporter assay and western blot analysis, we demonstrated that miR-132 could directly regulate the expression of Nurr1. Collectively, our data provide the first evidence that miR-132 is an important molecule regulating ES cell differentiation into dopamine neurons by directly targeting Nurr1 gene expression.
Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Dehua
D
Yang
Ting
T
Li
Yi
Y
Wang
Yuanjia
Y
Tang
Huijuan
H
Cui
Yu
Y
Tang
Xiaojie
X
Zhang
Degui
D
Chen
Nan
N
Shen
Weidong
W
Le
D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D059290:Dopaminergic Neurons / Q000166:cytology* / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D020869:Gene Expression Profiling; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction
C546976:MIRN132 microRNA, mouse; D035683:MicroRNAs; C539883:Nr4a2 protein, mouse; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2