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.