title: MicroRNA expression profiling of NGF-treated PC12 cells revealed a critical role for miR-221 in neuronal differentiation.
abstract: MicroRNAs (miRNAs) are small non-coding RNAs that control protein expression through translational inhibition or mRNA degradation. MiRNAs have been implicated in diverse biological processes such as development, proliferation, apoptosis and differentiation. Upon treatment with nerve growth factor (NGF), rat pheochromocytoma PC12 cells elicit neurite outgrowth and differentiate into neuron-like cells. NGF plays a critical role not only in neuronal differentiation but also in protection against apoptosis. In an attempt to identify NGF-regulated miRNAs in PC12 cells, we performed miRNA microarray analysis using total RNA harvested from cells treated with NGF. In response to NGF treatment, expression of 8 and 12 miRNAs were up- and down-regulated, respectively. Quantitative RT-PCR analysis of 11 out of 20 miRNAs verified increased expression of miR-181a(∗), miR-221 and miR-326, and decreased expression of miR-106b(∗), miR-126, miR-139-3p, miR-143, miR-210 and miR-532-3p after NGF treatment, among which miR-221 was drastically up-regulated. Functional annotation analysis of potential target genes of 7 out of 9 miRNAs excluding the passenger strands (*) revealed that NGF may regulate expression of various genes by controlling miRNA expression, including those whose functions and processes are known to be related to NGF. Overexpression of miR-221 induced neuronal differentiation of PC12 cells in the absence of NGF treatment, and also enhanced neuronal differentiation caused by low-dose NGF. Furthermore, miR-221 potentiated formation of neurite network, which was associated with increased expression of synapsin I, a marker for synapse formation. More importantly, knockdown of miR-221 expression by antagomir attenuated NGF-mediated neuronal differentiation. Finally, miR-221 decreased expression of Foxo3a and Apaf-1, both of which are known to be involved in apoptosis in PC12 cells. Our results suggest that miR-221 plays a critical role in neuronal differentiation as well as protection against apoptosis in PC12 cells.
authors.affiliation: Department of Longevity and Aging Research, Gifu International Institute of Biotechnology, Kakamigahara, Gifu, Japan.
authors.forename: Nanako
authors.initials: N
authors.lastname: Hamada
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Fujita
authors.forename: Toshio
authors.initials: T
authors.lastname: Kojima
authors.forename: Aya
authors.initials: A
authors.lastname: Kitamoto
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Akao
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Nozawa
authors.forename: Masafumi
authors.initials: M
authors.lastname: Ito
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000235:genetics*; D020869:Gene Expression Profiling; D035683:MicroRNAs / Q000235:genetics* / Q000502:physiology; D020932:Nerve Growth Factor / Q000494:pharmacology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects*; D016716:PC12 Cells; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D035683:MicroRNAs; D020932:Nerve Growth Factor; C525709:MIR221, human