MicroRNA expression profiling of NGF-treated PC12 cells revealed a critical role for miR-221 in neuronal differentiation

Neurochemistry International 60 (2012) 743–750

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MicroRNA expression profiling of NGF-treated PC12 cells revealed a critical role for miR-221 in neuronal differentiation Nanako Hamada a,e,1, Yasunori Fujita a,b,1, Toshio Kojima c,d, Aya Kitamoto d, Yukihiro Akao e, Yoshinori Nozawa a,f, Masafumi Ito a,b,⇑ a

Department of Longevity and Aging Research, Gifu International Institute of Biotechnology, Kakamigahara, Gifu 504-0838, Japan Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0015, Japan Research Equipment Center, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-3192, Japan d Computational Systems Biology Research Group, Advanced Science Institute, RIKEN, Yokohama, Kanagawa 230-0045, Japan e United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1193, Japan f Department of Food and Health, Tokai Gakuin University, Kakamigahara, Gifu 504-8511, Japan b c

a r t i c l e

i n f o

Article history: Received 22 September 2011 Received in revised form 16 March 2012 Accepted 17 March 2012 Available online 24 March 2012 Keywords: MicroRNA microarray Nerve growth factor Neuronal differentiation PC12 cells miR-221

a b s t r a c t 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 miR106b⁄, 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. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction MicroRNAs (miRNAs) are small non-coding RNAs of about 22–24 nucleotides in length and control protein expression through translational inhibition or mRNA degradation mainly by Abbreviations: miRNA, microRNA; 30 -UTR, 30 -untranslated region; AD, Alzheimer’s diseases; NGF, nerve growth factor; MAPK, mitogen-activated protein kinase; DMEM, Dulbecco’s modified Eagle’s medium; HS, horse serum; FBS, fetal bovine serum; GEO, Gene Expression Omnibus; Foxo3a, forkhead box O3a; Apaf-1, apoptosis protease activator protein-1; KSR, kinase suppressor of Ras. ⇑ Corresponding author at: Research Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 1730015, Japan. Tel.: +81 3 3964 3241; fax: +81 3 3579 4776. E-mail address: [email protected] (M. Ito). 1 These authors contributed equally to this work. 0197-0186/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2012.03.010

binding to the 30 -untranslated region (30 -UTR) of target mRNAs (Filipowicz et al., 2008). MiRNAs regulate diverse biological processes including development, proliferation, apoptosis and differentiation. In terms of differentiation, miRNAs have been shown to control differentiation of various types of cells such as neurons (Jing et al., 2011), adipocytes (Karbiener et al., 2011), myoblasts (Gagan et al., 2011) and osteoblasts (Eskildsen et al., 2011). Neurotrophic factors play critical roles in neuronal development and survival as well as the maintenance of synaptic plasticity and connections, and thus have been considered as potential therapeutic targets for neurodegenerative disorders such as Alzheimer’s diseases (AD). AD is characterized by cognitive decline, memory impairment and behavioral abnormalities due to progressive neuronal loss which is caused by extracellular amyloid

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plaques and intracellular neurofibrillary tangles. In addition, neurite dystrophy and synaptic dysfunction are another important pathological features associated with AD (Selkoe, 2002). Pure memory impairment observed in the earliest clinical stage of AD is ascribed to subtle alterations of synaptic efficacy prior to massive neuronal death. Since the hypofunction of the cholinergic system is linked to cognitive deficits (Bartus et al., 1982; Davies and Maloney, 1976; Whitehouse et al., 1982), and mature cholinergic neurons in the basal forebrain are highly dependent on the nerve growth factor (NGF) activity (Fischer et al., 1987; Hefti and Weiner, 1986), a clinical trial of NGF gene therapy was conducted. NGF delivery to the basal forebrain resulted in a mild but significant therapeutic benefit (Tuszynski et al., 2005). Rat pheochromocytoma PC12 cells have been popularly used as a cell culture model of neurons (Greene and Tischler, 1976). When treated with NGF, PC12 cells extend neurites and form synapse-like structures and neurite network, differentiating into neuron-like cells, which is associated with increased expression of neuronal specific genes (Das et al., 2004). NGF binding to its receptor, TrkA, results in its dimerization (Jing et al., 1992) and autophosphorylation (Stephens et al., 1994). TrkA receptor activation by NGF induces neuronal differentiation through activation of the mitogen-activated protein kinase (MAPK) pathway (Cowley et al., 1994; Obermeier et al., 1994; Pang et al., 1995; Stephens et al., 1994; Traverse et al., 1992), and also protects against apoptosis via activation of the PI3-K/Akt signaling pathway (Yao and Cooper, 1995). In the present study, we performed miRNA microarray analysis and identified NGF-regulated miRNAs in PC12 cells. Among miRNAs up- and down-regulated by NGF treatment, we focused on miR-221 whose expression was robustly increased in response to NGF, and examined its effects on neuronal differentiation and expression of apoptotic genes. Our results suggested that miR221 plays a critical role in neuronal differentiation as well as protection against apoptosis in PC12 cells. 2. Materials and methods 2.1. Cell culture and NGF treatment Rat pheochromocytoma PC12 cells were obtained from RIKEN Cell Bank (Tsukuba, Ibaraki, Japan) and cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% horse serum (HS, Invitrogen, Carlsbad, CA, USA), 5% fetal bovine serum (FBS, MP Biomedicals, Irvine, CA, USA), 100 U/ml penicillin and 100 lg/ml streptomycin at 37 °C under a humidified atmosphere of 5% CO2. Cells seeded on PolyD-lysine-coated plates were treated with murine NGF-7S (Invitrogen) in DMEM containing 1% HS and 0.5% FBS, and the medium was changed every other day.

processed, and raw data were collected using the Agilent Feature Extraction software. Expression data were analyzed using GeneSpring GX 11 (Agilent Technologies). The signal intensity of each probe was normalized by a percentile shift, in which each value was divided by the 75th percentile of all values in its array. Normalized expression values were used for further analysis. For pair-wise comparison analysis, only the probes which have present expression flags in at least one condition were considered. The microarray data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (GEO) and are accessible through the GEO Series accession number GSE32122 (http:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32122). Lists of predicted effectors of NGF-regulated miRNAs were generated using the TagetScan program (http://www.targetscan.org). The list was analyzed using the Ingenuity Pathways Analysis software (Ingenuity Systems, Redwood, CA, USA). 2.3. Quantitative reverse transcription-PCR Total RNA was extracted using the miRNeasy Mini Kit (Qiagen). Target miRNA was reverse transcribed to cDNA by a gene specific primer using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). TaqMan MicroRNA Assay was then performed with Premix Ex Taq (Takara, Shiga, Japan) using the Thermal Cycler Dice Real Time System (Takara). The relative quantification value of the target miRNA, normalized to a control, U6 RNA, was calculated by the comparative Ct method. 2.4. Transfection Cells were transfected with rno-miR-221 precursor, pre-miR miRNA precursor negative control #2, miR-221 antagomir or anti-miR miRNA inhibitor negative control #1 (Ambion, Austin, TX, USA) using Lipofectamine 2000 (Invitrogen) in Opti-MEM I reduced-serum medium for 4 h. Then, culture medium was changed to normal medium. 2.5. Quantification of neuronal differentiation Neurite outgrowth was examined 6 days after NGF treatment, because PC12 cell differentiation and neurite growth have been shown to reach a plateau after 6 days in culture (Das et al., 2004). Phase contrast microscopic pictures were taken using a digital microscopy, BZ-8100 (Keyence, Osaka, Japan). Cells extending at least one neurite with a length longer than the diameter of the cell body were assessed as differentiated cells. The neurite length was quantified by tracing the neurite using BZ-Analyzer software (Keyence). One hundred cells from each optical field were examined. For each group, six optical fields from three independent experiments, each in duplicate, were analyzed. 2.6. Western blot analysis

2.2. MiRNA microarray analysis and data processing Cells were treated with 100 ng/ml NGF for 0, 12, 24 and 48 h. Total RNA containing miRNA was extracted from cells using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). One hundred nanograms of total RNA were labeled using the Agilent miRNA Complete Labeling and Hybridization Kit (Agilent Technologies, Santa Clara CA, USA) according to the manufacturer’s instructions. The labeled RNA was hybridized to the Agilent Rat miRNA Microarray Release 10.1 (Agilent Technologies) in a rotating hybridization oven at 10 rpm for 20 h at 65 °C. After hybridization, the microarrays were washed according to the manufacturer’s instructions and scanned on an Agilent DNA Microarray Scanner with the Scan Control software (Agilent Technologies). The resulting images were

Antibodies against Apaf-1 and b-actin were purchased from Millipore (Bedford, MA, USA) and Sigma (St. Louis, MO, USA), respectively. Anti-Foxo3a and -GAPDH antibodies were from Cell Signaling Technology (Beverly, MA, USA). Cells were lysed in RIPA buffer (10 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 0.1% sodium deoxycholate) containing protease inhibitor cocktails (Sigma). Cell lysates were separated by SDS–PAGE and transferred onto PVDF membranes. After blocking in 5% skim milk, membranes were incubated with a primary antibody and then incubated with a horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Piscataway, NJ, USA). The immunoreactive proteins were visualized using the ECL Plus Western blotting detection reagents (GE Healthcare) and the

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LAS-4000 luminescent image analyzer (Fuji Film, Tokyo, Japan). Densitometric analysis of band intensity was performed using the Multi Gauge software (Fuji Film).

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changes at 12 and 24 h after NGF treatment were listed in Suppl. Tables 1 and 2. 3.2. Confirmation of miRNA expression changes by quantitative RTPCR analysis

2.7. Luciferase reporter assay The 30 -UTR of rat Foxo3a and Apaf-1 genes was amplified by PCR, which was then cloned into the pMIR-REPORT miRNA expression reporter plasmid (Ambion). In order to introduce mutations into the seed sequences of potential target sites within the 30 UTR, overlap extension PCR was performed. After construction of the reporter genes, DNA sequences of the 30 -UTR were verified. Cells were co-transfected with firefly reporter constructs containing the wild type or mutant 30 -UTR and Renilla expressing plasmid, phRL-TK, along with miR-221 precursor or control using Lipofectamine 2000. Firefly and Renilla luciferase activities were measured 24 h after transfection by the Pikkagene dual luciferase assay systems (TOYO B-Net, Tokyo, Japan) and firefly luciferase activity was normalized to Renilla luciferase activity. 2.8. Statistical analysis Statistical significance was determined by Student’s t-test or one way ANOVA and Bonferroni test using the SPSS software. A p-value less than 0.05 was considered to be statistically significant. 3. Results 3.1. Identification of NGF-regulated miRNAs in PC12 cells by miRNA microarray analysis In an attempt to identify NGF-regulated miRNAs in PC12 cells, we conducted miRNA microarray analysis using total RNA harvested from cells treated with NGF at 100 ng/ml for 0, 12, 24 and 48 h. We analyzed expression changes of 350 miRNAs. MiRNAs that showed more than 1.5-fold changes at 48 h as compared with before treatment were listed in Table 1. The expression levels of 8 and 12 miRNAs were up- and down-regulated, respectively. Temporal profile of their expression levels after treatment with NGF was also shown. Among them, miR-221 expression was markedly increased in response to NGF. MiRNAs with more than 1.5-fold

In order to confirm miRNA expression changes observed in a single microarray analysis (Table 1), we selected 11 miRNAs with greater fold changes from 20 miRNAs (rno-miR-181a⁄, -221, -326, -106b⁄, -126, -139-3p, -143, -210, 345-3p, -532-3p and -99b⁄) and performed quantitative RT-PCR analysis using total RNA newly isolated from 3 biologically independent experiments. Consistent with the microarray data, the expression level of miR-221 was markedly increased by 76-fold at 24 h and by 62-fold at 48 h after NGF treatment (Fig. 1). In a time-dependent manner, expression of miR-181a⁄ and miR-326 was up-regulated and that of miR-106b⁄, miR-126, miR-139-3p, miR-143, miR-210 and miR-532-3p was down-regulated. In accordance with the microarray data, expression of miR-345-3p and miR-99b⁄ and was reduced in response to NGF treatment, but which was not statistically significant (Suppl. Fig. 1). These results confirmed the overall accuracy of the microarray data. 3.3. Functional annotation of potential miRNA target genes predicted by the computational algorithm We conducted functional annotation analysis of potential target genes of 7 out of 9 miRNAs excluding the passenger strands, miR181a⁄ and miR-106b⁄, whose expression changes were verified by quantitative RT-PCR (Fig. 1). Specifically, the list of potential target genes predicted by the TargetScan computational algorithm for 7 miRNAs (Suppl. Table 3) was subjected to functional annotation analysis using the Ingenuity Pathways Analysis. It was found that potential target genes of miRNAs are those implicated in gene expression, tissue, cellular, embryonic and organ development, nervous system development and function, and neurological disease (Table 2) and those in NGF signaling, neurotrophin/TRK signaling, Wnt/b-catenin signaling, neuregulin signaling, IGF-1 signaling and ERK/MAPK signaling (Table 3). These results were largely consistent with physiological functions and processes known to be related to NGF. 3.4. Induction of neuronal differentiation by miR-221 in PC12 cells

Table 1 MicroRNA expression profiling of NGF-treated PC12 cells. miRNAs

Fold change Time 12 h

24 h

48 h

rno-miR-181a⁄ rno-miR-181b rno-miR-22 rno-miR-221 rno-miR-27a rno-miR-326 rno-miR-598-3p rno-miR-674-3p

1.36 1.19 1.35 99.54 1.46 1.12 0.98 1.46

1.19 1.39 1.54 124.93 1.61 1.44 1.17 1.71

1.55 1.54 1.55 130.81 1.59 1.78 1.52 1.92

rno-miR-106b⁄ rno-miR-126 rno-miR-139-3p rno-miR-143 rno-miR-193 rno-miR-210 rno-miR-219-5p rno-miR-345-3p rno-miR-378 rno-miR-378⁄ rno-miR-532-3p rno-miR-99b⁄

34.60 1.25 1.10 1.31 1.38 1.44 2.26 51.60 1.45 1.88 1.20 24.62

34.79 1.21 1.32 1.58 1.42 1.73 1.59 51.88 1.44 1.86 1.31 1.11

31.75 60.94 59.08 103.37 1.51 2.10 1.60 47.35 1.54 2.25 1.58 22.59

Since NGF plays a critical role in neuronal differentiation (Gotz, 2000), we examined the effect of robust increase in miR-221 expression seen within 48 h after NGF treatment on terminal differentiation of PC12 cells seen after 6 days culture. We hypothesized that miR-221 might regulate neuronal differentiation by itself or modulate NGF-induced differentiation. As shown in Fig. 2A and B, overexpression of miR-221 by transfection of its precursor increased the number of differentiated PC12 cells in the absence of NGF (1% vs. 14%). miR-221 also enhanced low-dose NGF (2, 5 and 10 ng/ml)-induced neuronal differentiation (4% vs. 27%, 11% vs. 55% and 45% vs. 96%, respectively). Furthermore, formation of neurite network was potentiated by miR-221 overexpression, which was most evident in cells treated with 10 ng/ml NGF. In the absence of exogenously administered miR-221, expression of synapsin I, a marker for synapse formation, was increased when cells were treated with 10 ng/ml NGF (Fig. 2C). In contrast, in the presence of miR-221, increased expression of synapsin I was observed in cells treated with even lower concentrations of NGF (2, 5 and 10 ng/ml), indicating that miR-221 induces synapsin I expression and enhances formation of neurite network. Finally, knockdown of miR-221 expression by transfection with antagomir attenuated NGF-mediated neurite outgrowth and thus decreased

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Fig. 1. Quantitative RT-PCR analysis of miRNAs whose expression was changed in response to NGF in miRNA microarray analysis. PC12 cells were treated with 100 ng/ml NGF for indicated time. Total RNA was harvested and subjected to quantitative RT-PCR for 9 miRNAs and U6 RNA. The relative expression level of miRNAs was expressed as a ratio to U6 RNA and the miRNA level at 0 h was set as 1. Data are expressed as mean ± SD from three biologically independent experiments. Asterisks indicate statistical significance as determined by one way ANOVA and Bonferroni test (p < 0.05).

Table 2 Function annotation of potential miRNA target genes.

Table 3 Pathway annotation of potential miRNA target genes.

Function

p-Value

Process

p-Value

Gene expression Tissue development Cellular development Embryonic development Organ development Organismal development Cancer Cellular assembly and organization Cellular function and maintenance Cardiovascular system development and function Cellular movement Endocrine system disorders Gastrointestinal disease Genetic disorder Metabolic disease Cellular growth and proliferation Nervous system development and function Cell morphology Cell death Behavior Neurological disease Organismal survival Immunological disease Connective tissue disorders Inflammatory disease

1.23E18 6.73E17 3.25E16 3.45E13 3.45E13 3.45E13 6.79E13 2.05E11 2.05E11 4.62E11 5.26E11 1.40E10 1.40E10 1.40E10 1.40E10 2.31E10 2.54E10 6.83E10 1.73E09 1.18E08 1.37E08 1.71E08 4.07E08 5.68E08 5.68E08

HGF signaling Axonal guidance signaling NGF signaling GNRH signaling Growth hormone signaling Neurotrophin/TRK signaling Wnt/b-catenin signaling Neuregulin signaling IGF-1 signaling ERK/MAPK signaling Glucocorticoid receptor signaling Molecular mechanisms of cancer PDGF signaling LPS-stimulated MAPK signaling Erythropoietin signaling PTEN signaling Corticotropin releasing hormone signaling IL-3 signaling CREB signaling in neurons Type II diabetes mellitus signaling Prolactin signaling IL-6 signaling Dopamine-DARPP32 feedback in cAMP signaling CXCR4 signaling Endothelin-1 signaling

9.33E08 6.31E07 1.00E06 1.02E06 1.15E06 1.41E06 1.51E06 1.66E06 2.09E06 6.46E06 9.77E06 1.00E05 1.05E05 1.48E05 2.63E05 2.88E05 4.17E05 4.90E05 5.89E05 6.61E05 6.76E05 7.08E05 8.32E05 9.12E05 1.00E04

the differentiated cell number (Fig. 3). These results suggested that miR-221 plays an important role in neuronal differentiation of PC12 cells. 3.5. Inhibition of apoptotic gene expression by miR-221 in PC12 cells In addition to enhancing neurite outgrowth, NGF protects against apoptosis (Haviv and Stein, 1999). It has been reported that

miR-221 targets genes that regulate apoptosis such as PTEN (Zhang et al., 2010b), Bim (Terasawa et al., 2009) and PUMA (Zhang et al., 2010a). In PC12 cells, forkhead box O3 (Foxo3a) (Cai and Xia, 2008) and apoptosis protease activator protein-1 (Apaf-1) (Ekshyyan and Aw, 2005) have been shown to be involved in apoptosis. Since we found potential binding sites of miR-221 in the 30 -UTR of Foxo3a and Apaf-1 genes using the computational algorithms and visual inspection, we examined the effects of miR-221 overexpression

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Fig. 2. Effects of miR-221 overexpression on differentiation of PC12 cells. After transfection with miR-221 precursor or negative control (NC), PC12 cells were treated with 0, 2, 5 and 10 ng/ml NGF for 6 days. (A) Phase contrast microscopic pictures were taken. (B) One hundred cells from each optical field were examined. For each group, six optical fields from three biologically independent experiments, each in duplicate, were analyzed. Cells extending at least one neurite with a length longer than the diameter of the cell body were taken as differentiated cells. The differentiation rate was calculated as a percentage of the differentiated cell number relative to that induced by treatment with 50 ng/ml NGF in mock-transfected cells (Lipofectamine 2000 alone). Data are expressed as mean ± SD from three independent experiments. Filled squares and triangles denote cells transfected with negative control and miR-221 precursor, respectively. (C) Cell lysates were prepared and subjected to Western blot analysis using antibodies against synapsin I and GAPDH. A representative immunoblot of three biologically independent experiments is shown.

Fig. 3. Effects of miR-221 knockdown on differentiation of PC12 cells. After transfection with anti-miR-221 or negative control (NC), PC12 cells were treated with 50 ng/ml NGF. (A) Phase contrast microscopic pictures were taken 6 days after treatment. (B) One hundred cells from each optical field were examined. For each group, six optical fields from three biologically independent experiments, each in duplicate, were analyzed. Cells extending at least one neurite with a length longer than the diameter of the cell body were taken as differentiated cells. The differentiation rate was calculated as a percentage of the differentiated cell number relative to that induced by treatment with 50 ng/ml NGF in mock-transfected cells (Lipofectamine 2000 alone). Data are expressed as mean ± SD from three independent experiments. Asterisks indicate statistical significance as determined by Student’s t-test (⁄p < 0.05; NS, non-significant difference).

on their expression. As shown in Fig. 4A, miR-221 decreased protein expression of Foxo3a and Apaf-1 in PC12 cells. In order to study if miR-221 could directly target Foxo3a and Apaf-1 genes, we constructed firefly luciferase reporter genes containing their 30 -UTR. miR-221 overexpression decreased the relative luciferase activity of the reporter genes containing the Foxo3a and Apaf-1 30 -UTR (Suppl. Fig. 2). However, introduction of mutations into the seed sequences of potential binding sites in the reporter genes did not affect the relative luciferase activity. We also examined the effects of antagomiR-221 on Foxo3a and Apaf-1 expression. The results showed that antagomiR-221 increased expression of Foxo3a, but did not change that of Apaf-1 (Suppl. Fig. 3). Taken together, these results suggested that miR-221 inhibits Foxo3a and Apaf-1 protein expression, but which is unlikely to be mediated by its di-

rect binding to the 30 -UTR of these two genes. Finally, we investigated the effects of NGF on Foxo3a and Apaf-1 expression and found that NGF decreased Foxo3a expression but not Apaf-1 expression (Fig. 4B). The inhibitory effect of miR-221 on Apaf-1 expression might be counteracted by as yet undefined mechanisms mediated by NGF under the experimental conditions used.

4. Discussion We performed microRNA expression profiling of NGF-treated PC12 cells and identified 8 and 12 miRNAs that were up- and down-regulated in response to NGF treatment, respectively. Quantitative RT-PCR analysis confirmed the miRNA expression changes

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Fig. 4. Effects of miR-221 overexpression on Apaf-1 and Foxo3a expression in PC12 cells. (A) Forty-eight hours after transfection with miR-221 precursor or negative control (NC), PC12 cell lysates were prepared and subjected to Western blot analysis using antibodies against Apaf-1, Foxo3a and b-actin. A representative immunoblot of three biologically independent experiments is shown in the upper panel, and densitometric data of three immunoblots are shown in the lower panel (mean ± SD). Asterisks indicate statistical significance as determined by Student’s t-test (⁄p < 0.05; NS, non-significant difference). (B) PC12 cells were treated with 100 ng/ml NGF for 24 and 48 h and then cell lysates were prepared and subjected to Western blot analysis using antibodies against Apaf-1, Foxo3a and GAPDH. A representative immunoblot of three biologically independent experiments is shown in the upper panel, and densitometric data of three immunoblots are shown in the lower panel (mean ± SD). Asterisks indicate statistical significance as determined by Student’s t-test (⁄p < 0.05; NS, non-significant difference).

detected by the microarray analysis. NGF up-regulated expression of miR-181a⁄, miR-221 and miR-326, and down-regulated that of miR-106b⁄, miR-126, miR-139-3p, miR-143, miR-210 and miR532-3p. Functional annotation analysis of potential target genes of 7 out of 9 miRNAs whose expression changes were verified by quantitative RT-PCR 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. For each miRNA, its physiological roles in NGF signaling and its target genes remain to be elucidated. In the present study, we further investigated miR-221 because of its robust induction in response to NGF treatment. miR-221 has been implicated in various biological processes including apoptosis, cell cycle and differentiation. miR-221 regulates apoptosis and cell cycle through targeting genes such as Bim (Terasawa et al., 2009), PUMA (Zhang et al., 2010a), PTEN (Zhang et al., 2010b), Bmf (Gramantieri et al., 2009) as well as p27 (Medina et al., 2008). In accordance with these findings, miR-221 expression has been shown to be elevated in various types of cancer (Chen et al., 2011; Fornari et al., 2008; Zhang et al., 2010b). miR-221 is also involved in differentiation of various types of cells such as chondrocytes (Kim et al., 2010), muscle cells (Cardinali et al., 2009) and dendritic cells (Kuipers et al., 2010). A previous report showed that miR-221 expression is up-regulated in NGF-treated PC12 cells and that miR-221 directly targets Bim (Terasawa et al., 2009). The authors also described that no detectable effect of NGF-induced neurite outgrowth was observed when miR-221 and/or 222 were overexpressed in PC12 cells. Indeed, when PC12 cells were treated with a higher concentration of NGF (50 ng/ml), miR-221 overexpression did not at all affect neuronal differentiation (data not shown), probably because miR-221 induction by 50 ng/ml NGF was so robust that exogenously administered miR-221 did not show any additional effect on neurite outgrowth. However, our results clearly demonstrated that miR-221 alone induces neuronal differentiation and that low-dose NGF-induced neuronal differentiation (2, 5 and 10 ng/ml) is significantly enhanced by miR-221 overexpression. Furthermore, miR-221 potentiated formation of neurite network. More importantly,

knockdown of miR-221 expression by antagomir attenuated NGF-mediated neurite outgrowth and thus decreased the differentiated cell number. These results indicate that miR-221 is capable of inducing neuronal differentiation of PC12 cells, which is likely to be consistent with previous findings that miR-221 plays an important role in the regulation of developmental and physiological processes during brain development (Natera-Naranjo et al., 2010; Podolska et al., 2011). It is of great importance that miR221 enhanced low-dose NGF-induced neuronal differentiation, because it suggests that miR-221 could compensate for defective NGF signaling in various pathological conditions such as AD. It is noteworthy that miR-221 has been shown to be a negative modulator of differentiation in some types of cells (Cardinali et al., 2009; Kim et al., 2010; Kuipers et al., 2010). For instance, miR-221 and miR-222 are strongly down-regulated upon differentiation of both primary and established myogenic cells (Cardinali et al., 2009). In myoblasts undergoing differentiation, miR-221 and miR-222 induces a delay in withdrawal from the cell cycle by targeting p27. Functional roles and target genes of miR-221 during neuronal differentiation may be different from those during differentiation of other types of cells. In an effort to identify miR-221 target genes that regulate neuronal differentiation, we searched for potential miR-221 target genes by the computational analysis. We examined if miR-221 could target kinase suppressor of Ras (KSR), which has been shown to inhibit Ras signaling by either blocking MEK and MAPK activation or inhibiting Elk-1 phosphorylation (Denouel-Galy et al., 1998; Joneson et al., 1998). However, KSR protein expression was not affected by miR-221 overexpression (data not shown). Further studies will be required to elucidate molecular mechanisms underlying the miR-221 effect on neuronal differentiation of PC12 cells. NGF protects against apoptosis (Haviv and Stein, 1999), and miR-221 has been demonstrated to directly target and regulate genes that are involved in apoptosis (Gramantieri et al., 2009; Terasawa et al., 2009; Zhang et al., 2010b). In the present study, we examined the effects of miR-221 on expression of Foxo3a and Apaf-1, which have been shown to be involved in apoptosis in PC12 cells (Cai and Xia, 2008). It was found that miR-221 inhibits

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Foxo3a and Apaf-1 protein expression, but which is likely due to its indirect effects via other target genes or its direct binding to other regions than the 30 -UTR of these two genes. Our results and others suggested that miR-221 may be capable of protecting against apoptosis in PC12 cells through regulating expression of Foxo3a and Apaf-1 as well as Bim (Terasawa et al., 2009). We have previously reported that metabolites of sesamin, a major lignan in sesame seeds, induce neuronal differentiation of PC12 cells through activating MAPK/ERK signaling (Hamada et al., 2009). In analogous to the effects of miR-221 on neuronal differentiation, the compounds induced neuronal differentiation by itself and also enhanced low-dose NGF-induced neuronal differentiation. Furthermore, they potentiated formation of neurite network in highdose NGF-treated PC12 cells. Since it has been shown that miR221 expression is positively regulated by the MAPK/ERK signaling (Ichimura et al., 2010; Stinson et al., 2011; Terasawa et al., 2009), small molecular weight compounds that activate the intracellular signaling like metabolites of sesamin may induce neuronal differentiation of PC12 cells in part by inducing miR-221 expression. In the present study, we provided global expression profiling data of NGF-induced miRNAs in PC12 cells and identified miRNAs whose expression was altered in response to NGF treatment. Among them, we focused on miR-221 which was robustly up-regulated by NGF, and demonstrated that miR-221 plays an important role in neuronal differentiation of PC12 cells. We also showed that miR-221 inhibits expression of Foxo3a and Apaf-1 involved in apoptosis. Our findings suggest that miR-221 has the potential as a promising therapeutic target for neurodegenerative diseases such as AD. Acknowledgments This work was supported by Grant for Biological Research from Gifu prefecture, Japan. We thank Dr. Ken Umemura for the initial work on this project and Ms. Mami Kishima (RIKEN OSC) for her technical advice. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neuint.2012.03.010. References Bartus, R.T., Dean III, R.L., Beer, B., Lippa, A.S., 1982. The cholinergic hypothesis of geriatric memory dysfunction. Science 217, 408–414. Cai, B., Xia, Z., 2008. P38 MAP kinase mediates arsenite-induced apoptosis through FOXO3a activation and induction of Bim transcription. Apoptosis 13, 803–810. Cardinali, B., Castellani, L., Fasanaro, P., Basso, A., Alema, S., Martelli, F., Falcone, G., 2009. Microrna-221 and microrna-222 modulate differentiation and maturation of skeletal muscle cells. PLoS ONE 4, e7607. Chen, Y., Zaman, M.S., Deng, G., Majid, S., Saini, S., Liu, J., Tanaka, Y., Dahiya, R., 2011. MicroRNAs 221/222 and genistein-mediated regulation of ARHI tumor suppressor gene in prostate cancer. Cancer Prev. Res. (Phila) 4, 76–86. Cowley, S., Paterson, H., Kemp, P., Marshall, C.J., 1994. Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell 77, 841–852. Das, K.P., Freudenrich, T.M., Mundy, W.R., 2004. Assessment of PC12 cell differentiation and neurite growth: a comparison of morphological and neurochemical measures. Neurotoxicol. Teratol. 26, 397–406. Davies, P., Maloney, A.J., 1976. Selective loss of central cholinergic neurons in Alzheimer’s disease. Lancet 2, 1403. Denouel-Galy, A., Douville, E.M., Warne, P.H., Papin, C., Laugier, D., Calothy, G., Downward, J., Eychene, A., 1998. Murine Ksr interacts with MEK and inhibits Ras-induced transformation. Curr. Biol. 8, 46–55. Ekshyyan, O., Aw, T.Y., 2005. Decreased susceptibility of differentiated PC12 cells to oxidative challenge: relationship to cellular redox and expression of apoptotic protease activator factor-1. Cell Death Differ. 12, 1066–1077. Eskildsen, T., Taipaleenmaki, H., Stenvang, J., Abdallah, B.M., Ditzel, N., Nossent, A.Y., Bak, M., Kauppinen, S., Kassem, M., 2011. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo. Proc. Natl. Acad. Sci. USA 108, 6139–6144.

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