Sustained activation of ERK1/2 by NGF induces microRNA-221 and 222 in PC12 cells Kazuya Terasawa 1 , Atsuhiko Ichimura 1 , Fumiaki Sato 2 , Kazuharu Shimizu 2 and Gozoh Tsujimoto 1 1 Department of Pharmcogenomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Japan 2 Department of Nanobio Drug Discovery, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Japan MicroRNAs (miRNAs) are evolutionally conserved small non-coding RNAs that regulate gene expres- sion at the post-transcriptional level and play impor- tant roles in a wide variety of biological functions, including cell differentiation, tumorigenesis, apoptosis and metabolism [1–3]. Approximately 30% of human protein-coding genes are predicted to be targets of miRNA [4,5]. Biogenesis of miRNA and the mecha- nism for regulation of target gene expression by miRNA are relatively well characterized. miRNA genes are initially transcribed mainly by RNA poly- merase II as long primary transcripts (pri-miRNAs), processed by the nuclear RNase Drosha to produce precursor miRNAs (pre-miRNAs), and then exported to the cytoplasm. Pre-miRNAs are further processed into mature miRNAs by the cytoplasmic RNase Dicer [6]. miRNAs recognize and bind to partially complementary sites in the 3¢ UTRs of target mRNAs, resulting in either translational repression or target degradation [7]. To further understand the functional significance of miRNA, the regulatory mechanism of miRNA expression needs to be better understood. The mitogen-activated protein kinase (MAPK) cascades play an essential role in transducing extra- cellular signals to cytoplasmic and nuclear effectors, and regulate a wide variety of cellular functions, including cell proliferation, differentiation and stress responses [8,9]. Extracellular signal-regulated kinases 1 and 2 (ERK1 ⁄ 2) were the first recognized members of the MAPK family of proteins. These kinases are primarily activated by mitogenic factors, differentiation Keywords Bim; ERK1 ⁄ 2; microRNA; NGF; PC12 Correspondence G. Tsujimoto, 45-29 Yoshida-Shimo-Adachi- cho, Sakyo-ku, Kyoto 606-8501, Japan Fax: +81 75 753 4523 Tel: +81 75 753 4544 E-mail: gtsuji@pharm.kyoto-u.ac.jp (Received 18 December 2008, revised 13 March 2009, accepted 6 April 2009) doi:10.1111/j.1742-4658.2009.07041.x MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by inhibiting translation and ⁄ or inducing degradation of target mRNAs, and they play important roles in a wide variety of biological func- tions including cell differentiation, tumorigenesis, apoptosis and metabo- lism. However, there is a paucity of information concerning the regulatory mechanism of miRNA expression. Here we report identification of growth factor-regulated miRNAs using the PC12 cell line, an established model of neuronal growth and differentiation. We found that expression of miR-221 and miR-222 expression were induced by nerve growth factor (NGF) stim- ulation in PC12 cells, and that this induction was dependent on sustained activation of the extracellular signal-regulated kinase 1 and 2 (ERK1 ⁄ 2) pathway. Using a target prediction program, we also identified a pro-apo- totic factor, the BH3-only protein Bim, as a potential target of miR- 221 ⁄ 222. Overexpression of miR-221 or miR-222 suppressed the activity of a luciferase reporter activity fused to the 3¢ UTR of Bim mRNA. Further- more, overexpression of miR-221 ⁄ 222 decreased endogenous Bim mRNA expression. These results reveal that the ERK signal regulates miR-221 ⁄ 222 expression, and that these miRNAs might contribute to NGF-dependent cell survival in PC12 cells. Abbreviations EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; miRNA, microRNA; NGF, nerve growth factor. FEBS Journal 276 (2009) 3269–3276 ª 2009 The Authors Journal compilation ª 2009 FEBS 3269 stimuli and cytokines such as nerve growth factor (NGF) and epidermal growth factor (EGF) [10–12]. Because both ERK signaling and miRNA function are involved in a variety of important biological responses, the significance of ERK signaling in terms of regulating miRNA expression is of great interest. To study the role of the ERK1 ⁄ 2 pathway in the regulation of miRNA expression, we first determined the expression profile of miRNAs by using the growth factor-induced neural differentiation process of PC12 as a model. It is well known that NGF, but not EGF, induces neural differentiation in PC12 cells, although both growth factors potently activate ERK1 ⁄ 2 [13–16]. We identified miR221 and 222 as differentially regulated miRNAs. Although the expression of these miRNAs was found to be ERK- dependent, the effect of NGF and EGF on their expression was different; thus, only NGF, but not EGF, can induce their expression. Further, our study showed that the sustained activation of ERK1 ⁄ 2by NGF, but not the transient activation of ERK, could effectively induce miR221 and 222 in PC12 cells. Finally we identified the BH3-only protein Bim, which is involved in NGF-dependent neuronal survival [17–19], as a potential target of miR-221 and 222. Results NGF stimulation induces miR-221/222 in PC12 cells Treatment of PC12 cells with NGF for 48 h induced readily detectable neurite outgrowth (Fig. 1A), so we selected two points at 0 and 48 h after stimulation to compare the expression of miRNAs. We used a TaqMan miRNA assay, featuring reverse transcription using stem-loop RT-PCR primers followed by real-time PCR using TaqMan probes [20], to examine the expression of 156 rat miRNAs. We identified only two miRNAs, miR-221 and 222, as drastically up-regulated 48 h after NGF stimulation (data not shown). These miRNAs are encoded in tandem on the chromosome X (Fig. 1B). Next, we examined the time course of expression of miR-221 and 222. Quantitative RT-PCR analysis showed that these miRNAs had a very similar profile (Fig. 1C). An alternative RT-PCR analysis, using a set of primers that amplify a fragment located between these two miRNAs, demonstrated transcriptional induction of this region upon NGF stimulation (data not shown). These data support the notion that miR- 221 and 222 derive from the same pri-miRNA [21]. Following NGF stimulation, the expression level of Fig. 1. NGF induces expression of the microRNAs miR-221 and 222 in PC12 cells. (A) NGF-induced differentiation of PC12 cells. (B) Schematic representation of the genomic structure of miR-221 and 222 and their corresponding sequences. (C, F) PC12 cells were treated with 100 ngÆmL )1 NGF or 30 n M EGF for the indicated times. Cells were harvested and total RNA was pre- pared. The RNA was subjected to quantita- tive RT-PCR to assess the levels of mature miR-221 ⁄ 222. The data represent means of the C t values (± SD, n = 3). In (C), *P < 0.01 for miR-221 versus 0 h point, and  P < 0.01 for miR-222 versus 0 h point. In (F), P < 0.01 for comparisons indicated by asterisks (Tukey’s test). (D, E) Cell extracts were subjected to immunoblotting with a-phospho-ERK1 ⁄ 2 and a-ERK1 ⁄ 2 IgGs. NGF induces miR-221 and 222 expression K. Terasawa et al. 3270 FEBS Journal 276 (2009) 3269–3276 ª 2009 The Authors Journal compilation ª 2009 FEBS both miRNAs rapidly increased and reached a maximum at 3–6 h, which was then sustained to the last time point assayed at 48 h. Over this time course, expression of an internal control U6 remained unchanged (Fig. 1C). Based on the threshold cycle (C t ) changes, we estimate that the expression of miR-221 and 222 had increased by approximately 2 6 -fold. NGF induced sustained activation of ERK1 ⁄ 2 over this time course (Fig. 1D). We further examined whether EGF stimulation also induces miR-221 ⁄ 222 in PC12 cells. Our analysis confirmed that EGF transiently activated ERK1 ⁄ 2, unlike NGF [13–16] (Fig. 1E). Because NGF-mediated expression of miR-221 ⁄ 222 peaked approximately 3 h after stimulation, we examined the expression of miR-221⁄ 222 at this time point in all further experiments. In contrast to NGF stimulation, EGF did not induce any detectable up-regulation of miR-221 and 222 at 3 h (Fig. 1F). Moreover, no such stimulation was found even after monitoring for up to 48 h (data not shown). Sustained activation of ERK1/2 is necessary for induction of miR-221/222 We next studied whether the NGF-induced expression of miR-221 ⁄ 222 depends on ERK1 ⁄ 2 activation. We first examined the effect of a specific inhibitor (U0126) for MAPK ⁄ ERK kinase (MEK) 1 ⁄ 2, which is a direct activator of ERK1 ⁄ 2 [22]. As shown in Fig. 2A, pre- treatment of U0126 potently inhibited NGF-induced ERK1 ⁄ 2 activation. The same pre-treatment with U0126 completely blocked induction of miR-221 and 222 (Fig. 2B). Moreover, we found that expression of miR-221 ⁄ 222 dramatically increased when constitu- tively active MEK1 (MEK1SDSE) [23] was transiently expressed in PC12 cells (Fig. 2C,D). Taken together, these results indicate that induction of miR-221 ⁄ 222 depends on the activation of ERK1 ⁄ 2. However, transient activation of ERK ⁄ 2 upon EGF stimulation did not induce miR-221 ⁄ 222 expression. This observation prompted us to hypothesize that induction of these miRNAs requires sustained activa- tion of ERK1 ⁄ 2. To verify this hypothesis, we blocked NGF-induced sustained ERK1 ⁄ 2 activation by add- ing U0126 10 min after NGF treatment (Fig. 3A). As shown in Fig. 3B, addition of U0126 completely inhib- ited the sustained activation of ERK1 ⁄ 2. In this situa- tion, the induction of miR221 ⁄ 222 was also completely suppressed (Fig. 3C). These results clearly demonstrate that sustained activation of ERK1 ⁄ 2 is required for induction of miR-221 and 222. However, the apparent induction of these miRNA molecules was only observed approximately 3 h after NGF stimulation (Fig. 1C), which implies that miR-221 and 222 are not the primary target genes regulated by NGF. We reasoned that this induction requires de novo protein synthesis. As shown in Fig. 4A, treatment with cycloheximide completely inhibited induction of these miRNAs, but had no significant effect on ERK1 ⁄ 2 activation (Fig. 4B). We also confirmed inhibition of protein synthesis by monitoring expression of c-Fos protein, which is a Fig. 2. Activation of ERK1 ⁄ 2 pathway is involved in miR-221 ⁄ 222 induction. (A, B) PC12 cells were pre-treated with 10 l M U0126 for 10 min before treatment with 100 ngÆmL )1 NGF for 3 h. Inhibition of ERK1 ⁄ 2 activity by U0126 was confirmed by immunoblotting (A). The expression levels of miR-221 ⁄ 222 were examined as described in Fig. 1C (*P < 0.05, Tukey’s test) (B). (C, D) PC12 cells were transfected either with empty vector or HA-tagged MEK1SDSE. The expression levels of miR-221 ⁄ 222 were examined 24 h after transfection as described in Fig. 1C (*P < 0.05 versus empty vec- tor, Student’s t test) (C). Expression of HA-tagged MEK1SDSE was confirmed by immunoblotting (D). DMSO, dimethylsulfoxide. K. Terasawa et al. NGF induces miR-221 and 222 expression FEBS Journal 276 (2009) 3269–3276 ª 2009 The Authors Journal compilation ª 2009 FEBS 3271 well-known NGF-induced immediate early gene [24]. Cycloheximide treatment completely blocked c-Fos protein synthesis (Fig. 4C, compare with control). These data indicate that de novo protein synthesis is required for the induction of miR-221 ⁄ 222. Pro-apototic Bim is a plausible target of miR-221/222 We used TargetScan [4] to identify the likely target genes of miR-221 ⁄ 222. Specifically, we focused on the pro-apototic Bim gene because Bim has been reported to be involved in NGF-dependent survival of PC12 cells [19]. The predicted target site for these miRNAs is conserved in human, mouse, rat, dog and chicken (Fig. 5A). The rat Bim gene had no annotated 3¢ UTR, and so in the TargetScan pro- gram this is computationally determined based on the human Bim 3¢ UTR sequence. Initially, we used RACE to verify whether the predicted 3¢ UTR region is transcribed. 3¢ RACE analysis detected products containing the terminal portion of the pre- dicted Bim 3¢ UTR. Moreover, RT-PCR analysis showed that a fragment containing the predicted tar- get site was amplified (data not shown). To examine whether these miRNAs can target the Bim gene, we generated a luciferase construct harboring a fragment of the Bim 3¢ UTR containing the target sequence (Fig. 5A). Co-expression of either miR-221 or miR- 222 significantly (P < 0.05) suppressed the reporter activity compared to the control (Fig. 5C, wt). Muta- tions introduced into the predicted binding site almost eliminated this suppression. These results sug- gest a direct interaction of these miRNAs with the predicted target site of the Bim 3¢ UTR (Fig. 5B,C, mt). Furthermore, we investigated the effect of expres- sion of miR-221 ⁄ 222 on endogenous Bim mRNA expression. Because the Bim gene has three alternative splice variants [25], we designed a set of primers to detect all three products. Overexpression of either miR-221 or miR-222 resulted in significant (P < 0.05) A B C Fig. 3. Sustained activation of ERK1 ⁄ 2 is required for NGF-induced miR-221 ⁄ 222 expression. (A) Schematic diagram of the experimen- tal design. (B, C) PC12 cells were pre-treated with 10 l M U0126 for 10 min after treatment with 100 ngÆmL )1 NGF for 3 h. Inhibition of sustained ERK1 ⁄ 2 activation by U0126 was confirmed by immu- noblotting (B). The expression levels of miR-221 ⁄ 222 were exam- ined as described in Fig. 1C (*P < 0.05, Tukey’s test) (C). DMSO, dimethylsulfoxide. Fig. 4. Protein synthesis is required for NGF-induced miR-221 ⁄ 222 expression. PC12 cells were pre-treated with 10 lgÆmL )1 cyclohexi- mide for 30 min before treatment with 100 ngÆmL )1 NGF for 3 h. The expression levels of miR-221 ⁄ 222 were examined as described in Fig. 1C (*P < 0.05, Tukey’s test) (A). ERK1 ⁄ 2 activation in the presence of cycloheximide (B) and inhibition of protein synthesis by cycloheximide (C) were confirmed by immunoblotting. a-Tubulin was used as a loading control. DMSO, dimethylsulfoxide. NGF induces miR-221 and 222 expression K. Terasawa et al. 3272 FEBS Journal 276 (2009) 3269–3276 ª 2009 The Authors Journal compilation ª 2009 FEBS down-regulation of Bim mRNA (Fig. 5D). To show that this down-regulation is a specific effect for Bim mRNA, we examined the mRNA level of an apoptosis-related gene, Bax, because the 3¢ UTR of Bax mRNA has no predicted target site for miR-221 ⁄ 222. We found that overexpression of either miR-221 or miR-222 had no significant effect on the Bax mRNA level (data not shown). Taken together, our data suggest that miR-221 and 222 can target the Bim gene. Discussion The present study has demonstrated that, in PC12 cells, miR-221 and 222 are transcriptionaly induced upon stimulation by NGF, and that this induction requires sustained ERK1 ⁄ 2 activation and de novo pro- tein synthesis. Presumably, sustained ERK1 ⁄ 2 activa- tion is required for the induction of transcriptional regulatory protein(s) that regulates miR-221 ⁄ 222 expression. Recently, miR-155 expression has been shown to be regulated by two MAPK pathways, the ERK1 ⁄ 2 and c-Jun N-terminal kinase pathways, through AP-1 signaling [26]. AP-1 family proteins are good candidates for mediating NGF-induced miR-221 ⁄ 222 expression in PC12 cells. A previous study showed that miR-221 and 222 are up-regulated in quiescent cells that have been stimulated to proliferate by serum stimulation [27]. ERK1 ⁄ 2 is known to play a critical role in growth factor-stimulated cell-cycle progression from G 0 ⁄ G 1 to S phase, and sustained activation is required for this progression [28]. Our finding that sustained ERK1 ⁄ 2 activation induces miR-221 ⁄ 222 expression is entirely consistent with this observation. miR-221 Fig. 5. The Bim 3¢ UTR is a target of miR-221 ⁄ 222. (A) Schematic representation of the reporter construct and conservation of the tar- get site of miR-221 ⁄ 222 in the Bim 3¢ UTR in vertebrates. The boxed region indicates the sites complementary to the seed sequence of miR-221 ⁄ 222. (B) Predicted base pairing between miR-221 and 222 and their target sites in the Bim 3¢ UTR. Underlin- ing indicate the seed sequences. The calculated free energy of hybridization of each miRNA with the target site is also indicated. These data were prepared using RNAhybrid. Lower-case letters indicate the sites introduced to the Bim 3¢ UTR by mutation. The indicated bases were substituted with complementary nucleotides in the mutation construct. (C) The indicated RNA oligonucleotides (10 pmol per well) and reporter plasmid (200 ng per well) were co-transfected with the internal control plasmid (20 ng per well) into PC12 cells. After 24 h, the cells were harvested, and the lysates were subjected to a luciferase assay. The results are the ratio of firefly to renilla luciferase activity (mean ± SD, n = 3). The lucifer- ase activity ratio for control RNA transfection (mock) for each repor- ter was set at 1 (*P < 0.01 versus mock; Student’s t test). (D) PC12 cells were transfected with the indicated RNA oligonucleo- tides. After 24 h, the cells were harvested and total RNA was pre- pared. The RNA was subjected to quantitative RT-PCR to assess the levels of Bim mRNA. The Bim mRNA expression was normal- ized against GAPDH mRNA expression (mean ± SD, n = 3). The normalized Bim expression of control RNA transfection (mock) was set at 1 (*P < 0.05 versus mock; Student’s t test). K. Terasawa et al. NGF induces miR-221 and 222 expression FEBS Journal 276 (2009) 3269–3276 ª 2009 The Authors Journal compilation ª 2009 FEBS 3273 and 222 have also been reported to be up-regulated in some cancer cell lines and to function as onco- genic miRNAs by targeting the cyclin-dependent kinase inhibitor p27 Kip1 [29–32]. These studies strongly suggest that miR-221 and 222 are involved in the regulation of cell growth. In PC12 cells, NGF stimulation of starved cells promotes cell survival and differentiation, and inhibits cell-cycle progression [16]. However, NGF-induced miR-221 and miR-222 expression in PC12 cells is probably not involved in cell-cycle progression. These apparently contradictory effects might be attributed to cell type-dependent differences. We could not observe any detectable effect of NGF-induced neurite outgrowth when miR-221 and ⁄ or 222 were overexpressed in PC12 cells (data not shown). However, we show that the pro-apototic protein Bim is a plausible target of miR-221⁄ 222. Bim is known to be regulated at both the transcrip- tional and post-transcriptional level [33]. Here, we have confirmed that Bim is regulated at the transla- tional level. In PC12 cells, the Bim gene is induced upon withdrawal of serum. When NGF stimulation occurs, activation of ERK1 ⁄ 2 causes phosphorylation of Bim proteins and inhibits their function, resulting in cell survival. This translational regulation of Bim ensures the down-regulation of Bim function and hence cell survival. These data indicate that NGF- induced miR-221 ⁄ 222 expression is involved in NGF- dependent cell survival. Interestingly, recent reports have shown that Bim is regulated by miR-32 and the miR-17–92 cluster of miRNAs, which are known to be up-regulated in several cancers [34–36]. Up-regulation of these miRNAs, which results in down-regulation of pro-apoptotic Bim mRNA, exerts an anti-apoptotic effect in some cancers [35,36]. The miR-17–92 cluster has also been shown to be involved in B-cell development and to target Bim mRNA [37,38]. In PC12 cells, the expression level of these miRNAs is moderate and remains unchanged upon NGF stimulation (data not shown). Thus, induced miR-221 and 222 might work cooperatively with these miRNAs. miR-221 and 222 are highly conserved in verte- brates and have the same seed sequence. Moreover, they are encoded in tandem on the same chromosome in human, mouse and rat, indicating that they have important roles in biological processes. In this study, we found that miR-221 ⁄ 222 expression is regulated by the ERK1 ⁄ 2 pathway in PC12 cells. This regula- tion might also function in different biological pro- cesses. Our findings provide new insights into the MAPK signaling pathway. Experimental procedures Cell culture and transfection PC12 cells were maintained in Dulbecco’s modified Eagle’s medium plus 10% fetal bovine serum, 5% donor horse serum and antibiotics at 37 °Cin5%CO 2 . The cells were seeded onto poly-l-lysine-coated 60 mm dishes (AGC Techno Glass Co. Ltd, Chiba, Japan) and incubated in a low concentration of serum (1% horse serum) for 24 h prior to treatment with 100 ngÆmL )1 NGF (Alomone Labs Ltd, Jerusalem, Israel) or 30 nm EGF (PeproTech EC Ltd, London, UK). Transfections were performed according to the manufacturer’s instructions using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA, USA). The miRNA precursors miR-221 and 222 and control RNA were purchased from Ambion (Austin, TX, USA). RNA isolation and RT-PCR analysis Total RNA was isolated using ISOGEN reagent (Nippon Gene Co. Ltd, Tokyo, Japan). miRNA expression was mea- sured using TaqMan MicroRNA Assays (Applied Biosys- tems, Lincoln, CA, USA) according to the manufacturer’s protocol, except that all reactions were carried out at half scale. The rat miRNAs assayed in this study are listed in the microrna assay index file version 1 (Applied Biosystems). U6 snRNA was used as an internal control. For detection of Bim mRNA, reverse transcription was performed using a QuantiTect reverse transcription kit (Qiagen, Hilden, Ger- many). Prepared cDNAs were then subjected to quantitative PCR analysis using Power SYBR Green PCR Master Mix (Applied Biosystems). The primers for the PCR analysis were 5¢-CTTCCATAAGGCAGTCTCAG-3¢ and 5¢-CGGAAGA TGAATCGTAACAG-3¢ for Bim, and 5¢-TTGCTGACAA TCTTGAGGGAG-3¢ and 5¢-GAGTATGTCGTGGAGTC TACTG-3¢ for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The data were obtained and analyzed using an ABI 7300 real-time PCR system (Applied Biosystems). Plasmid construction The plasmid pcDNA3-HA encoding Xenopus MEK1SDSE was supplied by E. Nishida (Graduate School of Biostudies, Kyoto University, Japan). A partial fragment of the 3¢ UTR of rat Bim was amplified by PCR using the primers 5¢-CCTGCCTCTTGAGGTACTGC-3¢ and 5¢-AGCTAGT CGCAAGTTTTA-3¢ following reverse transcription from total RNA isolated from PC12 cells, and cloned into the pCR-Blunt vector (Invitrogen). Mutagenesis of the Bim 3¢ UTR was performed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). An EcoRI site was introduced into the XbaI site of the lucifer- ase reporter vector pGL4.23 (Promega, Madison, WI, USA) by ligation with the oligonucleotides 5¢-CTAGACT NGF induces miR-221 and 222 expression K. Terasawa et al. 3274 FEBS Journal 276 (2009) 3269–3276 ª 2009 The Authors Journal compilation ª 2009 FEBS GAATTC-3¢ and 5¢-CTAGGAATTCAGT-3¢, yielding the pGL4.23EcoRI vector. EcoRI fragments of the wild-type (wt) and mutated (mt) Bim 3¢ UTR forms of the pCR- Blunt vector were ligated into the EcoRI site of the pGL4.23EcoRI vector. The identity of all constructs was confirmed by DNA sequencing. Immunoblotting Cells were harvested by scraping from culture dishes in hot 1· SDS sample buffer, and the lysates were separated by SDS–PAGE and analyzed by immunoblotting. Anti-HA (3F10) rat monoclonal IgG was purchased from Roche (Basel, Switzerland). Anti-p44 ⁄ 42 MAP kinase, anti-phos- pho-p44 ⁄ 42 MAP kinase IgGs (numbers 9101 and 9102, respectively) and anti-c-Fos IgG (number 4384) were obtained from Cell Signaling (Danvers, MA, USA). Anti-a- Tubulin (B-5-1-2) mouse monoclonal IgG was purchased from Sigma (St Louis, MO, USA). Peroxidase-linked sec- ondary antibodies were purchased from GE Healthcare (Chalfont St Giles, UK). An LAS3000 CCD imaging sys- tem (Fujifilm, Tokyo, Japan) was used for detection. Reporter assay Cells grown in 24-well plates (1.0 · 10 5 cells per well) were harvested for assays 24 h after transfection. The luciferase activity was measured using a dual-luciferase reporter assay system (Promega) with a Lumat LB9507 luminometer (Berthold Technologies, Bad Wildbad, Germany). As an internal control, a renilla luciferase vector pGL4.70 (Pro- mega) was used. The data represent means and standard deviations of three independent experiments. Statistical analysis The data were analyzed using Student’s t test or ANOVA followed by Tukey’s test as indicated. Acknowledgements This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K.T., F.S., K.S. and G.T.), the New Energy and Industrial Technology Develop- ment Organization (to K.T. and G.T.), and the Uehara Memorial Foundation (to G.T.). References 1 Ambros V (2004) The functions of animal microRNAs. Nature 431, 350–355. 2 Calin GA & Croce CM (2006) MicroRNA signatures in human cancers. 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Results NGF stimulation induces miR-221 /222 in PC12 cells Treatment of PC12 cells with NGF for 48 h induced readily