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
Trang 1microRNA-221 and 222 in PC12 cells
Kazuya Terasawa1, Atsuhiko Ichimura1, Fumiaki Sato2, Kazuharu Shimizu2and Gozoh Tsujimoto1
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.
Trang 2stimuli 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⁄ 2 by
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)1NGF 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 Ctvalues (± 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.
Trang 3both 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 (Ct)
changes, we estimate that the expression of miR-221
and 222 had increased by approximately 26-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)1NGF 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.
Trang 4well-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 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)1NGF 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)1NGF 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.
Trang 5down-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 inducactiva-tion of transcripactiva-tional 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 G0⁄ G1
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).
Trang 6and 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 p27Kip1 [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-transcriptranscrip-tional 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 funcregula-tion 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 37C in 5% CO2 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 fileversion 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
Trang 7GAATTC-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· 105cells 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.)
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