Biochemical and Biophysical Research Communications 394 (2010) 921–927
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
miR-7 and miR-214 are specifically expressed during neuroblastoma
differentiation, cortical development and embryonic stem cells differentiation,
and control neurite outgrowth in vitro
Hailan Chen a, Ruby Shalom-Feuerstein b,c,d, Joan Riley a, Shu-Dong Zhang a, Paola Tucci a,e,
Massimiliano Agostini a, Daniel Aberdam b,c,d, Richard A. Knight a, Giuseppe Genchi f, Pierluigi Nicotera a,g,
Gerry Melino a,e,*, Mariuca Vasa-Nicotera a,**
a
Medical Research Council Toxicology Unit, Hodgkin Building, Lancaster Road, PO Box 138, Leicester LE1 9HN, UK
INSERM U898, Nice, France
c
University of Nice-Sophia Antipolis, Nice, France
d
INSERTECH, Bruce Rappaport Institute of the Technion, Haifa, Israel
e
Biochemistry Laboratory, IDI-IRCCS, C/O Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘‘Tor Vergata”, 00133 Rome, Italy
f
Biochemistry Laboratory, Department of Pharmaco-Biology, University of Calabria, 87036 Rende (Cs), Italy
g
Deutsche Zentrum Fuer Neurodegenerative Erkrankungen (DZNE), Bonn, Germany
b
a r t i c l e
i n f o
Article history:
Received 9 March 2010
Available online 15 March 2010
Keywords:
Apoptosis
Cell death
microRNA
Neural differentiation
Central nervous system
Microarray
a b s t r a c t
The mammalian nervous system exerts essential control on many physiological processes in the organism and is itself controlled extensively by a variety of genetic regulatory mechanisms. microRNA (miR),
an abundant class of small non-coding RNA, are emerging as important post-transcriptional regulators of
gene expression in the brain. Increasing evidence indicates that miR regulate both the development and
function of the nervous system. Moreover, deficiency in miR function has also been implicated in a number of neurological disorders. Expression profile analysis of miR is necessary to understand their complex
role in the regulation of gene expression during the development and differentiation of cells. Here we
present a comparative study of miR expression profiles in neuroblastoma, in cortical development, and
in neuronal differentiation of embryonic stem (ES) cells. By microarray profiling in combination with real
time PCR we show that miR-7 and miR-214 are modulated in neuronal differentiation (as compared to
miR-1, -16 and -133a), and control neurite outgrowth in vitro. These findings provide an important step
toward further elucidation of miR function and miR-related gene regulatory networks in the mammalian
central nervous system.
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
Highly orchestrated programmes of gene expression act to
shape the developing nervous system. This tight regulation is mediated by a variety of transcriptional and post-transcriptional events
that control the expression of individual gene products. More recently, it has become clear that protein expression can also be
modulated by several classes of small RNAs, including small interfering (si), Piwi and microRNA (miR). The combination of these diAbbreviations: miR, microRNA; si, small interfering; CNS, central nervous
system; ES, embryonic stem; ESR1, oestrogen receptor alpha; RA, retinoic acid;
DIV, days in vitro; E, embryonic; P, postnatal day; W, weeks postnatal
* Corresponding author at: Medical Research Council Toxicology Unit, Hodgkin
Building, Lancaster Road, PO Box 138, Leicester LE1 9HN, UK.
** Corresponding author.
E-mail addresses: [email protected], [email protected] (G. Melino), mvn1@lei
cester.ac.uk (M. Vasa-Nicotera).
0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2010.03.076
verse regulatory processes on protein expression and activity
confer great plasticity to the cellular responses to changes in its local environment.
miRs are a recently discovered class of short non-coding RNA
genes that act post-transcriptionally as negative regulators of gene
expression [1]. A large body of research shows that animal miRs
play fundamental roles in many biological processes, including cell
growth and apoptosis, hematopoietic lineage differentiation, insulin secretion, brain morphogenesis, and muscle cell proliferation
and differentiation [2,3].
During development, many miRs are expressed in neurons or
show distinct expression patterns within the developing central
nervous system (CNS), suggesting their importance in brain
development and function. However, functional studies of miRs
in the vertebrate nervous system are still very limited. A number
of studies have begun to address the role of miR in neuronal differentiation. For example, a microarray comparison of the miR
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H. Chen et al. / Biochemical and Biophysical Research Communications 394 (2010) 921–927
profile in embryonic, postnatal, and adult brain revealed differential changes in nine miRs, including miR-9 and -124 [4,5]. In vitro,
the levels of both miRs increased sharply during the transition
from neuronal precursors to neurons in differentiating embryonic
stem (ES) cells [6]. Overexpression of both miRs shifted the differentiation of the precursors toward a neuronal fate, while inhibition had the opposite effect [7]. Another microarray study,
comparing miR expression profiles in rat neuronal progenitors between E11 and E13 also emphasised the importance of miR-9 and
-124 among the 21 miRs whose expression was increased and the
11 where reduced expression was found [8]. Moreover, ectopic
expression of miR-124 also increased the degree of retinoid-induced differentiation in a neuroblastoma cell line [9]. In neuroblastoma cells with enhanced expression of N-MYC, there is
correspondingly increased expression of miR-18a and -19a, which
repress oestrogen receptor alpha (ESR1), and overexpression of
ESR1 results in growth arrest and neuronal differentiation [10],
suggesting that a MYC/miR/ESR1 axis is important in development of the sympathetic nervous system. Moreover, links between miRs dysfunction and neurological diseases become more
and more apparent [11]. Despite the accumulating evidence that
miR play important roles in brain development and disorders,
our knowledge of miR function in the vertebrate nervous system
is still quite limited.
We have established a classic model of neuronal differentiation
and apoptosis treating neuroblastoma cells with retinoic acid (RA)
[12–16]. By combining microarray expression profiling with miRspecific real time PCR, we have compared the expression profiles
of miR in neuroblastoma cells induced to differentiate with RA,
in the development of mouse brain cortex, and in neuronal differentiation from mouse ES cells. While there are some differences in
the pattern of miR expression in the different models, we did not
identify a prominent expression for miR-9 and -124 in any system,
but did find additional miRs, namely miR-7 and miR-214, implicated in neuronal differentiation and in the control of neurite outgrowth in vitro, which will serve as an important basis for detailed
studies of individual miR, their target genes, and the miR-related
regulatory networks in the mammalian central nervous system.
2. Materials and methods
2.1. Human neuroblastoma cell line
SH-SY5Y cells (ATCC, UK) were maintained in non-differentiating
medium (DMEM, 10% FCS, 1% penicillin/streptomycin). Twenty-four
hours of post-seeding, the non-differentiating medium was replaced
with differentiating medium (DMEM, 1% FCS, 1% penicillin/streptomycin, 1% Glutamine, 10 lM RA) and cells incubated for further
48 h.
2.2. Primary culture of mouse cortical neurons
Mouse cortical neuronal cultures were prepared from E16–E17
mouse embryos and cultured on poly-D-lysine-coated cell culture
dishes in a defined serum-free medium (Neurobasal, 2% B-27 supplement, 2 mM glutamine, 1% penicillin/streptomycin). Cytosine
arabinoside (10 lM) was added at day 7 after plating. The cells
were then collected in Trizol (Invitrogen) every 2 days until day
in vitro (DIV) 12.
2.3. Mouse embryonic stem (ES) cells
Mouse ES cell lines CGR8 and CGR8/Sox1-GFP (a gift of Smith)
were routinely cultured in flasks coated with 0.1% gelatin in ES
medium (GMEM, 10% FCIII, 1% nonessential amino acids, 1 mM
sodium pyruvate, 0.1 mM b-mercaptoethanol and 103 U/ml LIF
(Leukemia Inhibitory Factor)). For neural differentiation, confluent
NIH-3T3 cells were fixed with 3% formaldehyde, washed with PBS
and incubated with glycine to saturate free formaldehyde sites. For
ES cells differentiation, the cells were cultured on fixed NIH-3T3
cells in differentiation medium (similar to ES medium but without
LIF and FCIII, and with 10% knock out serum).
2.4. Microarray printing, labelling, and hybridization
We use in-house made two-colour cDNA microarrays to measure the expression levels of miR. Briefly, 3.0 lg of total RNA was
labelled using FlashTag™ kits according to the manufacturers
instructions (Genisphere). Reference RNA (or control RNA, e.g.,
RNA from native SH-SY5Y cells, or DIV 0 for mouse cortical neurons, or differentiation day 0 for mouse ES cells), labelled with OysterÒ-550 was hybridized against RNA labelled with OysterÒ-650
from the experiment RNA (e.g., RNA from RA-differentiated SHSY5Y cells, or different DIV for mouse cortical neurons, or differentiation days for mouse ES cells), and reverse (Reference-OysterÒ650, experiment-OysterÒ-550) labelling reactions.
Hybridizations were performed overnight at 52 °C on microarrays printed in-house using the miRCURY LNA™ ready-to-spot
probe set version 208010V8.1 (Exiqon). Following hybridization,
the microarrays were washed at RT in 2 SSC containing 0.2%
(w/v) SDS for 5 min, 1 SSC for 5 min, 0.2 SSC for 5 min, and dried
by centrifugation.
2.5. Microarray data analysis
Microarray slides were scanned using an Axon 4200A scanner
to acquire the microarray images which were subsequently processed with GenePix Pro software (Molecular Devices) to generate
the expression data in GPR (Genepix Result File) format, all according to manufacture’s instructions. The median fluorescent intensity
of a feature spot on a microarray slide was used to represent the
expression level of the corresponding gene.
Each microarray was then normalized by globally shifting the
mode of log-ratio values to 0, implicitly making the assumption
that on each microarray slide most genes are not differentially expressed between the two samples. The normalized data were subsequently analyzed using the statistical method described by
Zhang and Gant [17] for gene expression experiments involving
both forward and reverse labelled microarrays. In the miR microarray data, the threshold p-value was set such that on average only
two of the genes (miRs) declared significant is expected to be false,
then genes (miRs) with p-values lower than the above set threshold are declared as statistically significant. The changes >1.6-fold
and p < 0.05 of up-regulated miRs and the changes <0.7-fold and
p < 0.05 of down-regulated miRs were used for further analysis.
Significant changes were verified by real time PCR as described
below.
2.6. Real time PCR
Total RNA from cells was isolated using Trizol (Invitrogen)
according to the manufacturer’s instructions. Then RNA was reverse transcribed using TaqMan MicroRNA Reverse Transcription
kit and qRT-PCR was performed with TaqMan universal master
mix (Applied Biosystem) and specific primers for miRs. SnoRNA202
(mouse) or RNU6B (human) were used as internal control (Applied
Biosystem). The expression of each miR was defined from the
threshold cycle (Ct), and relative expression levels were calculated
using the 2 DDC t method after normalization with reference to
expression of internal control.
H. Chen et al. / Biochemical and Biophysical Research Communications 394 (2010) 921–927
2.7. High-throughput neurite outgrowth assays
SH-SY5Y cells were maintained in non-differentiating medium.
Twenty-four hours of post-seeding, non-differentiating medium
was replaced with differentiating medium, containing 10 lM RA,
and the cells incubated for further 48 h. The cells were then transfected with 10 nM Pre-miR™ miRNA Precursors, or 75 nM AntimiR™ miRNA Inhibitors, or FAM-labeled Negative Control (Ambion), using the SiPORT neoFX transfection agent (Ambion) according
to the manufacturer’s instruction. After 48 h the cells were fixed
with 3% paraformaldehyde in PBS followed by treatment with
0.1% Triton X-100 for 5 min and 50% normal goat serum (NGS) in
PBS for 1 h at RT. Neuronal-specific anti-b III tubulin antibody
was used to stain the neurite processes (1:5000). Bound antibody
and nucleus were visualized using Alexa 546-conjugated secondary antibody (1:500) and Hoechst 33342 (1:1000), respectively.
2.8. Automated image analysis
Cells were imaged using a Cellomics Kinetic Scan Reader high
content microscope system and analysed using neurite outgrowth
for the outgrowth assay. Twenty images per well were taken at
10 magnification in a fully automated and blind manner. The total number of cell counts and the average of neurite length per
neuron in each experiment in duplicate were determined for neurite outgrowth assay.
3. Results
3.1. miR-7 and miR-214 are modulated during neuroblastoma cells
differentiation
In order to identify the miRs involved in the differentiation of
human SH-SY5Y cells, we analysed the expression profile of miR
in the cells induced to differentiate with RA (10 lM) using microarrays with 700 oligonucleotide probes complementary to mature
forms of miRs of human origin, based on version 10.1 of the Sanger
miRBase (http://microrna.sanger.ac.uk/sequences). Data from the
microarray (analyzed as described in Section 2) showed that 73
miRs were modulated during differentiation induced by RA (Table
S1).
To validate the microarray platform, we confirmed the expression of 12 miRs which were most strongly (statistical values are
shown in the table) up- or down-regulated (Table 1) by qRT-PCR,
using the same RNA samples that were used for the microarrays.
Real time PCR confirmed the modulation of several miRs, including
down-regulation of miR-7 (Fig. 1A), and up-regulation of miR-214
(Fig. 1B) after differentiation of the cells with RA for 48 h.
Table 1
miR regulated during RA-induced neuroblastoma differentiation.
Name
Fold
p-Value
Sanger mature miR-sequence
Up-regulated
hsa-miR-132
hsa-miR-16
hsa-miR-27b
hsa-mir-27a
hsa-miR-214
hsa-miR-197
2.67
2.62
2.45
1.99
1.80
1.65
0.0138848
0.0300957
0.0236875
0.0048806
0.0071070
0.0001151
59-uaacagucuacagccauggucg-80
14-uagcagcacguaaauauuggcg-35
51-uucacaguggcuaaguucugc-81
51-uucacaguggcuaaguuccgc-71
71-acagcaggcacagacaggcagu-92
48-uucaccaccuucuccacccagc-69
Down-regulated
hsa-miR-133a
hsa-miR-508-3p
hsa-miR-7
hsa-miR-1
hsa-mir-205
hsa-mir-20b
0.42
0.56
0.56
0.57
0.66
0.69
0.0001546
0.0005188
0.0500789
0.0076927
0.0098568
0.0001416
53-uuugguccccuucaaccagcug-74
61-ugauuguagccuuuuggaguaga-83
24-uggaagacuagugauuuuguugu-46
53-uggaauguaaagaaguauguau-74
34-uccuucauuccaccggagucug-55
6-caaagugcucauagugcagguag-28
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3.2. Effect of miR-7 and miR-214 on neurite outgrowth during
neuroblastoma cells differentiation
To investigate whether RA-induced modulation of these two
miRs has functional significance, we measured the neurite length
of SH-SY5Y cells transfected with either pre-miR precursors, or
anti-miR inhibitors, or with negative controls. We took advantage
of the high throughput technology to automatically visualize and
measure neurite outgrowth. We tested all miRs shown in Table
1, but only two showed a coherent behaviour, that is opposite effect of pre-miR versus anti-miR. In fact miR-1, -16 and -133a were
not able to show a direct coherent effect on neurite outgrowth. We
found that neurite length significantly increased when miR-214
(Fig. 1C and D) (whose expression was increased following RA,
Fig. 1B) was over-expressed. However, when the expression of
the miR-214 was inhibited, neurite length remained statistically
unchanged (Fig. 1D).
Conversely (Fig. 1C and D) we found a reduction in the neurite
outgrowth by transfecting the cells with the precursor of miR-7,
and an increase of the neurite outgrowth when the SH-SY5Y were
transfected with an inhibitor of the miR-7. Fig. 1D shows the statistical validation of results obtained by high throughput analysis.
These results indicate that not only miR-7 and miR-214 were
modulated during neuroblastoma differentiation, but also that
they had a relevant role during this phase, as they are able to modulate per se the outgrowth of neurites. Therefore, we decided to
investigate the involvement of these two miRs in other more physiological ex vivo cellular models, namely mouse cortical neurons
in vitro and cerebellar cortical neurons during late developmental
stages.
3.3. Expression of miR-7 and miR-214 during differentiation of mouse
cortical neurons ex vivo
miR expression levels were also evaluated in spontaneously differentiating primary mouse cortical neurons in vitro every 2 days
until DIV 12.
The miR-7 levels, whose expression after microarray and real
time PCR was found down-regulated during neuroblastoma differentiation, increased progressively during differentiation of mouse
primary cortical neurons in vitro (Fig. 2A).
In keeping with the expression pattern in RA-differentiated
neuroblastoma cells, the levels of miR-214 increased (Fig. 3A).
3.4. miR expression during development of mouse cerebral cortex
Figs. 2B and 3B shows the changes in expression levels of miRs7 and -214 during the development of mouse brain cortex from
E13 to 6 weeks postnatal. The data are expressed relative to their
respective values at E13. During embryonic and postnatal development the levels of miR-214 dropped significantly (Fig. 3B), while
expression of miR-7 did not change during the embryonic development but progressively increased during postnatal cortical development (Fig. 2B).
3.5. miR expression during neuronal differentiation of mouse ES cells
In vitro ES cells can be induced to differentiate, recapitulating
the physiological in vivo differentiation. Because of the high relevance of this model, we decided to evaluate in this model the regulation of miR-7 and miR-214.
In order to study the possible role of miRs in neural commitment, mouse ES cells were cultured on fixed feeder PA6 fibroblast
cells in the absence of serum. To quantify the efficacy of neural differentiation, we used ES cells with stable GFP gene expression under the control of the sox-1 promoter. Cells were collected at the
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H. Chen et al. / Biochemical and Biophysical Research Communications 394 (2010) 921–927
Fig. 1. Real time PCR and high-throughput screen of miR regulated during neuroblastoma cells differentiation. Cells were treated for 48 h with 10 lM RA and endogenous
levels of miR-7 (A) and miR-214 (B) were assayed by qRT-PCR in order to validate the results obtained from the microarray; the result were normalized to RNU6b. Data
represent mean ± SD of three different experiments analyzed in triplicate. (C) Example of neurite outgrowth evaluation by immunostaining, as described in Section 2, of SHSY5Y cells transfected with pre-miR, or anti-miR, or a negative control. (D) SH-SY5Y were transfected with pre-miRs, or with anti-miRs, or a negative control, and neurite
growth was measured as described in Section 2. The results were expressed as average of the neurite length per outgrowth neuron. Experiment was performed in triplicate,
and 25 or 40 images acquired per well. ***p < 0.001; **p < 0.01; *p < 0.05; t-test.
indicated times and neural differentiation was evaluated by FACS
analysis, immunofluorescence microscopy and real time PCR (data
not shown). ES cells were differentiated into large colonies of neural cells. Within 7 days of culture, 60% and 80% of the cells expressed the putative neural precursor markers, CD57 and sox-1,
respectively. Enhancement in the expression levels of the bIII-
tubulin and neurofilament, was demonstrated by immunofluorescence microscopy and real time PCR (data not shown), which together showed efficient neuronal differentiation. miR-214
showed an early increase in expression at day 1, which subsequently declined on days 4 and 7 (Fig. 3C). In contrast, expression
of miR-7 decrease although with an irregular fashion (Fig. 2C).
H. Chen et al. / Biochemical and Biophysical Research Communications 394 (2010) 921–927
Fig. 2. miR-7 regulation during neuronal differentiation. (A) Expression levels of
miR-7 during mouse cortical neuron development. Steady state expression levels
were evaluated by qRT-PCR in primary mouse cortical neuron in culture every
2 days until DIV 12 (DIV, day in vitro). (B) Expression of miR-7, by qRT-PCR, during
the development of mouse brain cortex for a period from E13 to adult (E,
embryonic; P, postnatal day; W, weeks postnatal) (N = 6 at E13, E16, E18 and P1;
N = 3 for P11, 3W and N = 3 at 6W). (C) Regulation of miR-7 during the ES cells
neuronal differentiation after 1, 4, and 7 days. The results were normalized to
SnoRNA202. Data represent mean ± SD of three different experiments analyzed in
triplicate.
925
Fig. 3. miR-214 regulation during neuronal differentiation. (A) Expression levels of
miR-214 during mouse cortical neuron development. Steady state expression levels
were evaluated by qRT-PCR in primary mouse cortical neuron in culture every
2 days until DIV 12. (B) Expression of miR-214, by qRT-PCR, during the development
of mouse brain cortex for a period from E13 to adult. Symbols as in Fig. 2. (C)
Regulation of miR-214 during the ES cells neuronal differentiation after 1, 4, and
7 days. The result were normalized to SnoRNA202. Data represent mean ± SD of
three different experiments analyzed in triplicate.
4. Discussion
3.6. Regulation of other miRs
Even though other miRs were not sufficient per se to cause
neurite outgrowth as discussed above (Table 1 and Fig. 1), we
evaluated the regulation of other miRs using our cellular models.
miR-1, as reported in Fig. 4, showed a consisted up-regulation in
all differentiation models tested. This indicates a relevant,
although not necessary not sufficient, role of miR-1 during neuronal differentiation. miR-133a (Fig. S1) showed a consistent induction in all models, despite that it was not significantly regulated
in RA-treated neuroblastoma cells. miR-16 was less consistent
(Fig. S2).
The nervous system undergoes extensive changes in patterning,
remodelling, and cell specification during development. In mature
mammals, it consists of networks of cells that reach every organ
and part of the body to conduct impulses back and forth to control
essential physiological responses to internal and external stimuli
in a timely fashion. To accomplish its tasks, the nervous system
uses a large number of cells with different properties to form
exceedingly complex structures and depends on an array of elaborate gene regulatory mechanisms for its development and function.
miRs are involved in a variety of physiological and pathological
processes in multicellular organisms [1–3], ranging from patterning (for example in the epidermis [18,19]) to cancer development.
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H. Chen et al. / Biochemical and Biophysical Research Communications 394 (2010) 921–927
Fig. 4. miR-1 regulation during neural differentiation. Expression level of miR-1 during in vitro differentiation of neuroblastoma cells treated with RA (A), and during mouse
cortical neuronal development (B). (C) Expression during the development of brain cortex in mouse from E13 to adult stages. (D) Regulation of miR-1 during the ES cells
neuronal differentiation. All technical details and symbols are as described in Fig. 2.
Since the central nervous system is a rich source of miRs that often
display a brain specific expression pattern, and since a single miR is
able to target up to a few hundreds of different mRNAs, it is hardly
surprising that the number of roles assigned to miRs during all
stages of central nervous system development and function is rapidly expanding. Moreover, the number of miR genes found to be
expressed in the nervous system seems to be larger than that in
many other organs, perhaps partly reflecting the fact that the nervous system contains many types and subtypes of cells.
Toward understanding the complexity of miRs expression, in
this study we have compared the expression profiles of miRs
involved in neuronal differentiation in three models: differentiation of neuroblastoma cells induced with RA, time kinetics of
expression in the developing mouse cerebral cortex, and neuronal
differentiation from ES cells. We have focused on two miRs (miR-7
and miR-214) in all three systems out of a total of 73 whose
expression was changed by microarray analysis during neuroblastoma differentiation. Even more interesting, these two miRs were
able to regulate neurite outgrowth per se, suggesting a pivotal role
in this process. These experiments have revealed that although
during neuronal differentiation and neurodevelopment distinct
areas of the central nervous system express similar miRs, the direction and degree of change relative miR levels vary significantly in
different regions or in different development stages.
When treated with RA the SH-SY5Y cells, neuronal MYCN-driven cancer cells, will terminally differentiate into neuron-like cells.
Accompanied with the classical morphological changes of neurite
outgrowth, expression of miR-214 is significantly induced over
time, as we confirmed both by microarray analysis and real time
PCR, suggesting that this miR may play a role in differentiation
or cell fate determination, in addition to its potential functions in
adults [20–22], as we saw an increase in the development of the
mouse cortical neurons and of brain cortex. Although expression
of anti-miR-214 had no significant effect on neurite outgrowth of
neuroblastoma cells, regulation of miR-214 not only is relevant
for cell lineage-specific differentiation (i.e., SH-SY5Y), but also
may influences ES cell commitment. Pluripotent ES cells, which
express very low levels of miR-214, readily activate miR-214
transcription when induced to differentiate. Indeed, miR-214 accumulation is evident at the very initial stages of cell differentiation
of the ES cells.
Thus, after RA treatment, miR-7 was found reduced both on the
array and by real time PCR, as well as in ES cells during the neuronal differentiation, which suggests that it has a more general influence on the process of differentiation and development. In
contrast, miR-7 expression increased during mouse cortical neuron
development and differentiation. Others [8] have also shown increases in miR-7 expression between days E11 and E13 of mouse
cortical development. These apparent differences in miR-7 kinetics
may, at least in part, reflect the fact that SH-SY5Y cells are dopaminergic, whereas neurons in the developing brain will contain a
variety of neurotransmitters. Moreover, overexpression of miR-7,
by pre-miR-7 precursor, reduced neurite outgrowth when the differentiation occurred in SH-SY5Y cells, whereas expression of antimiR-7 enhanced the size of the neural net. Although others have
shown a small increase in miR-7 expression 12 days after addition
H. Chen et al. / Biochemical and Biophysical Research Communications 394 (2010) 921–927
of RA to SH-SY5Y cells, changes in expression of miR were more
pronounced and occurred earlier in the differentiation process
[9]. Nevertheless, we believe that our findings provide a significant
evidence for a down-regulation and a functional involvement of
miR-7 at an early stage (48 h) of neuroblastoma differentiation.
All together these results suggest that miR expression profile
can serve as a marker of neuronal development and that specific
miR may contribute to the developmental process.
Acknowledgments
This work has been supported by the Medical Research Council,
UK; ‘‘Alleanza contro il Cancro” (ACC), MIUR/PRIN (RBIP06LCA9_0023), AIRC (2008-2010_33-08), ISS ‘‘Program Italia-USA”
N526D5, Italian Human ProteomeNet RBRN07BMCT_007, Telethon
(GGPO4110) and RF 06 73UO3, RF07EC57UO2 G.M.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2010.03.076.
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