MOLECULAR AND CELLULAR BIOLOGY, Oct. 2009, p. 5290–5305
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 29, No. 19
MicroRNA-125b Promotes Neuronal Differentiation in Human Cells
by Repressing Multiple Targets?†
Minh T. N. Le,1,2Huangming Xie,1,2,3Beiyan Zhou,3Poh Hui Chia,4‡ Pamela Rizk,4
Moonkyoung Um,3§ Gerald Udolph,4Henry Yang,5
Bing Lim,1,2,6* and Harvey F. Lodish1,3,7*
Computation and Systems Biology, Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore 117576, Republic of Singapore1;
Genome Institute of Singapore, 60 Biopolis Street, Genome, Singapore 138672, Republic of Singapore2; Whitehead Institute for
Biomedical Research, 9 Cambridge Center, Suite 601, Cambridge, Massachusetts 021423; Institute of Medical Biology,
8A Biomedical Grove, Immunos, Singapore 138648, Republic of Singapore4; Bioinformatics Group,
Singapore Immunology Network, 8A Biomedical Grove, Singapore 138648, Republic of Singapore5;
Beth Israel Deaconess Medical Center, Harvard Medical School, CLS 442, 330 Brookline Ave.,
Boston, Massachusetts 022156; and Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 021427
Received 1 November 2008/Returned for modification 14 December 2008/Accepted 11 July 2009
MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the posttranscrip-
of neurally enriched miRNAs remain poorly understood. We report here the expression profile of miRNAs during
during differentiation induced by all-trans-retinoic acid and brain-derived neurotrophic factor. We demonstrated
that the ectopic expression of either miR-124a or miR-125b increases the percentage of differentiated SH-SY5Y cells
with neurite outgrowth. Subsequently, we focused our functional analysis on miR-125b and demonstrated the
important role of this miRNA in both the spontaneous and induced differentiations of SH-SH5Y cells. miR-125b is
also upregulated during the differentiation of human neural progenitor ReNcell VM cells, and miR-125b ectopic
expression significantly promotes the neurite outgrowth of these cells. To identify the targets of miR-125b regula-
tion, we profiled the global changes in gene expression following miR-125b ectopic expression in SH-SY5Y cells.
miR-125b represses 164 genes that contain the seed match sequence of the miRNA and/or that are predicted to be
direct targets of miR-125b by conventional methods. Pathway analysis suggests that a subset of miR-125b-repressed
targets antagonizes neuronal genes in several neurogenic pathways, thereby mediating the positive effect of miR-
125b on neuronal differentiation. We have further validated the binding of miR-125b to the miRNA response
elements of 10 selected mRNA targets. Together, we report here for the first time the important role of miR-125b
in human neuronal differentiation.
MicroRNAs (miRNAs) represent an emerging class of small
noncoding RNAs that play important roles in the posttran-
scriptional regulation of gene expression (2). They are tran-
scribed initially as long RNAs and then processed by two
RNase complexes, Drosha and Dicer, into ?22-nucleotide (nt)
duplexes that are subsequently loaded into RNA-induced si-
lencing complexes (2). Mature miRNAs in the RNA-induced
silencing complexes usually bind to the 3? untranslated region
(UTR) of mRNAs, leading to the translational suppression or
destabilization of the target mRNAs or both (10). The inter-
action between a miRNA and its target mRNA does not re-
quire perfect complementarity. Hence, a single miRNA has the
potential to regulate multiple target mRNAs (10).
miRNAs have been demonstrated to be essential for neural
development. Recent reports highlighted the abundant and
diverse expression of miRNAs in the central nervous system
(15–17, 29, 33, 36). Mammalian brain tissues express about
70% of experimentally detectable miRNAs, many of which are
developmentally regulated (15–17, 29, 33, 36). In maternal-
zygotic zebrafish dicer mutants, a deficiency in Dicer-mediated
biogenesis of miRNAs leads to severe defects in brain mor-
phogenesis (11). Similarly, the loss of Dicer in sca3 mutant
Drosophila melanogaster enhances neurodegeneration (4). The
specific knockdown of Dicer in mouse midbrain dopaminergic
neurons resulted in a progressive loss of these cells (14). Re-
cent studies have also elucidated the contribution of individual
miRNAs to various aspects of neural development. For exam-
ple, miR-9a regulates the organizer function of the zebrafish
midbrain-hindbrain boundary (21). In Caenorhabditis elegans,
lsy-6 and miR-273 determine the cell fate of chemoreceptor
neurons (13). miR-7 regulates the differentiation of photore-
ceptor neurons in Drosophila (23). The miR-200 family regu-
lates the terminal differentiation of olfactory neurons in both
* Corresponding author. Mailing address for Harvey F. Lodish:
Whitehead Institute for Biomedical Research, 9 Cambridge Center,
Suite 601, Cambridge, MA 02142. Phone: (617) 258-5216. Fax: (617)
258-6768. E-mail: firstname.lastname@example.org. Mailing address for Bing Lim:
Genome Institute of Singapore, 60 Biopolis Street, Genome, Singapore
138672, Republic of Singapore. Phone: 65 6478 8186. Fax: 65 6478
9005. E-mail: email@example.com.
† Supplemental material for this article may be found at http://mcb
‡ Present address: Department of Biological Sciences, Stanford Uni-
versity, Stanford, CA 94305.
§ Present address: Boston Biomedical Research Institute, 64 Grove
Street, Watertown, MA 02472.
?Published ahead of print on 27 July 2009.
mouse and zebrafish (5). In addition, miRNAs play important
roles in neuronal function and survival. In Drosophila, the
miRNA bantam prevents neuronal apoptosis by suppressing
the proapoptotic gene hid (4). In mature rat neurons, miR-134
localizes to dendrites and regulates spine size (31). In C. el-
egans, miR-1 regulates MEF-2-dependent retrograde signaling
at neuromuscular junctions (34). Most functional studies of
miRNAs in neuronal development have been carried out using
animal models, and it remains to be proven if miRNAs play the
same role in human neurogenesis.
In this study we sought to understand the role of miRNAs in
the differentiation of human neural cells using simple in vitro
models, human neuroblastoma SH-SY5Y cells and human
neural progenitor ReNcell VM (RVM) cells. When sequen-
tially treated with all-trans-retinoic acid (RA) and brain-
derived neurotrophic factor (BDNF), SH-SY5Y cells give rise
to fully differentiated neuron-like cells (8). These differenti-
ated SH-SY5Y cells are withdrawn from the cell cycle, express
various neuronal markers, and exhibit carbachol-evoked nor-
adrenaline release (8). Moreover, as no glial cell is derived by
this process, it is a robust and homogenous model system for
investigating neuronal differentiation (8). Using microarrays
and Northern blots, we identified a group of miRNAs that are
significantly upregulated in differentiated SH-SY5Y cells. We
further showed that one of these miRNAs, miR-125b, signifi-
cantly enhances the differentiation and neuronal morphogen-
esis of SH-SY5Y cells. In addition, this miRNA also promotes
neurite outgrowth in human neural progenitor RVM cells.
miR-125b is a homolog of lin-4, which is the first miRNA
discovered and an important regulator of developmental tim-
ing in C. elegans (30). miR-125b is abundantly expressed in
animal brains and is upregulated during neurogenesis (17, 29,
33, 35). However, the function of miR-125b in neural devel-
opment has been unclear. For the first time, our report dem-
onstrates that miR-125b is important in regulating neuronal
differentiation. Furthermore, we identified a large number of
putative target genes repressed by miR-125b ectopic expres-
sion in SH-SY5Y cells. Data from computational analyses sug-
gests that 10 of these genes antagonize several neurogenic
pathways, especially extracellular signal-regulated kinase
(ERK) signaling, which is known to mediate the effect of RA
in neuronal differentiation.
MATERIALS AND METHODS
Cell culture and differentiation conditions. SH-SY5Y cells and HEK-293T
cells were maintained in Dulbecco’s modified Eagle medium (DMEM) contain-
ing 4,500 mg/liter glucose, 10% heat-inactivated fetal bovine serum (Gibco), 110
mg/liter sodium pyruvate (Gibco), 2 mM L-glutamine (Gibco), and 1% penicillin-
streptomycin (Gibco). This medium will be called hereafter “growth medium”
for SH-SY5Y cells. For differentiation, SH-SY5Y cells were seeded onto colla-
gen-coated plates (BD Biosciences) at an initial density of 104cells/cm2. RA
(Sigma) was added at a final concentration of 10 ?M the next day after plating.
After 5 days, the cells were washed three times with DMEM and incubated with
50 ng/ml BDNF (Sigma) in growth medium without serum for 7 days.
RVM cells were cultured in laminin-coated plates in DMEM-F12 (1:1) me-
dium (Invitrogen) supplemented with 10% B27 medium (Invitrogen), 10 ?g/ml
gentamicin (Gibco), 10 units/ml heparin (Sigma), 20 ng/ml epidermal growth
factor (EGF), and 10 ng/ml basic fibroblast growth factor (bFGF) (Invitrogen).
For differentiation, the growth medium was replaced with neurobasal medium
(Invitrogen) supplemented with 10% B27 medium (Invitrogen), 10 ?g/ml gen-
tamicin (Gibco), and 10 units/ml heparin (Sigma).
miRNA expression profiling. Total RNA samples were extracted from un-
treated SH-SY5Y cells, cells treated with RA for 5 days, and cells treated
subsequently with BDNF in serum-free medium for 7 days. Small RNA was
purified, labeled, and subjected to an oligonucleotide-based microarray as pre-
viously described (3). Briefly, two32P-labeled RNA markers of 18 nt and 24 nt
were coloaded with total RNA samples and used as indicators to identify the
small RNA population on the gel separating 100 ?g of total RNA. RNAs of 18
to 24 nt were gel purified and sequentially ligated to a 3?-endadaptor and a
5?-end adaptor. Ligated products were gel purified, reverse transcribed, PCR
amplified, and labeled with Cy3. The labeled sense strand was then gel purified
and applied onto the array. A set of synthetic reference oligonucleotides (with a
uniform amount of oligonucleotides corresponding to every probe) was pro-
cessed similarly but labeled with Cy5. These Cy5-labeled reference oligonucle-
otides were applied concurrently with the Cy3-labled samples onto a DNA
oligonucleotide-based array, serving as internal hybridization controls. This array
(provided by the Bartel laboratory at the Whitehead Institute) contains ?600
DNA probes, including probes for 175 human miRNAs (3). The obtained signals
were normalized to the total intensity of all noncognate probes (corresponding to
the nematode miRNAs that are not conserved in humans). Subsequently, signals
from the biological samples were normalized to the corresponding references as
Cy3/Cy5 ratios. The final reading was the average normalized intensity of four
replicates (two biological replicates each with two technical replicates).
Northern blot analysis. A total of 10 to 40 ?g of each total RNA sample and
a33P-labeled Decade RNA marker (Ambion) were separated on a 15% dena-
turing gel, transferred onto a Genescreen Plus membrane (Perkin-Elmer), UV
cross-linked, and baked at 80°C for 30 min. DNA probes with the sequences
complementary to the miRNAs were synthesized (Invitrogen) and labeled with
[?-32P]ATP (Amersham). U6 RNA and 5S RNA probes were used to determine
loading equity. The probe sequences are provided in Table S1 in the supplemen-
tal material. The membrane was prehybridized in PerfectHyb buffer (Sigma) with
1 mg of freshly added sheared salmon sperm DNA (Sigma) for 2 h at 48°C.
Subsequently, the labeled probes were added; hybridization was carried out
overnight at 48°C. The membrane was then washed and developed according to
the Bartel laboratory Northern blot protocol (http://web.wi.mit.edu/bartel/pub/).
Transfection of miRNA duplexes and antisense oligonucleotides. SH-SY5Y
cells (passage number of less than 25) were seeded at 80,000 cells/well in a
collagen-coated 12-well plate (BD Bioscience). On the next day, using 4 ?l
Lipofectamine 2000 reagent (Invitrogen) per well according to the manufactur-
er’s instructions, the cells were transfected with one of the following RNA
oligonucleotides at an 80 nM final concentration: BlockIT fluorescent oligonu-
cleotide (Invitrogen), scrambled duplex (Ambion PremiR negative control 1),
miRNA duplex (Ambion PremiR), or miRNA antisense (Ambion AntimiR).
After 5 h, the transfection medium was replaced with fresh growth medium
either with or without 10 ?M RA. Approximately 125,000 RVM cells were
transfected in suspension with 80 nM RNA oligonucleotides as described above
for SH-SY5Y cells. After 5 h of transfection, the medium was changed to fresh
RVM growth medium or differentiation medium, and the cells were plated into
Immunostaining and HCS. Four days after transfection, SH-SY5Y cells or
RVM cells were fixed with 4% paraformaldehyde for 15 min, followed by three
washes with phosphate-buffered saline (PBS). After a 1-h blocking with 0.2%
Triton X-100 and 3% goat serum in PBS, the cells were incubated with primary
antibodies overnight at 4°C. The primary antibodies used in this study include
mouse monoclonal ?III-tubulin antibody (1:1,000 dilution; Abcam), mouse
monoclonal Map2ab (1:1,000 dilution; Sigma), mouse monoclonal panaxonal
neurofilament antibody (1:1,500 dilution; Covance), goat monoclonal synapto-
tagmin V (Syt5) antibody (1:1,000 dilution; Santa Cruz), and rabbit polyclonal
Musashi-1 antibody (1:1,000 dilution; Abcam). Subsequently, the cells were
washed with PBS three times and then incubated with Alexa Fluor 488 goat
anti-mouse, Alexa Fluor 568 goat anti-rabbit, or Alexa Fluor 488 donkey anti-
goat secondary antibody (Invitrogen) for an hour. Hoechst dye (Invitrogen) was
added for 5 min. The cells were then washed with PBS three times. For high-
resolution imaging, the cells were observed with a Zeiss Duo inverted confocal
microscope (Carl Zeiss Vision GmbH). For quantitative imaging, fluorescent
images of the cells were collected automatically by use of the Cellomics high-
content screening (HCS) system using a 10? objective lens. For neurite out-
growth assays, images of ?III-tubulin and Hoechst staining were analyzed using
Neuronal Profiling BioApplication software (Cellomics). Differentiated SH-
SY5Y cells with neurite outgrowth were defined as ?III-tubulin-positive cells
with neurites longer than 30 ?m. For RVM cells, the percentage of differentiated
cells with neurite outgrowth was defined as the percentage of ?III-tubulin-
positive cells having neurites longer than 20 ?m (RVM cells are smaller than
SH-SY5Y cells, so we applied a lower threshold of neurite length). For quanti-
fications of neuronal marker staining, the images were analyzed by Target Ac-
tivation BioApplication (Cellomics). In all the HCS assays, a cell was considered
VOL. 29, 2009miR-125b PROMOTES NEURONAL DIFFERENTIATION5291
positive for a specific staining only if its fluorescent intensity was equal or higher
than the mean intensity plus two times the standard deviation of the respective
scrambled control replicates.
Quantitative real-time PCR. RNA was extracted from SH-SY5Y cells using
Trizol reagent (Invitrogen) and subsequently column purified with an RNeasy kit
(Qiagen). For quantitative real-time PCR of miRNA, 100 ng of total RNA was
reverse transcribed and subjected to a TaqMan miRNA assay (Applied Biosystems)
control. For quantitative real-time PCR of mRNAs, cDNA synthesis was performed
with 1 ?g of total RNA using the High Capacity cDNA archive kit (Applied Bio-
systems) and subjected to SYBR green or TaqMan gene expression assays (Applied
Biosystems) according to the manufacturer’s protocol.
Gene expression microarray and data analysis. Total RNA was extracted as
described above. A total of 750 ?g of total RNA was reverse transcribed,
converted to cRNA, labeled, purified, and applied onto the Illumina Ref-8 v2
human bead chip (Illumina) according to the manufacturer’s instructions. First,
the respective backgrounds were subtracted from all the raw data using Bead
Studio (Illumina) and then normalized using the cross-correlation method de-
scribed previously by Chua et al. (6). Subsequently, normalized data were pro-
cessed for the identification of differentially expressed genes using log21.5-fold
as the critical value for the mean of log2n-fold changes in expression between
miR-125b duplex (125b-DP)/miR-125b antisense (125b-AS)-transfected samples
and the scrambled controls.
Genes that were differentially expressed 4 days after the transfection of
125b-DP were subjected to Gene Ontology (GO) analysis by using BiNGO (25).
The percentage of these genes classified into each GO process was compared
with that of the whole genome. Statistically significant (P ? 0.05) classes were
selected. For the clustering of genes differentially expressed 2 days posttransfec-
tion, normalized and log2-transformed data were subtracted from the mean
values across all the arrays. Hierarchical clustering was then performed for these
processed data using average linkages.
Motif analysis by MEME. We checked if the 3? UTR sequences of the 388
primary effectors of miR-125b (selected by microarray analysis) were available in
the GenBank database. A total of 253 genes were found with the 3? UTR
sequences. Following the removal of the poly(A) tails, sequences were masked
for repeats using RepeatMasker (http://www.repeatmasker.org/) and analyzed by
MEME with the motif width from 4 to 9 and other MEME default parameters.
Sequence logos were constructed using WebLogo (http://weblogo.berkeley.edu).
Target prediction. The targets of miR-125b were predicted by four different
methods, TargetScan 4.2 (22), mirBASE target (12), rna22 (28), and miRNA
Viewer (9), using default parameters. To check the statistical significance of the
enrichment, we randomly selected 388 genes from either the whole genome or all
the differentially expressed genes (differentially expressed by at least one treat-
ment) and then applied the four methods to predict the targets of miR-125b. The
random selection and the target prediction were repeated 10,000 times. The
average percentages of predicted targets out of the selected gene lists were then
Pathway analysis. Ingenuity Pathway Analysis (IPA) (Ingenuity System) was
used to link the direct targets predicted either by MEME or by conventional
methods with the genes differentially expressed 4 days after the ectopic expres-
sion of miR-125b. We first compared the functional annotation of the two gene
groups and subsequently considered only the networks with differentially ex-
pressed neurogenesis-related genes. We extracted only the pathway links with
the direct targets as the starting points and with known functions related to
neurogenesis or differentiation.
Luciferase reporter assay. miRNA response elements (MREs) (Table 1) or
the whole 3? UTRs of the target genes were cloned into the psiCHECK-2
vector (Promega) between the XhoI and NotI sites immediately 3? down-
stream of the Renilla luciferase gene. The top (sense) and bottom (antisense)
strands of each MRE were designed to contain XhoI and NotI sites, respec-
tively. After synthesis, these were annealed and ligated into the psiCheck-2
vector. A 500-bp segment containing the miR-125b MRE in the 3? UTRs of
three selected target genes, TBC1D1, DGAT1, and SGPL1, were synthesized
as minigenes (first base) with or without seven mismatches (CTCAGGG was
mutated to GAGTCCC) in the seed region of miR-125b MREs and sub-
cloned into the psiCHECK-2 vector.
Ten nanograms of each psiCHECK-2 construct was cotransfected with 10 nM
125b-DP or scrambled duplex into HEK-293T cells in a 96-well plate using
Lipofectamine 2000 (Invitrogen). After 48 h, the cell extract was obtained, and
firefly and Renilla luciferase activities were measured with the dual-luciferase
reporter system (Promega) according to the manufacturer’s instructions.
Statistical analysis. A Student’s t test was used to determine the significance
of differences between the treated samples and the controls where values re-
sulted from quantitative real-time PCR, HCS assays, or permutation of target
prediction. Statistical analysis was performed using Microsoft Excel. For GO
analysis, the P value of any enrichment was calculated by BiNGO by using a
hypergeometric distribution with Bonferroni correction (25).
Microarray data accession number. Microarray data were deposited into the
Gene Expression Omnibus under accession number GSE14787.
Profiling miRNA expression in SH-SY5Y cells during dif-
ferentiation. To understand the regulation of miRNA expres-
sion in human neuronal differentiation, we induced the differ-
entiation of SH-SY5Y cells into neuron-like cells according to
methods described previously by Encinas et al. (8) and ob-
served the same morphological changes described by those
authors. Neurite outgrowth became apparent after a 5-day
treatment with RA and became profuse after a subsequent
7-day treatment with BDNF in serum-free medium (Fig. 1a).
TABLE 1. Target validation summarya
Specificity of miR-125b target
site in 3? UTR validated by
mutagenesis and luciferase
aTen target genes were selected from the microarray data, target prediction, and pathway analysis. Their expression pattern after a 2-day overexpression of miR-125b
in growth medium (GM) or in differentiation medium containing RA was validated by real-time PCR (Fig. 8a). The predicted MREs were validated for binding to
miR-125b by luciferase reporter assays (Fig. 8b and c); in three cases, the specificity of the response to miR-125b was validated by luciferase reporter assays in which
the predicted miR-125b target sites in the 3? UTRs were mutated (Fig. 8c). NT, not tested.
5292LE ET AL.MOL. CELL. BIOL.
Treated cells lost the expression of the neural progenitor
marker Musashi-1, while they increased the expression of the
mature neuronal marker Map2ab (Fig. 1b). Gene expression
changes profiled by cDNA microarrays indicated the acquisi-
tion of neuronal markers (upregulation of neuronal microtu-
bule-associated proteins, ion channels, and neurotransmitter
receptors), withdrawal from the cell cycle, and reduced metab-
olism (downregulation of proliferation and metabolic markers)
(see Table S2 in the supplemental material). These results are
consistent with a differentiation process from neural progeni-
tors to mature neurons. The upregulation of the neuronal
markers Syt5, cannabinoid receptor 1, and GABA type B re-
ceptor 1 (Gabbr1) was confirmed by quantitative real-time
PCR (Fig. 1c).
Next, we examined the miRNA expression profiles for un-
differentiated SH-SY5Y cells (day 0) and differentiated SH-
SY5Y cells after 5 days of RA treatment (day 5) and after an
additional 7 days of BDNF treatment in serum-free medium
(day 12). We profiled 175 human miRNAs using an miRNA
array designed previously by Baskerville and Bartel (3) and
applied stringent normalization by subtracting the signal from
a reference synthetic oligonucleotide for every miRNA (see
Fig. S1 in the supplemental material). Based on the change and
expression level, we selected 12 miRNAs that were significantly
FIG. 1. Neuronal differentiation of SH-SY5Y cells. (a) Representative pictures of undifferentiated cells (day 0) and cells undergoing differ-
entiation (day 5 and day 12). Differentiation was induced by RA for 5 days and subsequently induced by BDNF in serum-free medium for 7 days.
(b) Immunostaining of differentiating SH-SY5Y cells for the mature neuronal marker Map2ab (green) and the neural progenitor marker
Musashi-1 (Msi1) (red). Nuclei were counterstained with Hoechst dye (blue). (c) Microarray and quantitative real-time PCR (qRT-PCR) analysis
of representative marker genes including Syt5, cannabinoid receptor 1 (Cnr1), and GABA type B receptor 1 (Gabbr1) in differentiating SH-SY5Y
cells. The microarray data were normalized as described in Materials and Methods. Quantitative real-time PCR readings were normalized to the
expression level of ?-actin. All the readings are presented as average changes ? standard errors of the means (SEM) relative to the gene expression
level on day 0.
VOL. 29, 2009 miR-125b PROMOTES NEURONAL DIFFERENTIATION5293
and consistently regulated during the course of differentiation
(Fig. 2a). The expression of the selected miRNAs was vali-
dated by Northern blotting (Fig. 2b). We found that six
miRNAs, miR-7, miR-124a, miR-125b, miR-199a, miR-199a*,
and miR-214, were consistently upregulated during differenti-
ation (Fig. 2b and c). Other miRNA candidates were not de-
tected or showed no significant change in their levels of ex-
pression by Northern blotting (Fig. 2b).
Ectopic expression of six miRNA candidates and their ef-
fects on neurite outgrowth. Transfection using Lipofectamine
2000 was optimized to deliver double-stranded RNA into SH-
SY5Y cells. To examine the transfection efficiency, we first
transfected the cells with a fluorescent RNA duplex. After 1
day, more than 80% of the cells were positive for fluorescence,
and fluorescence persisted until 4 days after transfection (Fig.
3a). We then transfected the cells with miRNA duplexes and
found that by 4 days after transfection, the levels of the cor-
responding mature miRNAs were very high compared to those
of mock-transfected cells (Fig. 3b). Beyond 4 days posttrans-
fection, the cells often became confluent and unhealthy.
Hence, we performed all the experiments using cells collected
4 days after transfection. For gain-of-function (ectopic expres-
sion) studies, a duplex corresponding to each individual
miRNA candidate was transfected into SH-SY5Y cells, and
after 4 days, the cells were fixed and stained for ?III-tubulin, a
To quantify the degree of neuronal differentiation, neurite
outgrowth was analyzed by use of the Cellomics HCS system,
where a large number of images was acquired automatically,
and neurites were traced and measured in a uniform manner.
Since ?III-tubulin is an early neuronal marker, only ?III-tu-
bulin-positive cells that possessed neurites longer than 30 ?m,
FIG. 2. Analysis of miRNA expression in differentiating SH-SY5Y cells. (a) Microarray-detected absolute expression level of the miRNAs that
were selected based on their significant and consistent changes in expression during differentiation. (b) Northern blot validation of selected
miRNAs. The phosphorimages represent three blots (separated by solid lines), including two biological duplicates loaded onto lanes 1 to 3 and
lanes 4 to 6 respectively. U6 RNA and 5S RNA are shown as loading controls. (c) Northern analysis-detected expression changes of selected
miRNAs (with significant changes), normalized to the expression level of 5S RNA.
5294LE ET AL.MOL. CELL. BIOL.
about three times the diameter of the cell bodies, were counted
as neuronal cells. The percentage of SH-SY5Y cells meeting
this stringent criterion is very low, only ?1%, in growth me-
dium. By our stringent standards, we found that only the ec-
topic expression of miR-124a or miR-125b significantly in-
creased the percentage of differentiated cells with neurite
outgrowth, by approximately twofold, compared to the mock
and scrambled transfection controls (Fig. 3c and d). Our sub-
sequent analysis focused on miR-125b since the function of
miR-124a in neuronal differentiation was described previously
miR-125b is necessary and sufficient for neurite outgrowth
and neuronal marker gene expression. We assayed the effects
of the miR-125b gain and/or loss of function on neuronal
differentiation by measuring neurite outgrowth of SH-SY5Y
cells both in growth medium and in differentiation medium
containing RA. Synthetic 125b-DP was used for gain-of-func-
tion studies. For loss-of-function studies of miR-125b, we used
an antisense oligonucleotide (125b-AS) that is able to reduce
the level of synthetic miR-125b (when transfected together
with 125b-DP at the same concentration) as well as the level of
endogenous miR-125b (when transfected alone) (Fig. 4a).
FIG. 3. Ectopic expression of candidate miRNAs and neurite outgrowth assay in SH-SY5Y cells. (a) The transfection efficiency was tested
with 80 nM fluorescent double-stranded RNA in SH-SY5Y cells. Fluorescence (green) was observed every day until day 4 after transfection.
(b) Relative quantification of miR-7 and miR-125b levels in SH-SY5Y cells 4 days after a transfection with 80 nM synthetic miR-7 duplex
(7-DP) or 125b-DP. The obtained real-time PCR readings were normalized to the U6 RNA level and are presented as the changes ? SEM
relative to the respective miRNA level in mock-transfected controls (n ? 4). (c) Percentage of differentiated cells with neurite outgrowth,
defined as cells staining positively for ?III-tubulin and possessing neurites longer than 30 ?m. The error bars represent SEM of data from
at least three biological replicates. The percentage of neuron-like cells in each biological replicate was analyzed and averaged by at least 15
images; each image of 10? magnification typically captures 200 to 500 SH-SY5Y cells. The results of Student t tests are indicated as**(P ?
0.01 compared to the scrambled control). (d) Representative images of SH-SY5Y cells stained for ?III-tubulin (green) and Hoechst dye
(blue) 4 days after mock transfection or transfection with different miRNA duplexes. Images were acquired by a confocal microscope using
a 20? objective lens. Scale bar, 100 ?m.
VOL. 29, 2009 miR-125b PROMOTES NEURONAL DIFFERENTIATION5295
Consistent with the screening results, the ectopic expression
of miR-125b significantly increased the percentage of differ-
entiated cells with neurite outgrowth in both growth medium
and differentiation medium containing RA (Fig. 4b and c).
Specifically, the percentage of differentiated cells with neurite
outgrowth derived by spontaneous differentiation (of mock or
scrambled control transfection in growth medium) was ?1%.
miR-125b ectopic expression alone (in growth medium) in-
creased this fraction of differentiated cells by twofold, to ?2%.
Culture in differentiation medium with RA resulted in ?2.8%
of cells with neurite outgrowth, while miR-125b ectopic expres-
sion again doubled the percentage of cells with neurite out-
growth to 5.7%. This effect of miR-125b gain of function was
titrated by cotransfection with 125b-AS in both growth me-
dium and RA-containing medium (Fig. 4b and c). We also
considered the average neurite length (of neurites selected
with a minimum length of 30 ?m) as an indicator of SH-SY5Y
neuronal differentiation. miR-125b ectopic expression in-
FIG. 4. Function of miR-125b in SH-SY5Y cell differentiation. (a) Change in miR-125b levels in SH-SY5Y cells 4 days after transfection with
scrambled duplex (scrambled-DP), 125b-DP, or 125b-AS and maintained in growth medium (GM) or in differentiation medium containing RA.
Each oligonucleotide was transfected at an 80 nM final concentration. The obtained real-time PCR readings were normalized to the U6 RNA level
and are presented as the changes ? SEM relative to the respective miRNA levels of the scrambled transfected control (n ? 4). (b) Percentages
of differentiated SH-SY5Y cells with neurite outgrowth treated as described above (a) and their average neurite lengths. Automated image
acquisition and quantitative analysis were carried out by use of the Cellomics HCS system. Differentiated cells with neurite outgrowth were defined
as ?III-tubulin-positive cells with neurites longer than 30 ?m. (c) SH-SY5Y cells stained with ?III-tubulin (green) and Hoechst dye (blue) 4 days
after transfection. Transfection and treatments were applied as described above (a). The images were acquired by a confocal microscope using a
20? objective lens. Scale bar, 100 ?m. (d) SH-SY5Y cells stained with neuronal markers (green) and Hoechst dye (blue). The cells were transfected
as described above (a) and maintained in growth medium for 4 days. They were then fixed and stained for neuronal markers including Map2ab,
neurofilaments, and Syt5. The bar chart presents the percentage of positively stained cells quantified by the Cellomics HCS system. (e) Relative
quantification of marker gene expression. The cells were transfected as described above (a). RNA was isolated for quantitative real-time PCR 4
days posttransfection. The obtained readings were normalized to ?-actin expression levels (internal control) and are presented as average
changes ? SEM relative to the level of the transcripts in the scrambled control. In all cases, the error bars represent SEM of data from at least
three biological replicates. In all HCS experiments, the phenotype of each biological replicate was analyzed and averaged by at least 15 images;
each image of 10? magnifications typically captures 200 to 500 SH-SY5Y cells. The results of Student t tests are indicated as*for a P value
of ?0.05 and**for a P value of ?0.01 where the variation is compared to that of the scrambled control.
5296 LE ET AL.MOL. CELL. BIOL.
creased the average neurite length of SH-SY5Y cells by ?2.5
?m in growth medium and by ?6 ?m in differentiation me-
dium relative to the scrambled-duplex transfection control
(Fig. 4b and c). Together, these results indicate that miR-125b
alone is sufficient to stimulate neurite outgrowth.
Conversely, the specific knockdown of endogenous miR-
125b by an antisense oligonucleotide (125b-AS) reduced the
average neurite length by ?9 ?m (P ? 0.01), indicating that
endogenous miR-125b expression is necessary for neurite out-
growth (Fig. 4b and c). The cotransfection of 125b-DP with
125b-AS abrogated the increase in average neurite length due
to miR-125b ectopic expression, demonstrating the specificity
of the antisense oligonucleotide. Together, the results show
that miR-125b is both necessary and sufficient to stimulate
The role of miR-125b in promoting the differentiation of SH-
SY5Y cells was demonstrated further by staining for additional
neuronal markers. As quantified by use of the Cellomics HCS
system, miR-125b gain of function in growth medium significantly
increased the percentage of cells positive for mature neuronal
markers including Map2ab, neurofilament, and Syt5 (Fig. 4d).
These stimulatory effects were specifically abrogated by cotrans-
fection with the 125b-AS oligonucleotide. The transcript levels of
the mature neuronal markers Map2ab and Gabbr1 were also
increased by miR-125b ectopic expression (Fig. 4e). In contrast,
the level of expression of the neural progenitor marker Musashi-1
was reduced significantly (Fig. 4e).
miR-125b is upregulated during differentiation of RVM
cells, and miR-125b ectopic expression promotes neurite out-
growth in these cells. To elucidate the function of miR-125b in
non-cancer-derived cells, we used RVM cells, a neural progen-
itor cell line isolated from normal human brain and immortal-
ized by v-myc induction. EGF and bFGF were used to maintain
the cells in the undifferentiated state. The differentiation of
RVM cells into neurons and glial cells was induced by the
withdrawal of the growth factors. During this process, we ob-
served a continuous change in morphology marked by the
appearance of neurite outgrowth (Fig. 5a). By quantitative
real-time PCR, we found that miR-125b was gradually and
significantly upregulated during the 7-day differentiation of
RVM cells (Fig. 5b). The efficiency of transfection in RVM
cells was comparable to that in SH-SY5Y cells: following trans-
fection with a fluorescent RNA duplex, fluorescence was ob-
served in more than 80% of RVM cells by day 1 and remained
detectable until day 4 posttransfection (Fig. 5c).
We then transfected 125b-DP into RVM cells. Transfected
cells were maintained either in growth medium (containing
EGF and bFGF) or in differentiation medium (in the absence
of the two growth factors), and neurite outgrowth was assayed
as for SH-SY5Y cells. miR-125b ectopic expression signifi-
cantly promoted the neurite outgrowth of RVM cells in both
growth medium and differentiation medium, as indicated by
the percentage of differentiated cells with neurite outgrowth
(?III-tubulin-positive cells with neurites longer than 20 ?m)
(Fig. 5d and e). In addition, miR-125b ectopic expression sig-
nificantly increased the average neurite length of differentiated
neurons in growth medium but not in differentiation medium
(Fig. 5d and e). The effect of 125b-DP transfection was abro-
gated by cotransfecting 125b-AS at an equal concentration
(Fig. 5d and e). Hence, the effects of miR-125b on the differ-
entiation of RVM neural progenitor cells are similar to the
effects of miR-125b on neuroblastoma SH-SY5Y cells.
Profiling downstream effectors of miR-125b. To understand
the mechanism of the miR-125b-dependent differentiation of
neural cells, we studied the changes in the global gene expres-
sion profile of SH-SY5Y cells following miR-125b ectopic ex-
pression. First, the gene expression profile of SH-SY5Y cells 4
days after transfection with 125b-DP in growth medium was
compared with that of the scrambled-duplex transfection con-
trol. We found that the genes upregulated by miR-125b ectopic
expression were preferentially classified by GO into biological
processes related to development, especially nervous system
development, neurite growth, cell adhesion, cell morphology,
motility, and cytoskeleton organization (Fig. 6a). Specifically,
the percentage of miR-125b-upregulated genes classified into
each of these categories was statistically higher than the per-
centage of the whole genome sorted into the same category
(Fig. 6a). On the other hand, genes downregulated by miR-
125b ectopic expression were overrepresented by those related
to metabolism and transcriptional regulation (Fig. 6b). Note
that the changes in gene expression due to miR-125b ectopic
expression were profiled in transfected cells that had not nec-
essarily differentiated. Since the transfection efficiency was
very high, we assumed that most if not all the cells responded
to the elevated level of miR-125b. Thus, these responses indi-
cate a global transition of the cells from an undifferentiated
state to a differentiated state following miR-125b overexpres-
sion. Although not all the cells expressed mature neuronal
markers and/or exhibited neurite outgrowth by day 4 posttrans-
fection, many of them appear to have already acquired the
expression of neuron-related genes.
Second, we sought to identify the more direct effectors of
miR-125b by examining global gene expression profiles at an
earlier time point, 2 days posttransfection. Microarray profiling
was performed on SH-SY5Y cells transfected with scrambled
duplex and 125b-DP in growth medium or in differentiation
medium containing RA. We also included two other treat-
ments: miR-125b knockdown (125b-AS transfected) and neu-
tralization of 125b-DP (cotransfection of 125b-AS and
125b-DP at equal concentrations) in differentiation medium.
Strikingly, miR-125b ectopic expression downregulated a large
number of genes, both in growth medium and in differentiation
medium, forming a distinct cluster from all other treatments
(Fig. 6c). Unexpectedly, the knockdown of miR-125b did not
show an opposite effect. Since SH-SY5Y cells also express
miR-125a, which has the same seed sequence and can target a
set of genes similar to that targeted by miR-125b, knocking
down miR-125b alone may be insufficient to release the re-
pression of all its targets.
Identification of direct targets of miR-125b. In an attempt to
identify the direct targets of miR-125b in neuronal differenti-
ation, i.e., mRNAs whose expression is directly downregulated
by this miRNA, we selected 388 genes that were downregu-
lated by miR-125b ectopic expression in growth medium and in
RA-containing medium relative to all other transfection con-
ditions. To examine whether these genes might be directly
regulated by miR-125b binding, we applied two different bioin-
The first approach was to search for a common motif in the
3? UTR of the downregulated genes by using MEME motif
VOL. 29, 2009miR-125b PROMOTES NEURONAL DIFFERENTIATION5297
FIG. 5. Expression and function of miR-125b in RVM cells during differentiation. (a) Representative pictures of undifferentiated cells (day 0) and
was induced by the removal of growth factors and the addition of neurobasal medium. Scale bar, 100 ?m. (b) Expression pattern of miR-125b in RVM cells
during differentiation quantified by quantitative real-time PCR. The readings were normalized to U6 data and are presented as average changes ? SEM (n ?
cells 1 to 4 days after transfection with 80 nM fluorescent duplex. Scale bar, 100 ?m. (d) Representative images of RVM cells stained with ?III-tubulin (green)
and Hoechst dye (blue) 4 days after (i) mock transfection or transfection with (ii) 80 nM scrambled duplex (scrambled-DP), (iii) 80 nM 125b-DP, (iv) 80 nM
125b-DP and 80 nM 125b-AS. After transfection, the cells were maintained in growth medium (GM) or in differentiation medium (DM) until they were fixed
and stained. Images were acquired by a confocal microscope using a 20? objective lens. Scale bar, 100 ?m. (e) Percentage of differentiated RVM cells with
neurite outgrowth and their average neurite lengths. The cells were treated as described above (d). Automated image acquisition and quantitative analysis were
than 20 ?m. The error bars represent SEM (n ? 3). The phenotype of each replicate was analyzed and averaged by at least 15 images. Each image of 10?
discovery according to methods described previously by Lim et
al. (24). From the 388 candidate genes, we were able to obtain
the sequences of 253 3? UTRs from published data (135 can-
didate genes had no available 3? UTR sequence). A search by
MEME identified a 6-nt motif, “TCAGGG” in 129 of these
sequences, that is, 51% out of the 253 available 3? UTRs.
Importantly, this motif is perfectly complementary to the seed
sequence (nt 2 to 8) of miR-125b (Fig. 7a). Extensions of this
common motif to 7 to 9 nt also matched the seed sequence of
miR-125b in a significant proportion of these 129 3? UTR
sequences (Fig. 7a). As a control, we analyzed the 3? UTR
sequences of the genes upregulated 4 days after the ectopic
expression of miR-125b and found no enrichment in the “TC
AGGG” motif (data not shown).
The second approach is an integrated prediction of miR-
125b targets using four different conventional methods: Tar-
FIG. 6. Profiling of the downstream effectors of miR-125b. (a) GO classification of genes upregulated 4 days after transfection of 125b-DP. (b)
GO classification of genes downregulated 4 days after transfection of 125b-DP. In a and b, SH-SY5Y cells were transfected with 80 nM scrambled
duplex or 80 nM 125b-DP and then maintained in growth medium. RNA samples were collected 4 days after transfection and subjected to gene
expression profiling using a Ref-8 v2 Illumina bead chip. The percentage of differentially expressed genes (125b-DP versus scrambled duplex by
1.5-fold) belonging to each GO processes was compared to the percentage of the whole genome sorted into the same category. Categories enriched
for the differentially expressed genes are shown in the charts. P values (representing the difference between the percentage of differentially
expressed genes and the percentage of the whole genome in each category) are indicated as*for a P value of ?0.05 and**for a P value of ?0.01.
(c) Heat map representation of gene expression profiles for SH-SY5Y cells 2 days after transfection with scrambled duplex, 125b-DP, and/or
125b-AS in growth medium (GM) or in the presence of RA. Each oligonucleotide was transfected at a final concentration of 80 nM. Gene
expression was analyzed by use of a Ref-8 v2 Illumina bead chip. The colors indicate the average changes in intensity normalized to the mean of
all arrays, ranging from one-quarter-fold (green) to fourfold (red). Only genes downregulated by 125b-DP are shown. The tree diagram represents
the clustering of treatments based on their gene expression patterns.
VOL. 29, 2009 miR-125b PROMOTES NEURONAL DIFFERENTIATION5299
5300 LE ET AL.MOL. CELL. BIOL.
getScan 4.2 (22), mirBASE target (12), rna22 (28), and
miRNA Viewer (9). Different prediction methods identified
different numbers of targets with considerable overlap (Fig.
7b). In total, the four prediction methods identified 97 genes
(25%) among the 388 downregulated genes as being the direct
targets of miR-125b (Fig. 7b). When the same prediction meth-
ods were applied to genes randomly selected from the whole
genome or from unfiltered differentially expressed genes, only
4% and 11% of these genes were predicted to be targets of
miR-125b, respectively (Fig. 7c). Hence, our list of 388 candi-
date miR-125b targets is significantly enriched for predicted
direct targets. Furthermore, among the 97 predicted direct
miR-125b targets, we found that 81% of their 3? UTRs contain
the 6-nt motif that was identified by MEME, matching the seed
sequence of miR-125b. The list of targets predicted by both
approaches is provided in Tables S3 and S4 in the supplemen-
Pathway analysis and validation of direct miR-125b targets.
To understand how the predicted targets of miR-125b regulate
neuronal differentiation, we examined their known functions
and the signaling networks connecting them with other genes
(considered indirect effectors) that were differentially regu-
lated 4 days after the transfection of 125b-DP (shown in Fig. 6a
and b). The analysis was performed using the Ingenuity Sys-
tem, which maps biomolecular networks based on known sig-
naling pathways and known interactions with reliable data
curation. The predicted direct miR-125b targets (resulting
from MEME and the conventional predictions described
above) include genes of diverse functions. Compared to these
direct targets, the group of indirect effectors is significantly
enriched in genes involved in nervous system development and
function. Interestingly, many of the indirectly regulated neu-
ronal genes are extensively connected to the predicted direct
targets, forming a large network with hundreds of genes. To
simplify the network, we selected only the direct targets that
are placed upstream of the indirect effectors and filtered for
pathways that are relevant to neuronal differentiation. We
propose a model of regulation based on the resultant network
(Fig. 7d). In this model, miR-125b directly suppresses the ex-
pression of 10 key target genes that, in turn, repress pathways
that mediate neuronal differentiation. Pathways in the model
encompass both signaling transduction and gene regulation
with the key signaling molecules protein kinase C, JNK, ERK,
mitogen-activated protein kinase, and vascular EGF and the
important transcription factors SMAD2, SMAD4, and STAT3.
The final outcome of miR-125b upregulation during neurogen-
esis (Fig. 2) would then be the upregulation of many neuro-
nally important genes such as SCNBA, EPHB2, KCNQ2,
FLNA, SYN2, and NEFM (Fig. 7d).
Subsequently, we used real-time PCR to validate the expres-
sion of the 10 target genes used in our model. All 10 genes
were downregulated by a 2-day overexpression of miR-125b in
growth medium or differentiation medium except AP1M1,
which was downregulated only in the presence of RA (Fig. 8a).
Furthermore, the binding of miR-125b to the predicted MREs
in the 3? UTR of the 10 targets was also validated by a lucif-
erase reporter assay (Fig. 8b). In this assay, individual MREs
were cloned into the 3? UTR of a luciferase reporter gene. The
construct plasmids were transfected into HEK-293T cells, and
luciferase activity was quantified after 2 days. The cotransfection
of 125b-DP with the plasmids suppressed luciferase activity by 30
to 70% (P ? 0.01) in comparison to a scrambled-duplex-cotrans-
fected control. These data indicate that transfected miR-125b
bound to the target MREs and repressed the expression of lucif-
To confirm the specific interaction of miR-125b with the
target MREs, we selected the top three hits, TBC1D1,
DGAT1, and SGPL1, from the MRE-luciferase reporter
assay. A 500-bp segment containing the miR-125b MRE in
the 3? UTR of each gene was cloned after the luciferase
reporter gene; in these constructs, we also made seven mis-
matches in the predicted seed region of the binding sites
(MREs) for miR-125b. Figure 8c shows that miR-125b re-
duced the luciferase activity of the DGAT1, SGPL1, and
TBC1D1 reporters to ?82%, 65%, and 63% of the control
level, respectively. Importantly, the activity of the DGAT1
and SGPL1 luciferase reporters in the presence of miR-125b
was restored to 100% by the mutation of the predicted
miR-125b seed region. The mutation of the miR-125b seed
segment in the reporter for TBC1D1 resulted in a partial but
significant recovery of luciferase activity. These data suggest
that the predicted seed region is absolutely necessary for the
binding of miR-125b to the 3? UTR of DGAT1 and SGPL1
but that it is not the only factor that determines the binding
of miR-125b to the 3? UTR of TBC1D1. In summary, we
have shown that miR-125b is likely to directly target the 10
genes in the neurogenic pathway listed in Table 1, in par-
ticular DGAT1, SGPL1, and TBC1D1.
FIG. 7. Target prediction and pathway analysis. (a) MEME motif discovery searching for motifs containing 4 to 9 nt in the 3? UTRs of the
downregulated genes. The available 3? UTR sequences were collected for 253 out of the 388 genes downregulated exclusively by 2 days of miR-125b
ectopic expression. MEME identified significant motifs of 6 to 9 nt in a proportion of the 3? UTR sequences (shown as the percentage of the UTRs
with the consensus). The motifs (orange text) are perfectly complementary to the seed region of miR-125b (nt 2 to 8 [shaded blue text]). MEME
expectation represents the probability at which the same motifs can be found by chance. (b) Prediction of miR-125b targets by conventional
methods. Targets of miR-125b among the 388 genes downregulated exclusively by miR-125b gain of function were predicted by four methods,
TargetScan 4.2, mirBASE target, RNA22, and miRNA Viewer, using default settings. The numbers outside the circles represent the total numbers
of targets predicted by each method. The numbers inside the circles represent the numbers of overlapped and nonoverlapped targets. (c) Statistical
test elucidating the significance of our selection method. The targets of miR-125b were predicted (using the four methods described above) from
a list of 388 genes randomly selected from the whole genome and another list of 388 genes randomly selected out of 6,448 genes, which are
differentially expressed by at least one treatment. The random selection and prediction were repeated 10,000 times. The average percentage of
enriched targets was compared with the 97 enriched targets found in b. The P values computed by a Student’s one-sample t test indicate the
significance of the differences. (d) Modeled downstream network of miR-125b that mediates neuronal differentiation. Connections were mapped
using IPA. PI3K, phosphatidylinositol 3-kinase; IL-12, interleukin-12.
VOL. 29, 2009 miR-125b PROMOTES NEURONAL DIFFERENTIATION5301
In our study, we utilized a simple in vitro model of human
neuronal differentiation in which human neuroblastoma SH-
SY5Y cells were differentiated into a homogenous population
of cells with neuronal morphology. The advantages of this
model over other in vitro systems for human neuronal differ-
entiation include its robust differentiation capability (terminal
differentiation is obtained within 2 weeks of induction) and the
formation of neurons only and not other cell types such as glia
(8). In comparison to previous reports of miRNAs in human
neural differentiation (20, 33, 38, 40), which focused mainly on
the profiling of miRNAs, we have advanced well beyond ex-
pression profiling and established a number of reliable assays
to assess the biological functions of specific miRNAs in the
neuronal differentiation of SH-SY5Y cells as well as of human
neural progenitor RVM cells. We identified two miRNAs,
miR-124a and miR-125b, which promote neurite outgrowth.
We further demonstrated how the upregulation of miR-125b
during neurogenesis downregulates a set of direct mRNA tar-
gets. Since the proteins encoded by these mRNAs normally
repress neurogenesis, our model (Fig. 7d) suggests how miR-
125b induction causes an enhanced expression of multiple neu-
miR-125b is expressed in many types of tissues, but its high-
est level of expression is in the brain, especially in mature
neurons but not astrocytes (33, 35, 37). miR-125b is upregu-
lated during mouse neurogenesis (35), during the neural dif-
ferentiation of mouse embryonic stem cells (18), and upon RA
treatment of embryonic carcinoma cells (33) and of neuroblas-
toma SK-N-BE cells (19). Adding to these studies, our data
demonstrate that miR-125b is not only a marker of differenti-
ation but also a regulator of neuronal differentiation in SH-
FIG. 8. Target validation. (a) Quantitative real-time PCR validating the expression pattern of miR-125b target genes 2 days after transfection
of 125b-DP into SH-SY5Y cells in growth medium (GM) or in differentiation medium containing RA. The readings were normalized to the
expression level of ?-actin. All the readings are presented as average changes ? SEM relative to the gene expression level in the cells transfected
with scrambled duplex (DP). (b) Luciferase reporter assays validating the binding of miR-125b to the 3? UTRs of the target genes. Reporter genes
contain only the MREs that were predicted to bind to miR-125b. An MRE with perfect complementarity to miR-125b was used as a control. (c)
Reporter genes contain 500 bp of the 3? UTRs of selected target genes with wild-type sequence or with seven mismatches in the seed region of
the MREs. Plasmids carrying each reporter gene were cotransfected with 125b-DP into 293T cells. Luciferase readings were obtained 48 h after
transfection and are presented here as the average percentages of luciferase activity ? SEM (n ? 3) relative to the scrambled-duplex-cotransfected
control (100%). The results of Student t tests comparing the luciferase activities of each reporter gene in the presence of 125b-DP versus the
scrambled duplex are indicated as**for a P value of ?0.01. The results of Student t tests comparing the luciferase activity of the mutant reporter
genes with the activity of the corresponding wild-type reporter genes in the presence of 125b-DP are indicated as ## for a P value of ?0.01.
5302 LE ET AL.MOL. CELL. BIOL.
SY5Y cells. By quantifying the effect of miR-125b ectopic
expression and miR-125b knockdown on neurite outgrowth
and on the expression of neuronal markers, we demonstrate
that miR-125b is both necessary and sufficient to promote the
neuronal differentiation of SH-SY5Y cells.
In our functional assays, we examined the effect of miR-125b
ectopic expression on differentiation over a short time frame of
4 days and found that only a fraction of the cells differentiated.
Importantly, the percentage of “differentiated cells” varies de-
pending on the criteria used for quantification. In the neurite
outgrowth assay, we considered only the differentiated cells
with apparent neurite outgrowth. Because we used very strin-
gent parameters that allow us to identify only the most mature
neurons, ?III-tubulin-positive cells with neurites longer than
30 ?m, the percentage of the selected cells was rather small, 1
to 6% (Fig. 4b). Reducing the stringency by considering a
lower minimum neurite length would increase the percentage
of selected cells, but the neurite identification then becomes
less accurate since cell edges can be mistaken as short neurites.
In our immunostaining assay, where differentiation was deter-
mined based on the expression of the neuronal protein mark-
ers Map2ab, neurofilament, and Syt5, we observed a higher
percentage of differentiated cells, 5 to 16% (Fig. 4d). Hence,
the cells appeared to upregulate these markers earlier than the
onset of neurite outgrowth.
Because we were concerned with the abnormal karyotype
and tumor origin of SH-SY5Y cells, we examined the expres-
sion and the function of miR-125b in a more physiologically
relevant cell type, human neural progenitor RVM cells. Like
primary neural stem cells, RVM cells have a normal karyotype
and are able to differentiate into both neurons and glial cells
(7). We showed that, as in SH-SY5Y cells, miR-125b expres-
sion was gradually upregulated during the differentiation of
RVM cells. miR-125b ectopic expression significantly en-
hanced the neurite outgrowth of RVM cells in both growth
medium and differentiation medium. Thus, our data indicate
that miR-125b is important for neuronal differentiation in both
RVM cells and SH-SY5Y cells and suggest a common function
of miR-125b in neural progenitor cells. Potentially, miR-125b
gain of function may be useful to enhance the in vitro neuronal
differentiation of primary human neural stem cells for treat-
ments of neurodegenerative diseases. This approach would
probably be more advantageous than other types of gene ther-
apy since the miRNA is a small molecule that, in principle, can
be delivered more easily into a cell.
On the other hand, we also noted several differences in the
effects of miR-125b on SH-SY5Y and RVM cells. miR-125b
ectopic expression exhibited a stronger effect on the average
neurite length in RVM cells than in SH-SY5Y cells in growth
medium, but the reverse was observed for differentiation me-
dium. Hence, in RVM cells, miR-125b alone is sufficient to
promote the extension of neurite length, but in SH-SY5Y cells,
it requires the addition of RA. Furthermore, the knockdown of
miR-125b in SH-SY5Y cells significantly reduced the extension
of neurites induced by RA; however, the same effect was not
observed when miR-125b was knocked down in RVM cells
undergoing differentiation. Since the two cell lines were differ-
entiated by two different methods, the differences in the effects
of miR-125b may be more apparent than real, but it does
appear as if the role of miR-125b in neurite outgrowth is more
necessary for the RA-induced differentiation of SH-SY5Y cells
than it is for the differentiation of RVM cells upon the with-
drawal of EGF and bFGF. Additionally, the phenotype may
also be determined by the intrinsic differences between the two
cell lines: as they express different mRNAs, the genes directly
and indirectly affected by miR-125b regulation are likely to be
different. The physiological functions of miR-125b in vivo may
also depend on different extrinsic and intrinsic factors that are
regulated in a temporal and spatial manner. Interestingly, we
recently showed that the knockdown of miR-125b leads to
severe defects in zebrafish brain development, including the
malformation of axonal tracts in midbrain and hindbrain, sug-
gesting that miR-125b is required for neuronal differentiation
in vivo (our unpublished data). It would be interesting to fur-
ther study the cell-specific function of miR-125b in vivo.
To understand the mechanism mediating miR-125b func-
tion, we conducted global profiling to identify miR-125b-re-
sponsive genes. We chose to perform this experiment primarily
using SH-SY5Y cells because these cells are more responsive
to the modulation of miR-125b levels than RVM cells. Using
microarrays, we identified 388 genes repressed by miR-125b
ectopic expression and predicted that 164 of these genes are
the direct targets of miR-125b. This prediction is supported by
two lines of evidences: (i) MEME motif discovery identified a
6-nt motif in the 3? UTR of 129 genes that is perfectly com-
plementary to the seed sequence of miR-125b, and (ii) an
integrative search using four conventional miRNA target pre-
diction methods identified 97 direct targets among the 388
genes repressed by miR-125b. Moreover, we found that 57
(?35%) out of the 164 selected targets were downregulated by
RA- or BDNF-induced neuronal differentiation by ?1.5-fold.
The inverse expression pattern of these genes in comparison to
the endogenous expression of miR-125b implies that they are
targeted by miR-125b during differentiation. Although the ac-
tual number of endogenous targets is subject to a further
validation of our predictions, we do expect the complex func-
tion of miR-125b to be mediated by multiple mRNA targets.
Previous profiling studies of miRNA targets by microarrays
and proteomics demonstrated that miRNAs usually downregu-
late several hundred genes; the targets are mostly repressed at
both mRNA and protein levels, although a number of them are
regulated only at the protein level (1, 32). Our microarray data
for SH-SY5Y cells were able to identify only the targets reg-
ulated by miR-125b through mRNA degradation and/or dead-
enylation. In a separate study, we found that p53 is a bona fide
target of miR-125b; a modulation of miR-125b largely affects
the p53 protein level but did not show any significant change in
the transcript level of p53 in SH-SY5Y cells (19a). Besides p53,
it is possible that our microarray analysis also missed other
targets that are regulated only by translational inhibition.
We next asked how miR-125b mediates neuronal differen-
tiation by suppressing the 164 predicted targets. Data from
IPA suggest that a subset of these targets is connected to the
neuronal genes that were indirectly upregulated by miR-125b
gain of function. We propose a simple model to explain how
miR-125b enhances differentiation. In constructing the model,
we assumed that the direct targets of miR-125b inhibit path-
ways that promote the expression of neuronal genes. Hence,
from the network connecting the predicted downregulated di-
rect mRNA targets and the upregulated indirect neuronal ef-
VOL. 29, 2009miR-125b PROMOTES NEURONAL DIFFERENTIATION5303
fectors, we selected the pathways relevant to neurogenesis and Download full-text
the direct targets with known inhibitory effects or known bind-
ing to the components of these pathways. The model focused
on 10 predicted direct targets of miR-125b, and we validated
these both by real-time PCR analysis of mRNA expression
after the ectopic expression of miR-125b and by a luciferase
reporter assay (Table 1). IPA also revealed that many genes in
the modeled pathways are regulated by RA in the same man-
ner as by miR-125b. This relationship, and the fact that RA
upregulates miR-125b during differentiation, suggests that
miR-125b mediates RA-induced differentiation in SH-SY5Y
cells. Our proposed model of the miR-125b network supports
this hypothesis, since the ERK signaling pathway featured
prominently in our model is also known to mediate RA-in-
duced differentiation in SH-SY5Y cells (27). Indeed, the
model also predicts that miR-125b exerts positive feedback on
RXRA, the receptor for RA.
In addition, IPA shows that the predicted targets of miR-
125b are also connected to the repressed indirect effectors
(genes downregulated 4 days after the transfection of 125b-
DP), mainly with positive regulatory effects. These networks
are involved in metabolism, proliferation, and apoptosis; thus,
in part, miR-125b may enhance differentiation by reducing cell
metabolism and proliferation. Experimentally, we did not find
any significant effect of miR-125b gain of function on prolif-
eration (using Ki67 staining) (data not shown). Laneve et al.
also previously found that miR-125b alone has very little effect
on proliferation, although the ectopic expression of miR-125b
together with miR-125a and miR-9 inhibits cell cycling in neu-
roblastoma cells (19). Hence, the withdrawal of SH-SY5Y cells
from the cell cycle during differentiation may require a syner-
gistic effect between miR-125b and other miRNAs. On the
other hand, the negative regulation by miR-125b on a number
of apoptotic genes, including the four targets BAK1,
TP53INP1, PPP1CA, and PRKRA in the p53 pathway, sug-
gests that miR-125b has an antiapoptotic effect. In a separate
study, we found that miR-125b downregulates the p53 pathway
and that miR-125b gain of function represses apoptosis in-
duced by H7 in SH-SY5Y cells (19a). These results suggest
that miR-125b may promote the survival of differentiated neu-
ronal cells by suppressing apoptosis.
In conclusion, we report here several important and novel
functions of miR-125b in neuronal differentiation. Our results
demonstrate that this miRNA promotes the differentiation of
human neuroblastoma SH-SY5Y cells and human neural pro-
genitor RVM cells toward the neuronal phenotype. In SH-
SY5Y cells, we propose a model where the action of miR-125b
is mediated by 10 targets that repress multiple pathways in-
volved in neuronal differentiation.
We thank all our colleagues in Biopolis and the Whitehead Institute,
especially Philip Gaughwin, Boon Seng Soh, Wai Leong Tam, Yen Sin
Ang, Yvonne Tay, Yin Loon Lee, Andrew Thomson, Prakash Rao,
Shilpa Hattangadi, and Cheng Cheng Zhang for fruitful discussions
and Ng Shyh-Chang and Senthil Raja Jayapal for proofreading the
paper. We thank David Bartel and his laboratory for providing the
miRNA microarray and other reagents. We also thank the staffs at
the Biopolis High Content Screening Facility for providing the HCS
service and advices.
M.T.N.L. and H.X. were supported by an SMA graduate fellowship.
P.H.C., P.R., G.U., and H.Y. were supported by A*STAR, Singapore.
B.L. and H.F.L. were partially supported by SMA grant C-382-641-
001-091. B.L. was also supported by NIH grants DK047636 and
AI54973. H.F.L. and B.Z. were supported by NIH grant R01
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