The MicroRNA Cluster miR-106b~25 Regulates Adult Neural
Stem/Progenitor Cell Proliferation and Neuronal Differentiation
Jamie O. Brett1, Valérie M. Renault1, Victoria A. Rafalski1,2, Ashley E. Webb1, Anne Brunet1,2
1Department of Genetics; Stanford University School of Medicine; Stanford, CA 94305; USA
2Neurosciences Program; Stanford University School of Medicine; Stanford, CA 94305; USA
Key words: aging, neural stem cells, microRNAs, FoxO transcription factors, insulin signaling, neuronal differentiation
Received: 2/15/11; Accepted: 2/19/11; Published: 2/20/11
Corresponding author: Anne Brunet, PhD; Email: email@example.com
© Brett et al. This is an open‐access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract: In adult mammals, neural stem cells (NSCs) generate new neurons that are important for specific types of
learning and memory. Controlling adult NSC number and function is fundamental for preserving the stem cell pool and
ensuring proper levels of neurogenesis throughout life. Here we study the importance of the microRNA gene cluster miR‐
106b~25 (miR‐106b, miR‐93, and miR‐25) in primary cultures of neural stem/progenitor cells (NSPCs) isolated from adult
mice. We find that knocking down miR‐25 decreases NSPC proliferation, whereas ectopically expressing miR‐25 promotes
NSPC proliferation. Expressing the entire miR‐106b~25 cluster in NSPCs also increases their ability to generate new
neurons. Interestingly, miR‐25 has a number of potential target mRNAs involved in insulin/insulin‐like growth factor‐1 (IGF)
signaling, a pathway implicated in aging. Furthermore, the regulatory region of miR‐106b~25 is bound by FoxO3, a member
of the FoxO family of transcription factors that maintains adult stem cells and extends lifespan downstream of insulin/IGF
signaling. These results suggest that miR‐106b~25 regulates NSPC function and is part of a network involving the
insulin/IGF‐FoxO pathway, which may have important implications for the homeostasis of the NSC pool during aging.
New neurons are generated in the mammalian brain
throughout adult life. Slowly dividing and self-renewing
neural stem cells (NSCs) are present in the subvent-
ricular zone (SVZ) of the lateral ventricles and in the
subgranular zone (SGZ) of the hippocampal dentate
gyrus. NSCs generate rapidly proliferating neural
progenitor cells that ultimately differentiate to produce
thousands of new neurons each day in adult rats .
The progeny of SVZ NSCs migrate to the olfactory bulb
where they mature into inhibitory interneurons with
roles in olfactory learning and memory . SGZ NSCs
produce excitatory neurons that integrate into the
dentate gyrus and are critical for certain types of
hippocampus-dependent learning and memory [1,3].
Neurogenesis declines with age [4-6] and is impaired by
various types of stress  and brain inflammation .
Exercise and environmental enrichment increase
neurogenesis, and can reverse the effects of aging [9,10]
and stress . Excessive NSC proliferation, however,
can promote functional exhaustion of these cells [12-14]
and in some cases can lead to glioma, a form of brain
cancer [15-17]. Thus, regulation of NSC proliferation
and differentiation is pivotal for adult brain homeostasis
and is disrupted during aging.
Intrinsic and extrinsic factors regulate NSC function
largely by directing changes in gene expression. A
number of transcription factors and chromatin modifiers
control gene expression in adult NSCs, thereby
affecting NSC number and ability to differentiate into
multiple cell types. These regulators include the
polycomb member Bmi1 [18-20], the transcriptional
repressor Tlx [21,22], and the FoxO family of
transcription factors [13,14]. MicroRNAs (miRNAs)
represent an additional layer of gene expression control
and have recently emerged as key regulators of
embryonic and adult stem cells [23,24]. miRNAs are
single-stranded ~23-nucleotide RNA molecules that are
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usually derived from long primary host transcripts .
In the cytoplasm, miRNAs direct destabilization and
translational repression of target mRNAs by binding
sites usually in mRNA 3’ untranslated regions. This
miRNA-directed downregulation of gene expression
generally requires a complementary match between the
mRNA target site and the second to seventh nucleotides
of the miRNA 5’ end (the “seed sequence”). It also
depends on other regions of complementary pairing
between the mRNA site and the miRNA, the presence
of other miRNA-targeted sites, and the mRNA structure
at this region . Several hundred miRNAs have been
identified in humans and mice . As each miRNA
potentially targets hundreds of different mRNAs ,
miRNAs can coordinate cell behaviors by fine-tuning
gene expression [26,29].
A number of miRNAs recently have been found to
regulate adult NSCs in vivo and in culture . For
example, two miRNAs, let-7b and miR-9, inhibit NSC
proliferation and promote neuronal differentiation by
suppressing Tlx and the oncogenic chromatin regulator
Hmga2 [31-33]. In addition, miR-124 promotes
differentiation of SVZ NSCs into neuroblasts by
repressing the expression of the transcription factor
Sox9 . Finally, miR-184 and miR-137 trigger NSC
proliferation and inhibit differentiation by repressing the
NSC fate-regulator Numblike  and the polycomb
methyltransferase Ezh2 , respectively. Thus, miR-
124, miR-9, and let-7b elicit NSC differentiation, while
miR-184 and miR-137 increase proliferation at the
expense of differentiation potential. miRNAs that
promote the expansion of NSCs while maintaining their
ability to differentiate have not yet been identified.
The miRNAs in the miR-17 family are attractive
candidates for this function. Specific miR-17 family
members are overexpressed in a variety of cancers,
including glioma and glioblastoma brain cancers [37-
41], and promote cancer cell proliferation and survival
[42-45]. Furthermore, in embryonic stem cells, miR-17
family members are repressed by the REST neuronal
gene silencer , which negatively regulates neuro-
genesis . miR-17 member expression in the brain
declines between late embryonic and postnatal life ,
which correlates with the decline in neurogenesis that
occurs during this period [49,50]. These results suggest
that miR-17 members may be involved in promoting
both proliferation and neurogenesis.
The miR-17 family consists of three paralogous
polycistronic clusters on different chromosomes: miR-
17~92 (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-
1, and miR-92a-1), miR-106b~25 (miR-106b, miR-93,
and miR-25), and miR-106a~363 (miR-106a, miR-18b,
miR-20b, miR-19b-2, miR-92a-2, and miR-363).
Members of each cluster belong to one of four groups
with similar seed sequences and therefore similar
mRNA targets . Within the miR-17 family,
members of the miR-106b~25 cluster (miR-106b, miR-
93, and miR-25) appear to be the most strongly
expressed in the adult brain [27,52]. Further suggesting
a link between miR-106b~25 and neurogenesis,
expression of the host gene for miR-106b~25, Mcm7, is
reduced in a mouse model of Down syndrome with
diminished numbers of neural progenitor cells and
Interestingly, the miR-106b~25 genomic locus contains a
consensus binding sequence for the FoxO transcription
factors. FoxO factors are inhibited by the insulin/insulin-
like growth factor-1 (IGF) signaling pathway [54-56] and
have emerged as regulators of adult NSCs both in vitro
and in vivo [13,14]. The FoxO family promotes longevity
in a range of species [57-59] and is involved in nematode
lifespan regulation by the miRNA lin-4 . FoxO3, one
member of the FoxO family, has recently been associated
with extreme longevity in humans [61-65]. The presence
of a FoxO binding sequence in the miR-106b~25
genomic locus raises the possibility of an interaction
between this miRNA cluster and the insulin/IGF-FoxO
pathway in mammals.
Here we use primary cultures of neural stem/progenitor
cells (NSPCs) from adult mice to show that miR-
106b~25 promotes NSPC proliferation. Knocking down
miR-25 decreases NSPC proliferation, and ectopically
expressing miR-25 or the entire miR-106b~25 cluster
increases proliferation. In NSPCs induced to differen-
tiate, overexpressing miR-106b~25 enhances different-
iation toward the neuronal lineage. We find that
potential miR-25 target mRNAs are overrepresented in
insulin/IGF signaling. Furthermore, we show that
FoxO3 occupies a binding site near the promoter for
miR-106b~25 in NSPCs, raising the possibility of a
FoxO-miR-106b~25 feedback loop. Together, these
results suggest that miR-106b~25 modulates adult
NSPC proliferation and neuronal differentiation, which
may have crucial implications for the maintenance of
miR-106b, miR-93, and miR-25 are expressed in
adult NSPC cultures
We examined the expression levels of the miR-106b~25
cluster members (miR-106b, miR-93, and miR-25;
Figure 1A) in self-renewing or differentiating NSPCs
isolated from young adult (3 month-old) mice. After the
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first passage in culture, NSPCs were placed in self-
renewal conditions or in differentiation conditions
known to give rise to astrocytes, neurons, and
oligodendrocytes [14,66]. We confirmed differentiation
of NSPCs into these cell types by staining for markers
of astrocytes (GFAP-positive), neurons (Tuj1-positive),
and oligodendrocytes (O4-positive)  after seven
days of differentiation (Figure 1B). We then tested the
expression of miR-106b~25 by RT-qPCR in self-
renewing and differentiating NSPCs (Figure 1C). We
gene expression relative to self‐renewal conditions for 3 independent NSPC cultures (age 12 weeks, passage 2) are shown. One‐
sample two‐tailed t‐test, *: p<0.05.
found that miR-106b, miR-93, and miR-25 were all
expressed in self-renewing NSPCs. Expression of these
miRNAs was not significantly changed by multi-lineage
differentiation, although these miRNAs tended to be
slightly upregulated during differentiation. In contrast,
miR-9, a miRNA known to be induced by NSPC
differentiation , was significantly upregulated in
differentiating NSPCs. Together, these results indicate
that miR-106b~25 is expressed in both self-renewing
and differentiating adult NSPCs.
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Figure 1. The miR‐106b~25 cluster is expressed in adult NSPCs in culture. (A) Genomic locus of the mouse miR‐106b~25
cluster and its host gene, Mcm7. (B) NSPCs (age 12 weeks, passage 2) were grown in multi‐lineage differentiation conditions (no
EGF or bFGF, with 1% FBS) for 7 days and then stained for Tuj1 (a marker of neurons), GFAP (a marker of astrocytes), or O4 (a
marker of oligodendrocytes). Scale bar: 100 µm. (C) miRNA expression was determined by RT‐qPCR in NSPCs in self‐renewal
conditions (with EGF and bFGF, no FBS) or differentiation conditions (no EGF or bFGF, with 1% FBS) for 4 days. Mean and SEM of
miR-25 is important for adult NSPC proliferation
We next tested whether miR-106b~25 is important for
adult NSPC proliferation in self-renewal conditions. To
inhibit miR-106b~25, we transfected NSPCs with
locked nucleic acid (LNA)-modified oligonucleotides
antisense to miR-106b, miR-93, or miR-25, or with a
scrambled control LNA oligonucleotide. We assessed
incorporation of the thymidine analog 5-ethynyl-
deoxyuridine (EdU) in NSPCs transfected with LNA
probes antisense to each of the miRNAs in the miR-
106b~25 cluster or with control LNA probes. We found
that miR-25 knockdown decreased EdU incorporation
in NSPCs by 45% (p=0.005), whereas miR-106b or
miR-93 knockdown did not significantly affect EdU
incorporation in NSPCs (Figure 2). These results
indicate that within the miR-106b~25 cluster, miR-25 is
the most important for NSPC proliferation.
tailed t‐test, **: p<0.01.
Ectopic expression of miR-25 promotes proliferation
in adult NSPCs
To test if miR-25 could promote proliferation in adult
NSPCs, we ectopically expressed miR-25 in NSPCs
using a retroviral vector containing the miR-25
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Figure 2. miR‐25 is necessary for adult NSPC prolifer‐
ation. NSPCs were transfected to knock down miR‐106b,
miR‐93, or miR‐25 or were transfected with a scrambled
control oligonucleotide. Two days after transfection,
NSPCs were incubated with EdU for 1 hour and then
immediately fixed for analysis. (A) Representative photos
for control knockdown and miR‐25 knockdown. Scale bar:
100 µm. (B) Mean and SEM of the proportion of EdU+ cells
for each condition, for experiments on 5 independent
NSPC cultures (age 8‐14 weeks, passage 3‐7). Paired two‐
Figure 3. Expression of miR‐25 enhances adult NSPC
proliferation. NSPCs were infected with an empty control
retrovirus (expressing a GFP marker only) or a retrovirus
expressing miR‐25. NSPCs were grown to full neurospheres
for about 1 week after infection before miRNA expression
and proliferation were analyzed. (A) miR‐25 expression
was assessed with RT‐qPCR in control versus miR‐25‐
overexpressing NSPCs. Mean and SEM of 2 independent
NSPC cultures (age 12 weeks, passage 2‐5) are shown. (B)
Representative photos for each condition. Scale bar: 100
µm. (C) Control and miR‐25‐overexpressing NSPCs were
dissociated and incubated with EdU for 1 hour. Mean and
SEM of the proportion of EdU+ cells for each condition, for
experiments on 4 independent NSPC cultures (age 12
weeks, passage 3‐6), are shown. Paired two‐tailed t‐test,
precursor and green fluorescent protein (GFP). We
verified by RT-qPCR that miR-25 was overexpressed,
on average by 8-fold, in NSPCs after miR-25 retrovirus
infection (Figure 3A). We found that ectopic miR-25
expression increased NSPC incorporation of EdU by
18% compared to the GFP-only control (p=0.04; Figure
Expression of the entire miR-106b~25 cluster also
promotes adult NSPC proliferation
We next tested whether overexpressing the entire miR-
106b~25 cluster in adult NSPCs could further enhance
the proliferation increase caused by miR-25 over-
expression. We generated a retroviral construct
containing the 725-bp portion of the mouse gene
encoding the miR-106b, miR-93, and miR-25
precursors. We verified by RT-qPCR that each member
of miR-106b~25 was overexpressed in cells infected
with miR-106b~25 retroviruses: miR-106b~25 express-
ion was increased 10- to 30-fold in NSPCs infected with
retroviruses (Figure 4A). We assessed the proportion of
cells that incorporated EdU or bromodeoxyuridine
(BrdU), another thymidine analog, in miR-106b~25
expressing versus control NSPCs. Ectopic expression
of miR-106b~25 increased
incorporation by an average of 21% (p=0.03; Figure
4B,C), similar to miR-25 alone, supporting the idea that
miR-25 is the main miR-106b~25 member influencing
NSPC proliferation. Together, these results indicate that
miR-106b~25 promotes adult NSPC proliferation, and
this is likely due mainly to miR-25.
Expression of the miR-106b~25 cluster promotes
neuronal differentiation of adult NSPCs
We examined how miR-106b~25 influences the
generation of neurons from NSPCs during multi-lineage
differentiation in culture. Because the short-term nature
of LNA-mediated miRNA knockdown is not compatible
with the duration of NSPC differentiation, we examined
the effect of retrovirus overexpression of miR-106b~25
on neuronal differentiation. We infected NSPCs with
retroviruses expressing miR-106b~25 or control
retroviruses and then differentiated these cells for seven
days. We stained cells for Tuj1, a marker of neurons,
and determined the proportion of Tuj1-positive cells
(Figure 5). Although infected NSPCs formed relatively
few neurons – probably a consequence of the toxicity of
the infection – we found that compared to control
increased the proportion of Tuj1-positive cells, on
average from 0.3% to 0.9% (2.6-fold; p=0.005). These
results indicate that ectopic expression of miR-106b~25
compared to control
Figure 4. Expression of the entire miR‐106b~25 cluster
also enhances adult NSPC proliferation. NSPCs were
infected with an empty control retrovirus (expressing a GFP
marker only) or a retrovirus expressing miR‐106b, miR‐93,
and miR‐25 simultaneously (miR‐106b~25). NSPCs were
grown to full neurospheres for about 1 week after infection
before miRNA expression and proliferation were analyzed.
(A) miR‐106b, miR‐93, and miR‐25 expression was assessed
with RT‐qPCR in control versus miR‐106b~25‐overexpressing
NSPCs. Mean and SEM of 4 independent NSPC cultures (age
12‐14 weeks, passage 5‐14) are shown. (B) Representative
photos for each condition. Scale bar: 100 µm. (C) Control and
miR‐106b~25‐overexpressing NSPCs were dissociated and
incubated with EdU or BrdU for 1 hour. Mean and SEM of the
proportion of EdU+ or BrdU+ cells for each condition, for 6
experiments on independent NSPC cultures (age 12‐14
weeks, passage 3‐14), are shown. Paired two‐tailed t‐test,
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can enhance neurogenesis in culture.
miR-25 has a number of predicted targets in the
TGFβ and insulin/IGF-FoxO pathways
We next sought to identify the molecular networks
involving miR-25, the main miR-106b~25 member
controlling NSPC proliferation.
algorithms have been developed to predict miRNA
binding sites on target mRNA transcripts, based on
miRNA-target site complementarity, site context, and
site conservation . To examine miR-25 targets
through multiple bioinformatics approaches, we first
used the TargetScan program  to predict the
conserved mRNA targets of miR-25 (~600 targets) and
then used the gene classification programs PANTHER
[69,70] (Figure 6A) or GSEA  (Figure 6B) to
associate biological processes and gene sets with these
targets. In a parallel approach, we used the DIANA-
miRPath program  to predict miR-25 targets (~150)
with the DIANA-microT-3.0-Strict algorithm 
followed by comparison with the Kyoto Encyclopedia
of Genes and Genomes (KEGG) biological pathways
 (Figure 6C). A number of interesting molecular
networks were enriched for miR-25 targets, including
p53 signaling, hypoxia signaling, and nitric oxide
signaling, which are all important for NSC maintenance
and activity [75-77]. Two signaling pathways in
particular stood out from this target analysis:
transforming growth factor β (TGFβ)/bone morpho-
genic protein (BMP) signaling, which was enriched for
miR-25 targets in all three bioinformatics approaches,
and insulin/IGF signaling, which was enriched for miR-
25 targets in the TargetScan-PANTHER analysis
(Figure 6D). TGFβ signaling has been shown to inhibit
adult NSC proliferation and neurogenesis [78,79],
suggesting that miR-25
proliferation and neuronal differentiation by repressing
TGFβ signaling. Activation of the insulin/IGF pathway,
which inhibits FoxO factors , increases NSPC
proliferation and self-renewal [80-83], and FoxO factors
are necessary to maintain the relatively quiescent pool
of adult NSCs [13,14]. The observation that the
insulin/IGF-FoxO pathway is enriched for miR-25
targets is especially pertinent because the genomic locus
of miR-106b~25 contains a conserved FoxO binding
sequence (Figure 7A). Furthermore, there is crosstalk
between TGFβ signaling and the insulin/IGF-FoxO
pathway in nematode longevity, mammalian stem cells,
and cancer cells [84-86]. Taken together, these results
suggest that modulation of the TGFβ and insulin/IGF
signaling pathways may mediate part of the effects of
miR-25 in NSPCs.
The transcription factor FoxO3 binds to a site in the
first intron of miR-106b~25/Mcm7
The precursors of miR-106b~25 members are all
located in the thirteenth intron of the protein-coding
gene Mcm7, a member of a DNA helicase family
required for DNA replication . The first intron of
the Mcm7 gene contains a conserved core binding
sequence (TTGTTTAC) for the FoxO proteins [88,89]
(Figure 7A). As the FoxO factors, particularly FoxO3,
are important for NSC self-renewal, proliferation, and
differentiation [13,14], we tested whether FoxO3 could
bind to this site in the first intron of miR-
106b~25/Mcm7. We performed an electrophoretic
might promote NSC
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Figure 5. miR‐106b~25 enhances neurogenesis in
culture. NSPCs were infected with an empty control virus
or virus to overexpress miR‐106b~25. Three days after
infection, NSPCs were placed in differentiation conditions
for 7 days, and then stained for Tuj1, a marker of
neurons. (A) Representative photos for each condition.
Scale bar: 50 µm. (B) Mean and SEM of the proportion of
Tuj1+ cells (total Tuj1+ cells/total DAPI‐stained nuclei)
normalized to control infection, for experiments on 4
independent NSPC cultures (age 12 weeks, passage 2),
are shown. Paired two‐tailed t‐test, **: p<0.01.
mobility shift assay (EMSA) in which recombinant
FoxO3 was incubated with a 38-bp probe containing the
FoxO binding sequence in the miR-106b~25 genomic
locus. We found that FoxO3 caused a band shift of this
probe, showing that FoxO3 directly binds this site in
vitro (Figure 7B). To determine if FoxO3 is present at
the binding site at the miR-106b~25 locus in NSPCs in
the context of endogenous chromatin, we performed
FoxO3 chromatin immunoprecipitation (ChIP) on
NSPCs treated with brief growth factor removal and the
PI3K inhibitor LY294002, to activate endogenous
FoxO3 (Figure 7C). ChIP-qPCR showed that endogen-
ous FoxO3 occupies the binding site in the first intron
of miR-106b~25/Mcm7 in cultured adult NSPCs. This
enrichment was not present in FoxO3-null NSPCs,
verifying the specificity of the FoxO3 ChIP. These
results indicate that FoxO3 is bound at the genomic
locus of the miR-106b~25 cluster.
To test if FoxO3 could upregulate the transcription of
miR-106b~25/Mcm7, we generated a luciferase reporter
construct containing a minimal SV40 promoter and the
500 bp surrounding the FoxO binding site in the first
intron of miR-106b~25/Mcm7 (Figure 7A). We co-
Figure 6. miR‐25 targets genes involved in TGFβ and insulin/IGF signaling. (A) The PANTHER gene classification program
was used to analyze TargetScan‐predicted conserved targets for mouse miR‐25 (~600 targets total). Shown are the top 5
biological pathways (ordered by Bonferroni‐corrected binomial test p‐values). (B) The GSEA program was used to analyze the
same TargetScan‐predicted target list as in (A), using the Canonical Pathways and GO Gene Sets categories. Shown are the top 5
categories (ordered by hypergeometric distribution‐generated p‐values). (C) The DIANA‐microT program was used to generate a
stringent list of mouse miR‐25 targets. Shown are the top KEGG categories (ordered by Pearson’s chi‐square test p‐values). (D)
Pathway diagrams based on those in PANTHER Pathways for TGFβ and insulin/IGF‐Akt signaling pathways, modified for
simplicity and with select miR‐25 predicted targets listed.
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transfected HEK 293T cells with this reporter construct
and with plasmids to express wild-type FoxO3, a DNA-
constitutively active FoxO3. These luciferase assays
revealed that constitutively active FoxO3 enhanced
luciferase expression (p=0.002), and this was partly
abrogated by mutating the FoxO binding site
(p=0.08), indicating that FoxO3 acts as a transcriptional
activator at this genomic locus in HEK 293T cells
We next investigated whether
endogenous miR-106b~25 and Mcm7 expression in
NSPCs by comparing the expression of miR-106b~25
form of FoxO3, or
and Mcm7 in cultured NSPCs from wild-type versus
FoxO3-null adult mice (Figure 7E). FoxO3-null NSPCs
had decreased abundance of Mcm7 mRNA (p=0.01),
indicating that Mcm7 is a target gene of FoxO3.
However, FoxO3-null NSPCs did not display decreased
expression of the mature forms of miR-106b, miR-93,
and miR-25, suggesting that FoxO3 does not directly
upregulate miR-106b~25 and might even indirectly
repress the expression of this cluster. Together, these
results suggest a complex regulation in which FoxO3
activates the transcription of miR-106b~25/Mcm7, but
may repress miR-106b~25 abundance, perhaps by a
posttranscriptional mechanism or by acting at a separate
promoter for miR-106b~25.
5‐6 independent cultures (age 10‐13 weeks, passage 2‐5)
are shown. One‐sample two‐tailed t‐test, *: p<0.05.
Figure 7. FoxO3 binds to a site in the first intron of
miR‐106b~25/Mcm7. (A) Location of the FoxO binding
site (FHRE) within the first intron of the miR‐
106b~25/Mcm7 gene, and the sequence locations used for
EMSA, ChIP, and luciferase experiments. (B) EMSA with
recombinant FoxO3‐GST and a radioactively‐labeled (hot)
probe corresponding to the FoxO binding site in miR‐
106b~25/Mcm7 (FHRE WT). + Ctrl: FoxO3‐GST incubated
with a probe for a known FoxO binding site. The specificity
of the interaction was tested by increasing amounts of
unlabeled (cold) probe or cold probe with mutations in the
FoxO consensus binding sequence (FHRE Mut). (C) Wild‐
type and FoxO3‐null NSPCs were dissociated and the next
day treated with 4 hours growth factor removal followed
by addition of LY294002 for 1 hour. Antibodies to FoxO3 or
control IgG antibodies were used for ChIP. qPCR was used
to assess the enrichment of FHRE and of a negative control
site (‐ Ctrl). Shown is the relative enrichment for 1
experiment (age 12 weeks, passage 10). These results were
confirmed in ChIP‐Seq studies (Webb et al. submitted). (D)
HEK 293T cells were co‐transfected with a plasmid to
express FoxO3 (empty control, wild‐type FoxO3, FoxO3
lacking the DNA binding domain, or constitutively nuclear
FoxO3), a firefly luciferase reporter containing FHRE with
or without the FoxO consensus sequence mutated, and a
Renilla luciferase reporter to normalize for transfection
efficiency. As a positive control, a luciferase reporter
containing a known FoxO3‐activated site was used (+ Ctrl);
as a negative control, a luciferase reporter without an
enhancer site was used (‐ Ctrl). Luciferase activity was
assessed two days after transfection. Mean and SEM for 4
independent experiments (‐ Ctrl, + Ctrl, and FHRE WT) or 2
independent experiments (FHRE Mut) are shown.
Unpaired two‐tailed t‐test, **: p<0.01. (E) NSPCs from
wild‐type and FoxO3‐null mice were isolated and cultured.
Total RNA was collected, and the levels of mature miR‐
106b~25 members (relative to 5S RNA) and Mcm7 mRNA
(relative to β‐actin mRNA) were assessed by RT‐qPCR.
Mean and SEM of the FoxO3‐null/wild‐type fold change for
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miR-106b~25 members are known to promote cancer
cell proliferation and survival [42,44], modulate
embryonic stem cell differentiation , and promote
reprogramming of mouse embryonic fibroblasts into
induced pluripotent stem cells  – but the importance
of miR-106b~25 has not been investigated in an adult
stem cell population before. This study examined the
role of miR-106b~25 in adult NSPCs. We found that
miR-25 knockdown decreases NSPC proliferation, miR-
25 or miR-106b~25 overexpression increases adult
NSPC proliferation, and miR-106b~25 overexpression
promotes neuronal differentiation. Furthermore, FoxO3
binds near the promoter for the host gene of miR-
106b~25 and thus has the potential to influence miR-
106b~25 expression. These results add to our
understanding of the factors regulating NSPC activity
and suggest that oncogenic miRNAs could have
physiological functions in adult stem cells.
miR-106b~25 and NSPC proliferation
The effects of miR-106b~25 on adult NSPC
proliferation are modest: miR-106b~25 or miR-25
overexpression increased NSPC proliferation by about
1.2-fold, miR-25 knockdown reduced proliferation by
about 1.4-fold, and individual miR-106b and miR-93
knockdowns did not affect NSPC proliferation. While
these proliferation changes are somewhat smaller than
those seen by miR-106b~25 manipulation in carcinoma
cells (up to 1.8-fold in similar assays) [42,44], a modest
effect of miR-106b~25 on NSPC proliferation could
still be important physiologically. While an enforced
large increase in NSC proliferation rate could result in
tumor initiation [15,17], a weaker increase in
proliferation rate could lead to premature stem cell
exhaustion [12,13]. On the other hand, fewer divisions
could reduce NSC number and neurogenesis [32,92].
Therefore, it is possible that miR-106b~25 overex-
pression or underexpression, both of which alter NSPC
proliferation in culture, could affect long-term NSC
function in vivo.
Redundancy within the miR-17 family could dampen
the influence of miR-106b~25 on NSPC proliferation in
vitro. Knockdown of miR-106b or miR-93, which share
the same mRNA-targeting seed sequence, did not affect
proliferation, while knockdown of miR-25, which has a
different seed sequence, reduced proliferation. miR-
106b and miR-93 might be able to compensate for each
other in NSPCs, which could be tested by inhibiting
both miRNAs simultaneously. Furthermore, it is
possible that NSPCs buffer miR-106b~25 alteration by
expressing miR-17 family members from the other
paralogous clusters, thereby lessening the relative
importance of one or even three miRNAs within this
family, or allowing NSPCs to react to changes in miR-
106b~25 expression with compensatory changes in
miR-17~92 or miR-106a~363 expression. Our findings
suggest the idea that compared to cancer cells, stem
cells may be more resilient against oncogene
perturbation, and therefore more tolerant of certain
gene-specific anti-cancer therapies. This may be
particularly true for miRNAs, which have been
duplicated during animal evolution and tend to have
overlapping targets and functions. Such redundancy
may have evolved not only so that duplicated miRNAs
can be controlled by distinct cis regulatory elements,
but perhaps also so that stem cells can absorb
fluctuations in gene expression.
miR-106b~25 in neuronal differentiation
We found that miR-106b~25 promotes both NSPC
proliferation in self-renewal conditions and neuron
production in differentiation conditions, whereas other
miRNAs previously studied in adult NSCs seem to
promote one function while inhibiting the other. The
mechanism of this effect is still unknown: miR-106b~25
could affect NSPC tendency to produce neurons instead
of glia, neuronal progenitor proliferation and survival,
and neuron survival. Thus, it remains to be determined
whether miR-106b~25 influences neurogenesis by
directing cell fate or by regulating cell division and
survival in specific cell types.
Adult NSCs decline in number and proliferation,
neurogenesis, and self-renewal abilities during aging
. Activities that restore NSC activity, such as
exercise or environmental enrichment, also restore
cognitive performance in aged mice [93,94]. As NSC
decline may contribute to cognitive aging, investigating
how miR-106b~25 affects neurogenesis will improve
our understanding of the molecular mechanisms
involved in cognitive aging. While miR-106b~25
knockout mice have no apparent phenotype ,
neurogenesis and learning have not been examined in
these mice. It would be worthwhile to investigate how
NSCs lacking or overexpressing miR-106b~25 in vivo
preserve their numbers and sustain neurogenesis
Potential signaling pathways regulated by miR-25
Deciphering how stem cells sense and respond to tissue
integrity and nutrient supply is key to understanding
how stem cells maintain tissue homeostasis and how
this function changes with age [83,95,96]. Analyzing
candidate targets of miR-25 revealed that miR-25 might
www.impactaging.com 116 AGING, February 2011, Vol.3 No.2
modulate TGFβ or insulin/IGF signaling at multiple
points in each pathway. As TGFβ signaling negatively
regulates adult NSC proliferation and neurogenesis
[78,79], one way miR-106b~25 might promote these
behaviors is by repressing TGFβ signaling in NSPCs.
TGFβ Receptor-2 is directly repressed by miR-106b in
neuroblastoma cells  and by miR-106b and miR-93
in mouse embryonic fibroblasts ; thus, one enticing
possibility is that TGFβ Receptor-2 is targeted by all
miR-106b~25 members in NSPCs. While inhibitory
Smads (Smad6 and Smad7) are also predicted miR-25
targets, Smad7-deficient mice have increased adult
NSPC proliferation and numbers, which may be due to
TGFβ-independent mechanisms . The net functional
effect of miR-25 regulation of TGFβ signaling in
NSPCs will depend on the relative expression, degree of
miR-25 repression, and network connections of each
member of the TGFβ pathway in NSPCs.
Activation of the insulin/IGF pathway is sufficient to
increase NSPC proliferation and self-renewal [80-83],
while FoxO factors are necessary to prevent
overproliferation, abnormal differentiation, and long-
term depletion of NSCs [13,14]. Thus, another way
miR-25 might increase NSPC proliferation is by de-
repressing insulin/IGF signaling. Given that PTEN can
be a major inhibitor of insulin/IGF signaling [99,100]
and is a known target of miR-25 in prostate cancer cells
, miR-25 may target PTEN to increase insulin/IGF
signaling and repress FoxO activity. We cannot exclude
the possibility, however, that miR-25 negatively
regulates insulin/IGF signaling under some circum-
stances, such as by repressing Akt or PI3K.
There may even be crosstalk between the different
pathways targeted by miR-25. In nematodes the TGFβ
pathway has been shown to genetically interact with the
insulin/IGF-FoxO pathway to regulate lifespan . In
mammals TGFβ promotes hematopoietic stem cell
quiescence by downregulating Akt activity and
upregulating FoxO3 activity , and in glioblastoma
cells TGFβ signaling induces Smad-FoxO transcript-
tional activation complexes that suppress proliferation
. In human keratinocytes, FoxO factors are required
for the induction of a number of genes by TGFβ,
particularly cytostatic and stress response genes .
Thus, it is possible that miR-25 regulate NSPCs by
Regulation of miR-106b~25 by FoxO proteins
Our experiments suggest that FoxO3 regulates miR-
106b~25 in a complex manner. FoxO3 binds to a site in
the first intron of miR-106b~25/Mcm7 in NSPCs. In
insulin/IGF and TGFβ
FoxO3-null NSPCs, while Mcm7 mRNA abundance
was decreased, the levels of mature miR-106b~25
members were not decreased, and were even slightly
increased. Thus, FoxO3 might transcriptionally activate
miR-106b~25/Mcm7, but act to repress miR-106b,
miR-93, and miR-25 at a different promoter or at
posttranscriptional steps like precursor cleavage,
nuclear export, base editing, and degradation.
Other factors complicate our ability to define the
regulation of miR-106b~25 by FoxO3. It is possible that
in self-renewal culture conditions FoxO3 is bound near
the promoter of miR-106b~25 but exerts control over
miR-106b~25 expression only in other conditions such
as differentiation, low nutrient levels, oxidative stress,
or low oxygen tension. As NSPC cultures are
heterogeneous, containing mixtures of stem cells,
progenitor cells, and even some differentiated progeny
[103,104], FoxO3 might also alter miR-106b~25
expression differently in different cell types. Such
differential regulation would be consistent with FoxO3
and miR-106b~25 both
differentiation but having opposite effects on NSPC
proliferation [13,14]. In these scenarios, FoxO3 would
serve as one component of a “coincidence detector”
regulating miR-106b~25, which in turn might indirectly
influence FoxO activity.
This study shows that miR-106b~25 members modulate
NSPC proliferation and differentiation and could
potentially be regulated
transcription factor FoxO3 under some circumstances.
These results suggest a role for miR-106b~25 in normal
adult stem cell function, in addition to a known role in
cancer cells. Understanding how miR-106b~25 and
FoxO3 function in NSPCs could reveal new strategies
for preventing the loss of neurogenesis in adults,
particularly during aging.
Constructs. For miRNA overexpression, the 725-bp
segment of the mouse Mcm7 gene containing the miR-
106b, miR-93, and miR-25 precursors was cloned
between the XhoI and PmeI sites of the MDH1-PGK-
GFP 2.0 vector  using the primers F: 5’-
CAAGC-3’. The 350-bp segment of the mouse Mcm7
gene containing the miR-25 precursor only was cloned
between the XhoI and EcoRI sites of the MDH1-PGK-
GFP 2.0 vector using
by the pro-longevity
the primers F: 5’-
www.impactaging.com 117 AGING, February 2011, Vol.3 No.2
For luciferase assays, the 500-bp region of the mouse
Mcm7 intron containing the FoxO3 binding site was
cloned between the KpnI and XhoI sites of the pGL3-
SV40 vector (Promega) using the primers F: 5’-
-3’. Mutations in the FoxO binding sequence were made
using the primers
AGAGCGG-3’, and this mutated enhancer was
subcloned into a new pGL3-SV40 backbone. The
positive control plasmid, pGL3-SV40 containing three
repeats of the FoxO3 binding site in the FasL promoter,
and the FoxO3 expression plasmids were described
Antibodies. For immunocytochemistry, the primary
antibodies used were rat anti-BrdU (AbD Serotec;
1:500), goat anti-GFP (Rockland; 1:500), rabbit anti-
Tuj1 (Covance; 1:1000), rat anti-GFAP (Calbiochem;
1:1000), and mouse anti-O4 (a gift from Ben Barres;
1:1000). Fluorescent secondary antibodies were from
Jackson Immuno-Research and Molecular Probes
(Invitrogen) and were used at 1:400 dilutions. The
antibodies for ChIP were rabbit anti-FoxO3 “NFL”
(Brunet laboratory) and rabbit IgG (Zymed).
NSPC isolation and culture. Each NSPC culture was
generated from four to eight FVB/N mice (1:1 male-
female ratio). Whole brain was extracted from each
animal, and the olfactory bulbs, cerebellum, and
brainstem were discarded. To dissociate the forebrain
tissue, brains were diced, treated at 37°C for 30 min
with HBSS (Invitrogen) containing 2.5 U/ml Papain
(Worthington), 1 U/ml Dispase II (Roche), 250 U/ml
DNase I (Sigma), and 1X penicillin-streptomycin-L-
glutamine (PSQ; Invitrogen), and then mechanically
dissociated in DMEM/F12 (Invitrogen) containing 10%
fetal bovine serum (FBS; Invitrogen) and 1X PSQ.
NSPCs were purified from myelin with a 22.5% Percoll
gradient (GE Healthcare) and then from red blood cells
with a 58.5% Percoll gradient. Freshly isolated NSPCs
were considered “passage 1.”
NSPCs were grown at 5% CO2 in a 37°C incubator at
50,000 cells/ml in Neurobasal A Medium (NBA;
Invitrogen) supplemented with 1X PSQ, 1X B-27
Supplement Minus Vitamin A (B27; Invitrogen), 20
ng/ml recombinant human bFGF (PeproTech), and 20
ng/ml recombinant human EGF (PeproTech). Cells
were fed every 2 days by replacing half the media and
replenishing bFGF and EGF; cells were transferred to a
new plate every 4 days. NSPCs grew to full neurosphere
colonies every 5-8 days, and were passaged using
Accutase (Millipore) for dissociation.
miRNA overexpression by retroviral infection. HEK
293T cells were co-transfected with the expression
vector MDH1-PGK-GFP 2.0 containing either miR-
106b~25 or no insert (empty control) and the pCL-Eco
viral packaging vector in a 2:1 ratio, using the calcium
phosphate transfection method. The media was changed
to NBA containing 1X PSQ and 1X B27 6-8 h later.
The next day, NSPCs were dissociated and plated at
50,000 cells/ml on plates coated with 50 µg/ml poly-D-
lysine (Sigma). The following day, NSPCs were
infected by replacing half the media with 0.45 µm-
filtered virus-containing supernatant from the 293T
cultures and replenishing the growth factors. Sixteen
hours later, the infection was stopped by replacing all
the media with NSPC-conditioned media and fresh
media in a 1:1 ratio and replenishing growth factors.
NSPCs were fed every other day until they were 80%
confluent, and then detached with Accutase and grown
in suspension. After NSPCs had grown to full
neurospheres, RNA and protein were collected, and
cells were plated for proliferation assays.
miRNA knockdown. NSPCs were plated at 100,000
cells/ml in 0.5 ml NBA containing 1X L-glutamine
(Invitrogen) and 1X B27 with growth factors in a poly-
D-lysine-coated well of a 24-well plate. The next day,
45 nM locked nucleic acid (LNA) oligonucleotide
(Exiqon) was diluted with 100 µl Opti-MEM
(Invitrogen), incubated with 1 µl Lipofectamine PLUS
reagent (Invitrogen) per 1 µg nucleic acid for 5 min, and
then incubated with 6 µl Lipofectamine LTX reagent
(Invitrogen) per 1 µg nucleic acid for 30 min before
being added to cells. The media was changed to 1 ml
NBA containing 1X PSQ and 1X B27 with growth
factors 4-6 h later.
Proliferation assays. One week after retroviral infection
(when NSPCs had grown to full neurospheres), NSPCs
were dissociated and plated on nitric acid-treated glass
coverslips (Bellco) coated with poly-D-lysine. Two
days later, BrdU (EMD Biosciences) was added to a
final concentration of 10 µM, or EdU (Invitrogen) was
added to a 5 µM final concentration. One hour later,
NSPCs were fixed in 4% paraformaldehyde in PBS for
12 min. The coverslips were blocked for 1 h with 10%
donkey serum and 0.1% Triton in PBS and then
incubated with goat anti-GFP antibody for 2 h. The
www.impactaging.com 118 AGING, February 2011, Vol.3 No.2
coverslips were then refixed with 4% paraformaldehyde
for 10 min and incubated with 0.4% Triton for 30 min.
DNA was denatured with 2 N HCl for 10 min. After 1 h
of blocking, coverslips were incubated with rat anti-
BrdU antibody for 2 h. The coverslips were incubated
with Texas Red donkey anti-rat and FITC donkey anti-
goat secondary antibodies for 1 h. The coverslips were
mounted on slides using Vectashield with DAPI (Vector
Two days after transfection with LNA probes, EdU was
added to a final concentration of 5 µM. One hour later,
NSPCs were fixed in 4% paraformaldehyde and 2%
sucrose for 12 min. Cells were permeabilized with 0.4%
Triton in PBS for 30 min and blocked with two 3%
BSA (USB) rinses. Cells were then incubated in 1X
Click-iT Reaction Buffer, 4 mM CuSO4, 1:400 Alexa
Fluor 594 azide, and 200 nM Click-iT EdU Buffer
Additive (Invitrogen) for 30 min. Cells were then
washed with 3% BSA, rinsed with PBS, and mounted
on slides using Vectashield with DAPI.
Coverslips were examined using a Zeiss Axioskop 2
Plus microscope and digital camera with AxioVision 4
software. For quantification, 3-6 random fields (about
1000-2000 cells) were counted in a blinded manner,
using Metamorph 7.0 software.
Differentiation assays. NSPCs were dissociated and
plated on nitric acid-treated coverslips coated with poly-
D-lysine at a density of 25,000 cells/ml. NSPCs were
infected the next day, and the infection was stopped
after 16 h. Two days later, NSPCs were differentiated
by changing the media to NBA containing 1X PSQ, 1X
B27, and 1% FBS. The media was replaced every other
day. After 7 days of these differentiation conditions,
NSPCs were stained for GFAP, Tuj1, or O4. For GFAP
and Tuj1 staining, NSPCs were fixed in 4%
paraformaldehyde and 2% sucrose. The coverslips were
blocked for 1 h with 10% donkey serum and 0.1%
Triton in PBS, and then incubated with rabbit anti-Tuj1
antibody for 2 h. After rinsing with PBS containing
0.01% Tween and blocking for another 15 min,
coverslips were incubated with Texas Red donkey anti-
rabbit or anti-rat secondary antibody for 1 h. For O4
staining, NSPCs were blocked with 5% goat serum and
7.5% BSA in PBS for 1 h and then incubated with
mouse anti-O4 antibody (in 10% goat serum, 1% BSA,
and 100 mM L-lysine in PBS) for 2 h. After rinsing
with PBS, cells were fixed in 4% paraformaldehyde and
2% sucrose, blocked for another 15 min, and then
incubated with Alexa Fluor 546 goat anti-mouse
secondary antibody for 1 h. Coverslips were mounted
on slides using Vectashield with DAPI. The total
number of neurons on each coverslip was counted in a
blinded manner, and the total number of nuclei was
estimated by counting 5 random fields (about 300-600
cells) in a blinded manner.
Target prediction. TargetScan (www.targetscan.org,
version 5.1) was used to predict all conserved targets for
mouse miR-25. This target list was analyzed using
PANTHER (www.pantherdb.org, version 7) to compare
Biological Process associations for genes in this list and
the reference list, “NCBI: M. musculus genes,” or
analyzed with GSEA Molecular Signatures Database
(www.broadinstitute.org/gsea/msigdb/, version 3.0) to
compute overlaps for genes in this list and “CP”
(Canonical Pathways) and “C5” (GO Gene Sets).
DIANA-microT-3.0-Strict was used to predict and
analyze conserved targets for mouse miR-25 in the
RT-qPCR. Total RNA was extracted from NSPCs using
the miRVana kit (Ambion). RNA was treated to remove
genomic DNA in a reaction containing 100 ng/μl RNA,
1 U/μl RNase OUT (Invitrogen), and 10 U/μl DNase I
(Invitrogen) at 37°C for 15 min and 75°C for 15 min.
miRNA expression was quantified using the miRCURY
LNA miRNA PCR system or the miRCURY LNA
Universal RT miRNA PCR system, according to the
manufacturer’s instructions (Exiqon). Samples were run
in triplicate on a C1000 Thermal Cycler with the
CFX96 Real-Time software (Bio-Rad), and miRNA
expression was normalized to 5S RNA expression.
To quantify Mcm7 mRNA expression, RT was carried
out using the High Capacity cDNA Reverse
Transcription kit (Applied Biosystems). Each reaction
contained 1X RT Buffer, 4 mM each dNTP, 1X
Random Hexamers, 1 U/µl RNase OUT, 2.5 U/µl
MultiScribe Reverse Transcriptase, and 45-90 ng/µl
RNA. RT was performed at 25°C for 10 min, 37°C for 2
h, and 85°C for 5 min. Each 20-µl qPCR reaction
contained 0.25 µM forward (F) Primer, 0.25 µM reverse
(R) Primer, 10 µl iQ SYBR Green Supermix (Bio-Rad),
and 0.625 µl RT reaction. The program used was 95°C
for 10 min; 40 cycles of 95°C for 20 sec, 55°C for 20
sec, and 72°C for 45 sec. Samples were run in triplicate,
and Mcm7 expression was normalized to β-actin
expression. The Mcm7 primers
primers were F: 5’-TGTTACCAACTGGGACGACA-
3’ and R: 5’-CTCTCAGCTGTGGTGGTGAA-3’.
Chromatin immunoprecipitation. ChIP was performed
www.impactaging.com 119 AGING, February 2011, Vol.3 No.2
as described  using IgG or FoxO3 antibodies.
Immunoprecipitated chromatin was analyzed with
qPCR: each 20-µl reaction contained 2.5 µl DNA, 10 µl
iQ SYBR Green Supermix, 0.25 µM F primer, and 0.25
µM R primer. Triplicate reactions were run with the
following program: 94°C for 3 min; 40 cycles of 95°C
for 20 sec, 57°C for 30 sec, and 72°C for 30 sec. The
primers to amplify the region surrounding the FHRE
FoxO3 binding site in the Mcm7 first intron were F: 5’-
control primers to amplify an intergenic region lacking
a Forkhead binding sequence
chromatin sample, a standard curve using five 5-fold
dilutions of input chromatin was used to quantify
binding at each target site in the ChIPs: linear
regression (y=-ax+b) was performed on Ct versus
log5(input), and the amount of a site in the FoxO3 ChIP
relative to the IgG ChIP was calculated as 5-ΔCt/a, with
Electrophoretic mobility shift assay. Complementary
oligonucleotides (20 µM) were annealed in 100 mM
NaCl by heating at 80°C for 5 min and then cooling
slowly to room temperature. Annealed probe (1 µM)
was labeled with 20 µCi/µl 32P-γ ATP and 1 U/µl T4
PNK at 37°C for 1 h. Annealed probes were purified on
15% polyacrylamide and resuspended in 1X TE pH 8.
Each binding reaction was performed in Binding Buffer
(200 mM Tris-HCl pH 7.5, 200 mM KCl, 200 mM
MgCl2, 2% NP-40, 10% glycerol, 5 mM DTT, and 500
ng/μl salmon sperm DNA) and contained 50 ng/µl GST
or human FoxO3-GST, 1000 cpm/μl hot probe (5 nM
FHRE probe; 3 nM positive control probe), and 0, 5, 50,
or 500X competing cold probe. The reactions were
incubated at room temperature for 20 min and then
resolved on 4% non-denaturing PAGE at 4°C. The gels
were dried and then autoradiographed for 4 days. The
positive control oligonucleotides for a site bound by
FoxO3 near its own promoter  were F: 5’-
GTTATTT-3’. The oligonucleotides for the FHRE site
within the Mcm7 intron region bound by FoxO3 in
GGGCC-3’. The oligonucleotides for this FHRE site
containing mutations in the FoxO consensus binding
were F: 5’-
Luciferase assays. HEK 293T cells were plated in 24-
well plates at 150,000 cells/ml. The next day, they were
transfected using the calcium phosphate method with
400 ng each of FoxO3 expression plasmid, pGL3-SV40
firefly luciferase plasmid, and pRL-null Renilla
luciferase plasmid. Two days after transfection, cells
were lysed with 0.5 ml Passive Lysis Buffer (Promega)
and luciferase activity was measured with the Dual
Luciferase Reporter Assay
transfections were averaged within each experiment,
and firefly luciferase activity was normalized to Renilla
Statistical analysis. Gene expression (RT-qPCR
experiments) was analyzed using one-sample two-tailed
t-tests. NSPC phenotype
differentiation assays) was analyzed using paired two-
tailed t-tests. The luciferase assay experiments were
analyzed with unpaired two-tailed t-tests.
J.O.B. designed, performed,
experiments. V.M.R. trained J.O.B., helped with
experimental design and analysis, and generated
FoxO3-null NSPC cultures. V.A.R. helped with
experimental design and analysis and with neuronal
differentiation experiments. A.E.W. performed the
FoxO3 ChIP and helped with the bioinformatics
analysis. A.B. helped with the design and analysis of the
experiments. J.O.B and A.B. wrote the paper.
CONFLICTS OF INTEREST STATEMENT
The authors have no conflicts of interest to declare.
We thank Chang-Zheng Chen for providing the MDH1-
PGK-GFP plasmid and for his advice on miRNA
expression and manipulation studies. We thank Dervis
Salih for generating the FoxO3-GST and GST proteins
for EMSA experiments. We thank Dena S. Leeman for
critically reading the manuscript and for stimulating
discussion, and Elizabeth Pollina for stimulating
discussion. We thank Camille Guillerey, T. Richard
Parenteau, and Christopher Itoh for conducting
preliminary experiments. This work was funded by an
NIH/NIA grant R01 AG026648, a CIRM grant, a Brain
Tumor Society grant, a Klingenstein Fellowship, and an
and analyzed all
www.impactaging.com 120 AGING, February 2011, Vol.3 No.2
AFAR grant (A.B.), a Stanford University Major Grant
(J.O.B.), a Stanford University Dean’s fellowship
(V.M.R.), an NSF graduate fellowship (V.A.R.), and an
NIH/NRSA 5T32 CA09302 (A.E.W.).
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