Tài liệu Báo cáo khoa học: Bioimaging of the unbalanced expression of microRNA9 and microRNA9* during the neuronal differentiation of P19 cells doc

Bioimaging of the unbalanced expression of microRNA9
and microRNA9* during the neuronal differentiation of
P19 cells
Mee Hyang Ko
1,2,3
, Soonhag Kim
2,4,
*, Do Won Hwang
1,2,3
, Hae Young Ko
2,3
, Young Ha Kim
2,3
and
Dong Soo Lee
2,4,
*
1 Programs in Neuroscience, Seoul National University, Korea
2 Department of Nuclear Medicine, Seoul National University College of Medicine, Korea
3 Laboratory of Molecular Imaging and Therapy of Cancer Research Institute, Seoul National University College of Medicine, Korea
4 Medical Research Center, Seoul National University College of Medicine, Korea
MicroRNAs (miRs) are a class of small non-coding
RNA molecules, encoded as short inverted repeats in
the genomes of plants and animals. miRs are believed
to modulate the post-transcriptional regulations of
their targets in diverse biological regulatory systems
including cellular development [1,2], cell differentiation
[3], fat metabolism [4], cell proliferation and cell death
[5]. Hundreds of miRs have been isolated from mam-
malian species and a dozen of these, including
miR124a, miR9, miR128, miR131, miR178 and

Keywords
bioimaging; Luciferase; microRNA;
microRNA9 and microRNA9*; neurogenesis
Correspondence
S. Kim, Department of Nuclear Medicine,
Medical Research Center, Seoul National
University College of Medicine, 28 Yongon-
dong, Jongno-gu, Seoul 110 744, Korea
Fax: +82 2 3668 7090
Tel: +82 2 3668 7028
E-mail:
D. S. Lee, Department of Nuclear Medicine,
Medical Research Center, Seoul National
University College of Medicine, 28 Yongon-
dong, Jongno-gu, Seoul 110 744, Korea
Fax: +82 2 3668 7090
Tel: +82 2 2072 2501
E-mail:
*These authors contributed equally to this
work
(Received 18 January 2008, revised 10
March 2008, accepted 17 March 2008)
doi:10.1111/j.1742-4658.2008.06408.x
Generally, the 3¢-end of the duplex microRNA (miR) precursor (pre-miR)
is known to be stable in vivo and serve as a mature form of miR. However,
both the 3¢-end (miR9) and 5¢-end (miR9*) of a brain-specific miR9 have
been shown to function biologically in brain development. In this study,
real-time PCR analysis and in vitro ⁄ in vivo bioluminescent imaging demon-
strated that the upstream region of a primary miR9-1 (pri-miR9-1) can be
used to monitor the highly expressed pattern of endogenous pri-miR9-1

during neurogenesis, and that the Luciferase reporter gene can image the
unequal expression patterns of miR9 and miR9* seen during the neuronal
differentiation of P19 cells. This demonstrates that our bioimaging system
can be used to study the participation of miRs in the regulation of neur-
onal differentiation.
Abbreviations
Dicer, RNase III endonuclease; FLuc, Firefly Luciferase; GLuc, Gaussia Luciferase; miR, microRNA; miR9*, microRNA9*, 5¢-end of pre-miR9;
miR9, microRNA9, 3¢-end of miR9 precursor; piRNA, Piwi-interacting RNA; pre-miR, precursor microRNA; pre-miR9, miR9 precursor or
precursor miR9; pri-miR9-1, primary miR9-1; ROI, region of interest analysis.
FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS 2605
miR125b, have been found to be associated with polyr-
ibosomes in primary neurons [6,7]. These studies have
shown that both microRNA9 [miR9, 3¢-end of miR9
precursor (pre-miR9)] and microRNA9* (miR9*,
5¢-end of pre-miR9) originate from the hairpin-loop
structure of the same pre-miR9, and are highly
co-expressed and neuron-specific during brain devel-
opment.
In general, intergenic or intragenic miRs are tran-
scribed into primary miRNA (pri-miR) by RNA
polymerase II and processed into a 70-nucleotide hair-
pin-structured pre-miR in the nucleus by Drosha
[8–10]. Pre-miRs are transported to the cytoplasm by
exportin-5 (a member of the Ran transport receptor
family) and another factor Ran [11]. Pre-miR hairpin
is further processed into a 19- to 23-nucleotide single-
stranded mature miR by RNase III endonuclease
(Dicer) [12]. During Dicer cleavage, duplex pre-miRs
are uncoiled by helicase into two single strands,
mature miR (from the 3¢-end of duplex pre-miR) or

miR* (opposite strand, from the 5¢-end of duplex
pre-miR), although miR* is generally rapidly
degraded by an unknown enzyme nuclease [13].
Mature miRs are then incorporated into the RNA-
induced silencing complex and bound to the 3¢-UTR
of its target mRNA to induce either mRNA degrada-
tion or translational inhibition [2,14,15]. Interestingly,
unlike most miRs, which have a single mature miR,
miR cloning and sequencing from the human and
miRNAMap database (u.
edu.tw) showed that a few miRs, including miR302b,
miR302c, miR373 and miR9, have two types of
mature form, miR and miR* [16,17]. This is similar
to the final functional forms of Piwi-interacting RNA
(piRNA), which are also small RNA molecules
although distinct in size from miR. Even though
25- to 31-nucleotide long piRNAs are not generated
by Dicer, both sense and antisense strands of the
piRNA hairpin are involved in formation of the piR-
NA-interacting complex (piRC) and function in the
transcriptional gene silencing of retrotransposons and
genetic elements in germline cells [18].
Investigations into the gene expression of endoge-
nous miR in cells or tissues are useful for understand-
ing cellular metabolism, disease diagnosis and the
effects of therapies related to miR. However, current
methods of monitoring endogenous miR levels, such as
northern blotting, RT-PCR, and microarrays are time-
consuming, laborious, and non-reproducible. In a pre-
vious study, we successfully imaged miR23a biogenesis

in small animals to noninvasively monitor the expres-
sion patterns of endogenous miR23a in different cells
[19]. This type of bioluminescence imaging technology
may be clinically relevant and could be applied to the
real-time analysis of miR biogenesis in living animals.
Currently, the most widely used bioluminescent pro-
teins in living animals are Gaussia Luciferase (GLuc)
and Firefly Luciferase (FLuc). GLuc emits light at a
480 nm by oxidizing its substrate coelenterazine [20],
FLuc emits at 562 nm when it oxidizes d-luciferine
[21].
We cloned the upstream region of miR9 and studied
the expression pattern of endogenous pri-miR9-1 to
try to understand miR9 biogenesis during the neuronal
differentiation of P19 cells using RT-PCR and biolu-
minescent imaging. The unbalanced biogenesis of
mature miR9 and miR9* during neurogenesis was
monitored by real-time PCR and in vitro and in vivo
Luciferase reporter gene systems.
Results
Detection of the endogenous level of the
pri-miR9s
To study miR9 biogenesis during neurogenesis, we
first investigated the primary transcript level of miR9
in P19 cells that had differentiated into neuronal cells
following retinoic acid treatment. Mouse and human
genomes from the UCSC database showed three dif-
ferent loci that can be processed into mature miR9
and ⁄ or miR9*. Three different primary transcripts
of miR9 in mouse are located at chromosome 3

(pri-miR9-1), chromosome 13 (pri-miR9-2), and chro-
mosome 7 (pri-miR9-3; Fig. 1A). The gene-expression
levels of the primary transcripts of miR9 were moni-
tored by sequence-specific RT-PCR analysis using
total RNA from P19 cells induced to differentiate by
retinoic acid. PCR primers were designed by aligning
the sequences of three different pri-miR9s to match
their unique pri-miR9s, but not to amplify alternative
sequences (Fig. 1B). Gene-expression analysis of P19
cells treated with retinoic acid for 6 days showed a
gradual increase in MAP2 transcript levels, a neuro-
nal marker gene, which was expected to occur during
neuronal differentiation (Fig. 1C). The gene expres-
sions of the three different pri-miR9s exhibited vari-
ous transcript patterns during neuronal differentiation
of P19 cells. Pri-miR9-1 showed a dramatic change in
gene expression immediately after treatment with reti-
noic acid, i.e. a gradual increase in primary transcript
level until the third day, followed by a sudden
decrease. By contrast, pri-miR9-3 was relatively highly
expressed even before neuronal differentiation,
increasing gradually during neurogenesis until the
fourth day and then completely disappeared. Unlike
Bioimaging of miR9 and miR9* during neurogenesis M. H. Ko et al.
2606 FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS
the other two pri-miR9s, pri-miR9-2 expression
was barely detectable in undifferentiated and P19 cells
differentiated by retinoic acid. This suggests that the
transcript level of pri-miR9-1 is a good bioindicator
for the neuronal differentiation of P19 cells treated

with retinoic acid.
Bioimaging of the highly expressed pri-miR9-1
during neurogenesis
To monitor the endogenous expression of pri-miR9s
using the bioluminescent Luciferase reporter gene
system during neuronal differentiation in P19 cells,
A
B
C
Fig. 1. Detecting the gene-expression pat-
terns of the three different pri-miR9s during
neurogenesis. (A) Chromosomal locations of
the three different pri-miR9s in mice from
the UCSC database. (B) Primer positions
and sequences used to amplify pri-miR9-1,
9-2 and 9-3. Arrows indicate the position
and orientation of the primers. Bold font
represents the sequence of mature miR9*
and italic fonts the sequence of mature
miR9. (C) RT-PCR analysis of pri-miR9s dur-
ing the neuronal differentiation of P19 cells
treated with retinoic acid (RA). pri-miR9-1
and -9-3 were gradually increased during
neuronal differentiation, whereas the pri-
miR9-2 transcript was barely detected.
MAP2, a neuronal marker gene, showed
that retinoic acid treatment efficiently
induced neuronal differentiation of P19 cells.
B-Actin was used as an internal control.
M. H. Ko et al. Bioimaging of miR9 and miR9* during neurogenesis

FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS 2607
1343 bp of the upstream region of the pri-miR9-1 from
human genomic DNA was cloned and fused into a pro-
moterless reporter vector, pGL3_Basic, which contained
the ORF of the FLuc reporter gene (Fig. 2A). FLuc
activity was measured to determine the promoter activ-
ity of pri-miR9-1 during retinoic acid-induced neuronal
differentiation. The upstream region of the pri-miR9-1
was randomly split into five different segments by PCR,
)1387 to )44 bp (miR9-1PN1_Fluc), )846 to )44 bp
(miR9-1PN2_Fluc), )530 to )44 bp (miR9-1PN3_Fluc),
)236 to )44 bp (miR9-1PN4_Fluc), and )135 to )44 bp
(miR9-1PN5_Fluc; Fig. 2B). These five different con-
structs were then transfected into P19 cells and their pro-
moter activities monitored using an in vitro Luciferase
assay over 2 days following treatment of P19 cells with
retinoic acid (Fig. 2C). Most of the constructs from P19
cells treated or not with retinoic acid had equal or lower
promoter activities than did the pGL3_Basic vector used
as a negative control. However, miR9-1PN3_Fluc
showed a relatively stronger FLuc signal and a higher
expression level after neuronal differentiation than the
other segments, which indicated an increased endoge-
nous level of pri-miR9-1 during neurogenesis. This indi-
cates that negative promoter elements of pri-miR9-1
transcription may be involved in the upstream region
between -846 and -531 bp and that positive elements
may be involved between -530 and -237 bp. These find-
ings indicate that the miR9-1PN3_Fluc construct could
be used for in vivo imaging of gene expression of endoge-

nous pri-miR9 during neurogenesis.
To image in vivo the endogenous expression of the
pri-miR9 in small animals, 2.5 · 10
6
of P19 cells bear-
ing the miR9-1PN3_Fluc construct were subcutane-
ously implanted into mice and region of interest
(ROI) analysis was performed on the basis of the
resultant bioluminescent signals obtained 2 days after
inducing neuronal differentiation with retinoic acid
(Fig. 2D). All the Luciferase signals of the
CMV_Fluc, a positive control, from the right shoul-
der, showed constant and high FLuc expression at 0,
18, 24, and 48 h after retinoic acid treatment. The
negative control, pGL3_Basic, in left shoulders, was
found to show weak or undetectable FLuc expression
throughout the investigation. FLuc intensities of
miR9-1PN3_Fluc (right thighs; normalized versus
CMV_Fluc) showed a gradual increase in the presence
of retinoic acid compared with left thighs which were
not treated with retinoic acid. ROI analysis showed
that miR9-1PN3_Fluc showed an almost fivefold
increase in FLuc activity 1 day after retinoic acid
treatment. The findings of our in vitro and in vivo
Luciferase assays showed that miR9-1PN3_Fluc biolu-
minescence reflects elevated endogenous pri-miR9-1
levels during the neuronal differentiation of P19 cells
treated with retinoic acid.
Mature miR9 was relatively higher expressed
than mature miR9* during neurogenesis

Mature miR9 and miR9*, which may be processed
from the same pre-miR9, are known to be highly
expressed at the same time during neuronal develop-
ment [17]. To quantify their relative expression levels
during neurogenesis, we conducted real-time PCR
using small RNAs extracted from the neuronal differ-
entiation of P19 cells at 0, 1, 2, 3, 4, 5 and 6 days after
treatment with retinoic acid. The amplicons produced
using pairs of specific primers for mature miR9 and
miR9* were quantified and normalized using U6 small
RNA. Endogenous mature miR9 and miR9* were
barely detectable prior to the neuronal differentiation
of P19 cells (Fig. 3A). However, these mature miRs
showed a similar and gradually increased expression
pattern during neuronal differentiation of P19 cells.
Interestingly, mature miR9 was consistently expressed
at a  40% higher level than mature miR9* during
differentiation.
To image the endogenously unequal expressions of
mature miR9 and miR9* during neurogenesis, a GLuc
reporter gene vector was first designed containing the
following components in order: a CMV promoter, an
ORF of GLuc, three copies of perfectly complemen-
tary sequences of mature miR9 (designated as
CMV ⁄ Gluc ⁄ 3xPT_mir9) or miR9* (designated as
CMV ⁄ Gluc ⁄ 3xPT_mir9*) (Fig. 3B). When mature
miR9 or miR9* is present in cells, the GLuc activities
of CMV ⁄ Gluc ⁄ 3xPT_mir9 andCMV ⁄ Gluc ⁄ 3xPT_mir9*
are repressed by cognate mature miR9 and miR9*,
respectively. To demonstrate the specificity of the bio-

luminescent reporter system to monitor both mature
miR9 and miR9*, CMV ⁄ Gluc ⁄ 3xPT_mir9 or CMV ⁄
Gluc ⁄ 3xPT_mir9* with a negative control vector,
CMV_Gluc, were transfected into HeLa cells which do
not express mature miR (Fig. 3C). The CMV_Gluc
construct, which was not repressed by exogenous pre-
miR was used to normalize the GLuc activities of the
CMV ⁄ Gluc ⁄ 3xPT_mir9 and CMV ⁄ Gluc ⁄ 3xPT_mir9*
in HeLa cells treated with various concentrations (0,
2.5, 5, 10, 20 nm) of exogenously derived pre-miR9 or
pre-miR9*. The GLuc expressions of both CMV ⁄
Gluc ⁄ 3xPT_mir9 and CMV ⁄ Gluc ⁄ 3xPT_mir9* showed
a dramatic decrease in response to exogenous
pre-miR9 and pre-miR9*, respectively. The
CMV ⁄ Gluc ⁄ 3xPT_mir23a vector, which has previously
been reported to monitor mature miR23a [19], was
transfected into HeLa cells and found not to change
Bioimaging of miR9 and miR9* during neurogenesis M. H. Ko et al.
2608 FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS
A
B
C
D
Fig. 2. In vitro and in vivo GLuc expression
of pri-miR9-1 during neuronal differentiation.
(A) The chromosomal region of has-pri-
miR9-1. (B) Schematic diagram of the
upstream region of pri-miR9-1 fused to a
promoterless FLuc reporter vector. The pro-
moter sizes of the construct are indicated

by numbers in parentheses. )44 means
located 44 bp upstream of the first base pair
at the 5¢-end of pre-miR-9-1 defined as +1.
FLuc is the ORF of the Firefly Luciferase
reporter gene. (C) In vitro bioluminescent
assay of pri-miR9-1 during neuronal differen-
tiation of P19 cells. Five different upstream
regions of pri-miR9-1 were transfected into
P19 cells, miR9-1PN3_Fluc produced a
strong FLuc signal after neuronal differentia-
tion of P19 cells, pGL3_Basic vector was
used to normalize the FLuc activities
obtained from the five different constructs.
Transfections were performed in triplicate
and results are expressed as mean ± SD.
(D) Bioluminescence image of pri-miR9-1
expression in nude mice. P19 cells
(2.5 · 10
6
) were transiently transfected with
miR9-1PN3_Fluc and injected into nude
mice. In right thighs, neuronal differentiation
was induced by retinoic acid, whereas left
thighs were not treated with retinoic acid.
The pGL3_Basic vector in the right shoul-
ders was used as a negative control and
CMV_Fluc in right shoulders as a positive
control, and were used to normalize FLuc
activities acquired on each day. Biolumines-
cence intensities in right thighs, expressing

pri-miR9-1 were increased during neuronal
differentiation of P19 cells compared with
the left thigh (n = 3 mice ⁄ group). The lower
panel shows ROI analysis of the biolumines-
cence image. Fold ratios of FLuc activities
were normalized versus FLuc intensity on
day 0. Experiments were performed in tripli-
cate and results are expressed as
mean ± SD.
M. H. Ko et al. Bioimaging of miR9 and miR9* during neurogenesis
FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS 2609
GLuc expression significantly after treatment with
exogenous pre-miR9 or pre-miR9*. Both CMV ⁄ Gluc ⁄
3xPT_mir9 and CMV ⁄ Gluc ⁄ 3xPT_mir9* reporter sys-
tems demonstrated a great specificity of monitoring its
cognate mature miR9 and miR9*, respectively.
In vitro bioluminescent Luciferase assays of the
unequally expressed mature miR9 and miR9* during
neurogenesis were conducted in P19 cells treated with
retinoic acid for 4 days. The CMV ⁄ Gluc ⁄ 3xPT_mir9
or CMV ⁄ Gluc ⁄ 3xPT_mir9* construct was transfected
into P19 cells and GLuc activities, representing the
endogenous levels of mature miR9 or miR9*, were
measured and normalized versus CMV_Gluc
(Fig. 3D). As observed for mature miR9 or miR9*
during the neuronal differentiation of P19 cells by
real-time PCR, GLuc expressions of CMV ⁄ Gluc ⁄
3xPT_ mir9 and CMV ⁄ Gluc ⁄ 3xPT_mir9* were both
significantly lower in neuronally differentiated than in
undifferentiated P19 cells and were observed to gradu-

ally decreased during the neuronal differentiation. In
addition, the GLuc signal of CMV ⁄ Gluc ⁄ 3xPT_mir9
was relatively smaller than that of CMV⁄ Gluc ⁄ 3xPT_
mir9* throughout the investigation, which implies a
A
B
C
D
Fig. 3. Gene expressions of mature miR9 and miR9* during neuro-
nal differentiation. (A) Using real-time PCR. The gene expressions
of endogenous mature miR9 and miR9* during the neuronal differ-
entiation of P19 cells treated with retinoic acid for 6 days were
determined. The expressions of mature miR9 and miR9* gradually
increased during neurogenesis, but mature miR9 was found to be
expressed 1.4-fold more than mature miR9*. The line with a trian-
gular head represents the fold ratio of miR9 to miR9* expression
values (right y-axis). The left y-axis shows the real-time PCR intensi-
ties of each mature miRNAs normalized versus U6 snRNA
(C
T
=C
T-before
–C
T-day,
C
T
=C
T-miRNA
–C
T-U6RNA

). Experiments were
performed in triplicate and results are expressed as mean ± SD.
(B) Schematic diagram of the reporter genes used to monitor
mature miR9 and miR9*. The black and gray boxes indicated three
copies of perfectly complementary sequences of mature miR9 and
miR9*, respectively. These three copies were located between the
stop codon and the polyadenylation sequence of the GLuc gene.
These systems were designed to repress GLuc expression when
mature miR9 and miR9* were present. (C) Specification of the
recombination constructs CMV ⁄ Gluc ⁄ 3xPT_mir9 and CMV ⁄ Gluc ⁄
3xPT_mir9* regulated by miR9 and miR9*, respectively. The GLuc
activities of CMV ⁄ Gluc ⁄ 3xPT_mir9, CMV ⁄ Gluc ⁄ 3xPT_mir9*, or
CMV ⁄ Gluc ⁄ 3xPT_mir23a constructs were normalized using
CMV_Gluc vector and determined at five different concentrations
(n
M on the x-axis) of exogenous pre-miR9 or pre-miR9* in HeLa
cells. Experiments were performed in triplicate and results are
shown as means ± SDs. (D) In vitro Luciferase assay of endoge-
nous mature miR9 and miR9* during the neuronal differentiation of
P19 cells. CMV ⁄ Gluc ⁄ 3xPT_mir9 or CMV ⁄ Gluc ⁄ 3xPT_mir9* were
transfected and endogenous levels of mature miR9 and miR9* in
P19 cells treated with retinoic acid were determined. The y-axis
represents the fold ratio of GLuc expression during neuronal differ-
entiation versus GLuc expression from undifferentiated P19 cells;
the white bar represents mature miR9 and the black bar mature
miR9*, and the line with a triangular head indicates the ratio
(numerical values shown on the top of bar) of mature miR9 to
miR9*. Experiments were performed in triplicate and results are
expressed as mean ± SD.
Bioimaging of miR9 and miR9* during neurogenesis M. H. Ko et al.

2610 FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS
higher endogenous level of mature miR9 than of
mature miR9*.
In vivo visualization of unbalanced expressions of
mature miR9 and miR9* during neurogenesis
To monitor in vivo the endogenously unequal expres-
sion of mature miR9 and miR9* during neuronal dif-
ferentiation in P19 cells, CMV ⁄ Gluc ⁄ 3xPT_mir9,
CMV ⁄ Gluc ⁄ 3xPT_mir9* or CMV_Gluc (a negative
control), were transfected into 2.5 · 10
6
of P19 cells
and subcutaneously implanted into nude mice in the
presence or absence of retinoic acid (Fig. 4A). In addi-
tion to in vivo imaging of endogenous mature miR9 or
miR9* during neurogenesis, CMV_Fluc vector, which
expressed constant FLuc activity regardless of the pres-
ence of mature miR9 or miR9* or retinoic acid, was
cotransfected with CMV ⁄ Gluc ⁄ 3xPT_mir9 or CMV ⁄
Gluc ⁄ 3xPT_mir9* into P19 cells as an internal control.
FLuc activities of left thighs not treated with retinoic
acid and of treated right thighs showed no significant
change after CMV ⁄ Gluc ⁄ 3xPT_mir9 or CMV ⁄ Gluc ⁄
3xPT_mir9* transfection (Fig. 4B,C, lower). GLuc
signals, as determined by ROI analysis, from
A
B
C
Fig. 4. In vivo Luciferase imaging of the
mature miR9 and miR9* during the neuronal

differentiation. (A) The strategy used to
implant P19 cells into mice to image mature
miR9 and miR9* during neurogenesis.
CMV_Gluc tranfected into right shoulders
was used to normalize GLuc signals and
CMV_Fluc transfected into left and right
thighs was used as an internal control. (B,C)
In vivo imaging of mature miR9 and miR9*
during the neuronal differentiation of P19
cells treated with retinoic acid. FLuc signals
of CMV_Fluc in the lower panel showed
similar strong expressions in the presence
(right thigh) or absence (left thigh) of retinoic
acid. ROI analysis results in the right panel
and bioluminescence imaging results in the
left upper panel show that the GLuc activi-
ties of CMV ⁄ Gluc ⁄ 3xPT_mir9 in right thighs
(B) and left shoulders (C) were more
repressed during the neuronal differentiation
of P19 cells than CMV ⁄ Gluc ⁄ 3xPT_mir9* in
left shoulders (B) and right thighs (C). ROI
fold ratios were normalized versus GLuc
intensity at 0 h. Experiments were per-
formed in triplicate and results are
expressed as mean ± SD.
M. H. Ko et al. Bioimaging of miR9 and miR9* during neurogenesis
FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS 2611
CMV ⁄ Gluc ⁄ 3xPT_mir9 with retinoic acid in right
thighs were dramatically reduced compared with
CMV ⁄ Gluc ⁄ 3xPT_mir9 without retinoic acid in left

thighs, and had almost disappeared 2 days after neuro-
nal differentiation (Fig. 4B,C, upper). Similarly,
CMV ⁄ Gluc ⁄ 3xPT_mir9* in right thighs showed signifi-
cant GLuc repression during the neuronal differentia-
tion of P19 cells treated with retinoic acid compared
with left thighs not treated with retinoic acid. Fold
ratios of ROI of CMV⁄ Gluc ⁄ 3xPT_mir9 and
CMV ⁄ Gluc ⁄ 3xPT_mir9* on day 1 between pre- and
post differentiation were 6- and 14-fold, respectively.
The bioluminescent signals and ROI analysis in
Fig. 4B,C showed that CMV ⁄ Gluc ⁄ 3xPT_mir9 had
higher repression of the GLuc intensity during neuro-
genesis than CMV ⁄ Gluc ⁄ 3xPT_mir9*, indicating that
mature miR9 are relatively more expressed than
miR9* during neuronal differentiation of P19 cells
treated with retinoic acid.
Discussion
Thousands of miRs proven by the cloning of hundreds
of miRs from various species have been identified by
bioinformatics analysis [22] and tens of miRs have
been reported to be related to specific tissue develop-
ment, cellular differentiation, proliferation, apoptosis,
and various diseases including cancers, cardiovascular
diseases, neurological diseases and metabolic disorders
[4,5]. Even though the regulation and functions of
miRs are unclear, the basic molecular mechanisms of
miR biogenesis in cells have been shown to be pro-
cessed into the primary, precursor, and mature form of
miRs by RNA polymerase II, Drosha, exportin-5,
Dicer and RNA-induced silencing complex [1,11,12].

However, cellular gene-expression analysis of miRs has
been restricted to laborious and irreproducible meth-
ods like in situ hybridization and northern blotting.
Moreover, these methods have been used to detect
only endogenous mature miRs in cells [17,23]. Few
studies have examined miRs to determine initial gene
expression associated with miR biogenesis using the
upstream region of miRs, which is considered a pro-
moter. Using the developed bioluminescent imaging
system to monitor pri-miR9-1 we found that pri-miR9-
1 is highly and specifically expressed in neurons during
the retinoic acid-induced differentiation of P19 cells.
The upstream region of the pri-miR9-1 from )530 to
)44 bp was found to show substantial promoter activ-
ity during neurogeneis, whereas other constructs with
longer or shorter fragments were not found to be effec-
tive enough to monitor differences in endogenous
pri-miR9 expression during the neuronal differentia-
tion of P19 cells. Recently, a number of important
transcription factors, such as repressor element silenc-
ing transcription factor, cAMP response element-bind-
ing protwin, Nanog, and Octamer4 have been
suggested to be involved in the transcriptional regula-
tion of neuronal miRs [24–26]. Unfortunately, the
upstream region of the pri-miR9-1 does not have any
homologue-binding sequence for the transcription
factors that are required to maintain the neuronal
differentiation of stem cells.
Moreover, the molecular mechanisms of the bioge-
neses of a number of miRs are equivocal. miR23a,

which was previously reported upon by our laboratory,
showed unbalanced biogenesis in pri-miR23a and
resultant mature 23a in HEK293 cells, but not in
HeLa cells and P19 cells [19]. Highly expressed pri-
miR23a produced a relatively low endogenous level of
mature miR23a in HEK293 cells, indicating a slow
turnover from pri-miR23a to mature miR23a. In this
study, real-time PCR and in vitro and in vivo biolumi-
nescent imaging demonstrated relatively higher expres-
sion levels of mature miR9 than of miR9* during the
neuronal differentiation of P19 cells treated with reti-
noic acid, even though both mature miRs probably
originated from the hairpin sequences of the same pre-
miR. For strand selection from the secondary structure
of pre-miRs to be a single-stranded mature miR, ther-
modynamic profiling of duplex pre-miR hairpin
showed that in general, the 5¢ terminal sequence of
pre-miR hairpin has less internal stability than the 3¢
terminal sequences of the pre-miRs, which implies that
the mature miRs prefer miRs to miR*s [13,27]. How-
ever, this hypothesis is not applicable to several miRs
cloned from several species. miR18, miR106, miR16
and miR105 have a 5¢-end of precursor form in the
mature form and miR142, miR17, miR302, miR373
and miR9 have both ends in mature forms
[7,16,17,24]. Even though the molecular mechanism of
miR biogenesis is still unclear, interestingly, northern
blotting and microarray analysis using human brain
tissues also demonstrated that miR9 is more highly
expressed than miR9* [6,17]. Our previously reported

dual Luciferase system will provide clearer and simul-
taneous imaging of this phenomenon during miR bio-
genesis [19].
The endogenous expression of mature miR9 has
been recently reported to contribute to the develop-
mental shift from neuron generation to glial cell gener-
ation, and to be related to the expression of
granuphilin ⁄ slp4 in insulin-producing cells [28]. More-
over, miR9 and other neuronal miRs including
miR125b and miR128, are involved in the Alzheimer’
disease [29].
Bioimaging of miR9 and miR9* during neurogenesis M. H. Ko et al.
2612 FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS
Our noninvasive bioluminescent imaging systems
devised to monitor miR9 can also be usefully applied
to study and monitor the biogenesis of other miRs
related to neuronal development, differentiation, and
neuronal diseases. In addition, the in vitro and in vivo
imaging systems will undoubtedly provide information
about the molecular patterns and mechanisms of miR
biogenesis in various heterogeneous cells.
Experimental procedures
Recombinant constructions of reporter gene to
monitor primary and mature form of miR9
To detect the transcript level of pri-miR9, the upstream
region of pri-miR9-1 was isolated from the genomic DNA
of HeLa cells and cloned into a promoterless vector,
pGL3_Basic vector (Promega, Madison, WI) containing the
ORF of FLuc. Five different fragments, which were fused
into the HindIII site of the reporter gene and designated

miR9-1PN1_Fluc, miR9-1PN2_Fluc, miR9-1PN3_Fluc,
miR9-1PN4_Fluc, and miR9-1PN5_Fluc, were amplified
using the primer pairs listed in Table 1, and then sequenced
to determine the orientation of the fragments in the repor-
ter vector. These constructs were then transfected into P19
cells by liposome-mediated transfection using a Lipofectin
reagent kit (Invitrogen, Grand Island, NY, USA) and FLuc
activity was monitored during the neuronal differentiation
of P19 cells treated with retinoic acid (retinoic acid).
To study the mature forms miR9 and miR9*, mature
sequences of has-miR-9 and mature has-miR-9* were
obtained from the MirnaMap database (http://mirna-
map.mbc.nctu.edu.tw) and oligonucleotides containing
three copies of a perfectly complementary sequence of
mature miR9 or miR9* were synthesized (Table 1). Each
pair of sense and antisense oligos was annealed in annealing
buffer (·1 TE buffer + 50 mm NaCl) for 10 min at 60 °C
and ligated into the XhoI and XbaI sites of CMV_Gluc vec-
tor (Targeting Systems, San Diego, CA, USA) to create
CMV ⁄ Gluc ⁄ 3xPT_mir9 and CMV ⁄ Gluc ⁄ 3xPT_mir9*.
Cell culture and neuronal induction of P19 cells
P19 (a mouse embryonic carcinoma cell line) was purchased
from the American Type Culture Collection (Manassa, VA,
USA). P19 cells were grown in a-MEM (Gibco, Grand
Island, NY, USA) supplemented with 2.5% fetal bovine
serum (Cellgro, Herndon, VA, USA), 7.5% bovine calf
serum (Gibco), and 1% antibiotics–antimycotic (Cellgro)
[30]. To induce neuronal differentiation, P19 cells were cul-
tured under serum-free conditions in Dulbecco’s modified
Eagle’s medium ⁄ 12(1 : 1) media (Gibco) supplemented with

insulin, transferring, and selenium (ITS; Gibco) and then
treated with 5 · 10
)7
m all-trans retinoic acid (Sigma, St
Louis, MO, USA) for 3 days. HeLa cells (an adenocarci-
noma cell line) were cultured routinely in RPMI (Jeil Bio-
techservices Inc, Daegu, Korea) containing 10% fetal
bovine serum and 1% antibiotics–antimycotic.
Transfection of CMV
⁄
Gluc
⁄
3xPT_mir9
& CMV
⁄
Gluc
⁄
3xPT_mir9* and precursor
miR9 & miR9*
P19 cells were seeded at 0.6 · 10
5
(undifferentiated),
0.8 · 10
5
(1 day), 0.6 · 10
5
(2 days), 0.4 · 10
5
(3 days),
and 0.2 · 10

5
(4 days) in a six-well plate 24 h prior to
Table 1. Primers used to amplify pri-miR9-1 promoter and clone the perfect target sequence of mature miR9 and miR9*.
Name of primer Sequence (5¢-to3¢) Primer applications
miR9-1PN1 GCA CAA GCT TGG AGT GTG AAA GGA TGA Amplify 1343mer promoter fragment
miR9-1PN2 GCA CAA GCT TCT TTC CTC CCC TCC GCC CCT CTC AT Amplify 802mer promoter fragment
miR9-1PN3 GCA CAA GCT TCA CCG CGG CTC CCC ATT TCC ATC Amplify 486mer promoter fragment
miR9-1PN4 GCA CAA GCT TGG TCA TCG CGT CCT TTC CAC GCC Amplify 192mer promoter fragment
miR9-1PN5 GCA CAA GCT TTC ACC CTC CCC CTC AAC TCC ACT AG Amplify 91mer promoter fragment
miR9-1P reverse GCA CAA GCT TGC CGC CGC CGC CAG CAC CTC Amplify N1–N5 promoter fragments
Perfect target seq.
of miR9 sense
TCGAGAATCTAGT TCA TAC AGC TAG ATA ACC AAA GA
TAGTA TCA TAC AGC TAG ATA ACC AAA GA TAGTA TCA
TAC AGC TAG ATA ACC AAA GAT
Construction of CMV ⁄ Gluc ⁄ 3xPT_mir9
Perfect target seq.
of miR9 anti-sense
CTAGA TCT TTG GTT ATC TAG CTG TAT GA TACTA TCT
TTG GTT ATC TAG CTG TAT GA TACTA TCT TTG GTT ATC
TAG CTG TAT GA ACTAGATTC
Construction of CMV ⁄ Gluc ⁄ 3xPT_mir9
Perfect target seq.
of miR9* sense
TCGAGAATC TAG TAC TTT CGG TTA TCT AGC TTT A TAGTA
ACT TTC GGT TAT CTA GCT TTA TAGTA ACT TTC GGT TAT
CTA GCT TTAT
Construction of CMV ⁄ Gluc ⁄ 3xPT_mir9*
Perfect target seq.
of miR9* anti-sense

CTA GAT AAA GCT AGA TAA TTG AAA GT TACTA TAA AGC
TAG ATA ACC GAA AGT TACTA TAA ACG TAC ATA ACC
GAA AGT ACTAGATTC
Construction of CMV ⁄ Gluc ⁄ 3xPT_mir9*
M. H. Ko et al. Bioimaging of miR9 and miR9* during neurogenesis
FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS 2613
tranfection. Transient transfections were performed using
1 lg of DNA using lipofectamine (Invitrogen). HeLa (a
miR9 non-producing cell line) was seeded at 1 · 10
5
cells to
determine the expressions of miR9 and miR9*.
Luciferase assays for FLuc and GLuc activities
P19 and HeLa cells were washed with NaCl ⁄ P
i
and treated
with lysis buffer (200 lLÆwell
)1
) for Luciferase assays.
Lysed cells were transferred to a 96-well white microplate
and Luciferase activities were measured using luminometer
(TR717; Applied Biosystems, Foster City, CA, USA) and
an exposed time of 1s. All data are presented as
means ± SD calculated from triplicate wells.
RT-PCR analysis in undifferentiated and
differentiated P19 cells
Total RNA was isolated from cultured cells using Tri-
zol reagent (Invitrogen). Reverse transcription to synthesize
first-strand cDNA was carried out using random-hexamer
primer and SuperScript II reverse transcriptase (Invitrogen),

according to the manufacturer’s instructions, and used as a
template for PCR amplification. PCR amplifications of
MAP2 and b-actin cDNA were performed using i-Taq
DNA polymerase (Table 2) (iNtRON; Korea). PCR prod-
ucts were loaded on agarose gels containing ethidium
bromide, and bands were revealed under UV. For pri-
miR9-1, pri-miR9-2 and pri-miR9-3, first-strand cDNA
synthesis was carried out using random-hexamer primer
and promoter primers (Table 2).
Quantitative RT-PCR of mature miR9 and miR9*
Small RNA was isolated from cultured cells using mirVana
miRNA isolation kits (Ambion, Austin, TX, USA), and
qRT-PCR was performed using mirVana
TM
qRT-PCR miR
detection kits (Ambion) using a has-miR-9 or a has-miR-9*
primer set (Ambion) according to the manufacturer’s
instructions. To normalize experimental samples for RNA
content, the U6 snRNA primer set (Ambion) was used as a
control.
In vivo visualization of primary miR9 expression
or mature miR9 and miR9* expressions in
undifferentiated and differentiated P19 cells
The miR9-1PN3_Fluc construct was transfected into P19
cells, which were divided into retinoic acid-treated and non-
retinoic acid treated groups for in vivo imaging. At 48 h
after transfection, 1 · 10
6
P19 cells were harvested with
100 lL NaCl ⁄ P

i
, and resuspended with retinoic acid for the
neuronal differentiation group. P19 cells were then subcuta-
neously injected into each thigh of 6-week-old male Balb ⁄ c
nude mice, 3 mg of d-luciferin was administered intraperi-
toneally [31]. This study was approved by the IACUC
(Institutional Animal Care and Use Committee) of Clinical
Research Institute, Seoul National University Hospital
(AAALAC accredited faculty). Bioluminescence images
were acquired using an IVIS100 (In vivo Imaging System;
Xenogen, Alameda, CA, USA) with the integration time of
5 min. For in vivo GLuc imaging, nude mice were imaged
using the IVIS100 system after direct administering 50 lg
of coelenterazine.
Acknowledgements
This study was supported by Nano Bio Regenomics
Project of Korean Science and Engineering Founda-
tion and by Innovation Cluster for Advanced Medical
Imaging Technology. This study was made easier using
KREONET, Korean Research Network, a nationwide
giga-bps network system.
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Mouse 9-2 lower AGC CTG GCC CCT TTA GAT TTC RT-PCR for expression of pri-miR9-2
Mouse9-3 upper GAG TGC CAC AGA GCC GTC ATA AA RT-PCR for expression of pri-miR9-3
Mouse9-3 lower CTG GGT GGC GTG GGC TTC TCT GG RT-PCR for expression of pri-miR9-3
MAP2 forward CCC AAG AAC CAA CAA GAT GAA RT-PCR for neuronal differentiation
MAP2 reverse AAT CAA GGC AAG ACA TAG CGA RT-PCR for neuronal differentiation
b-actin forward TGA CGG GGT CAC CCA ACT GTG CCC ATC TA Control
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