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
Trang 1and microRNA9* during the neuronal differentiation of
P19 cells
Mee Hyang Ko1,2,3, Soonhag Kim2,4,*, Do Won Hwang1,2,3, Hae Young Ko2,3, Young Ha Kim2,3and Dong Soo Lee2,4,*
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: kimsoonhag@empal.com
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: dsl@plaza.snu.ac.kr
*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.
Trang 2miR125b, 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 transladegrada-tional inhibidegrada-tion [2,14,15] Interestingly,
unlike most miRs, which have a single mature miR,
miR cloning and sequencing from the human and
miRNAMap database (http://mirnamap.mbc.nctu
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
Trang 3the 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.
Trang 41343 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· 106 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
Trang 5B
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.
Trang 6GLuc 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
(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.
Trang 7higher 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· 106 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.
Trang 8CMV⁄ 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 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
[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]
Trang 9Our 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)7m 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· 105 (undifferentiated), 0.8· 105
(1 day), 0.6· 105
(2 days), 0.4· 105
(3 days), and 0.2· 105
(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*.
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*
Trang 10tranfection Transient transfections were performed using
1 lg of DNA using lipofectamine (Invitrogen) HeLa (a
miR9 non-producing cell line) was seeded at 1· 105
cells to determine the expressions of miR9 and miR9*
Luciferase assays for FLuc and GLuc activities
P19 and HeLa cells were washed with NaCl⁄ Piand 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 mirVanaTMqRT-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· 106 P19 cells were harvested with
100 lL NaCl⁄ Pi, 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 InnovaFounda-tion Cluster for Advanced Medical Imaging Technology This study was made easier using KREONET, Korean Research Network, a nationwide giga-bps network system
References
1 Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR & Ruvkun G (2000) The 21-nucleotide let-7 RNA regulates
Table 2 Primers used in real-time polymerase chain reaction (RT-PCR).