Báo cáo khoa học: A study of microRNAs in silico and in vivo: bioimaging of microRNA biogenesis and regulation doc

MINIREVIEW
A study of microRNAs in silico and in vivo: bioimaging of
microRNA biogenesis and regulation
Soonhag Kim
1,2,
*, Do W. Hwang
2,3
and Dong S. Lee
2,3,
*
1 Medical Research Center, Seoul National University College of Medicine, Korea
2 Department of Nuclear Medicine, Seoul National University College of Medicine, Korea
3 Programs in Neuroscience, Seoul National University, Korea
MicroRNA (miRNA) has been recognized as a critical
regulatory gene involved in various biological
processes, such as development, cellular proliferation
and differentiation, in mammalian cells. Recently, the
progress in miRNA research has accelerated the pace
of molecular diagnostics and therapeutics for clinical
application [1–3].
To date, the detection and analysis of endogenous
miRNA production has been conducted with micro-
arrays and fluorescence in situ hybridization using opti-
cal probes [4–6]. However, these techniques require the
fixation or lysis of cells and thus cannot be used to
study miRNA production in living cells. A noninvasive
monitoring method capable of real-time image acquisi-
tion is needed to assess the miRNA production pattern
in vivo. Remarkable advances in molecular imaging
techniques have resulted in the ability to not only
provide noninvasive information and repetitive image

Keywords
bioimaging; microRNA; primary RNA;
luciferase
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,
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 25 August 2008, revised 8
December 2008, accepted 21 January 2009)
doi:10.1111/j.1742-4658.2009.06935.x
Many recent studies have reported that microRNA (miRNA) biogenesis
and function are related to the molecular mechanisms of various clinical
diseases. Several methods, including northern blotting and DNA chip anal-
yses, are capable of assessing miRNA-production patterns in cells. How-
ever, the development of repetitive monitoring of the miRNA-production
profile in a noninvasive manner is demanded for the application of

miRNAs to human medicine. Here, we describe a noninvasive system for
monitoring miRNA biogenesis, from the stage of primary transcripts to
that of mature miRNA regulation. We review the optical methods that
have been developed to image miRNA production at each step of the
miRNA-processing pathway in living subjects. We propose that an optical
miRNA-imaging strategy, based on molecular imaging, can be used as
an miRNA imaging detector to monitor various miRNAs, by using differ-
ent reporters, simultaneously, for high-throughput screening, and will
provide potential application for the diagnosis and therapeutics of multiple
diseases.
Abbreviations
CMV, cytomegalovirus; DGCR8, DiGeorge syndrome critical region gene 8; miR, microRNA; pre-miR, precursor microRNA; pri-miR, primary
microRNA.
FEBS Journal 276 (2009) 2165–2174 ª 2009 The Authors Journal compilation ª 2009 FEBS 2165
aquisition, but also to carry out imaging over an
extended period of time without having to kill experi-
mental subjects. Several reporter-based imaging
probes, including luciferase for optical imaging and
sodium iodide symporter and herpes simplex virus
1-thymidine kinase for radionuclide imaging, have
been widely used to track the distribution of implanted
stem cells and to evaluate endogenous gene expression
[7–10]. The luciferase optical reporter genes, which
include firefly, Renilla and Gaussia luciferases, have
been widely used to visualize bioluminescence signals
in living animals. While the firefly luciferase generates
bioluminescence energy (emission wavelength: 480 nm)
by catalyzing the oxidation of d-luciferin to oxylucifer-
in, Gaussia luciferase catalyzes the oxidation of its
substrate, coelenterazine, to produce bioluminescence

light (emission wavelength: 560 nm).
Although newly identified miRNAs in mammalian
cells have been intensively studied to establish their
role in human disease, including cancers, there has
been limited research on miRNA imaging. Therefore,
active investigation of the production pattern and
functional action of miRNAs is needed. The several
available imaging strategies used to detect endogenous
miRNA production can be used to monitor both the
primary transcript and the mature form of miRNA
(Fig. 1) [11–13].
As miRNAs are essential in all biological areas, a
noninvasive technique for monitoring miRNA biogene-
sis would help to eluciate the versatile functions and
production patterns of miRNAs relative to genetic
modulation, cell development and multiple diseases,
in vivo. In addition, the bioimaging techniques for
miRNAs based on molecular imaging methods could
be applied as target imaging indicators to help under-
stand the developmental process, in the development
of cancer biomarkers and to evaluate the therapeutic
effects, in terms of cancer therapy, in medicine. Here,
we review the currently available miRNA imaging sys-
tems that are used to create a better understanding of
the production and function of miRNA in vivo as well
as for monitoring the therapeutic potential of miRNAs
in cancer.
Bioimaging of microRNA biogenesis
The molecular mechanisms involved in miRNA gener-
ation are complex, and at least several processing steps

in the nucleus and cytoplasm should be monitored, by
imaging, as follows (Fig. 1A): (a) imaging of a primary
miRNA (pri-miRNA) that is transcribed from the gen-
ome by RNA polymerase II in the nucleus (Fig. 1B),
(b) imaging the miRNA precursor (pre-miRNA) that
is cleaved from pri-miRNA by Drosha and DiGeorge
syndrome critical region gene 8 (DGCR8) (Fig. 1C),
(c) imaging a partially double-stranded miRNA
complex (miRNA–miRNA*) that is released from the
pre-miRNA by Dicer (Fig. 1D), (d) imaging a single-
stranded mature miRNA (Fig. 1E) and (e) imaging
miRNA function that either destabilizes mRNA or
inhibits the translation of target genes by binding to
target genes (Fig. 1F).
First, imaging of the generation of pri-miRNA
revealed that the 5¢ upstream region of genomic
miRNA controls the long primary transcripts of
miRNA that shape a single or a large family of
miRNA gene clusters. Interestingly, several miRNAs,
including miRNA9 and miRNA124, are located at
multiple loci, each of which can produce pri-miRNA,
pre-miRNA and mature miRNA [12]; this implies that
the 5¢ upstream region from a few different loci should
be investigated concurrently to provide an accurate
reflection of the generation of a pri-miRNA. Like
other eukaryotic mRNAs, the primary transcript of
miRNA is controlled by RNA polymerase II, and sev-
eral transcription factors, including Oct4, c-Myc and
Nanog, regulate the expression of primary miRNAs by
binding them to the 5¢ terminal regulatory region of

the miRNAs that participate in critical molecular
and ⁄ or cellular processes during the developmental
stage [14–16]. Similarly to the reporter gene assay of
the eukaryotic promoter, the 5¢ upstream region of a
miRNA that proportionally reflects the endogenous
expression level of a pri-miRNA can be fused into the
cassette of a promoterless optical reporter gene vector
(Fig. 1B). The cloned miRNA-specific reporter-imaging
vector can be transfected into cultured cells, and the
cell lines can be collected and implanted (e.g. into the
thighs of a mouse). The expression level of the pri-
miRNA transcript can be obtained from living animals
by imaging the in vivo bioluminescence signals.
The optical reporter gene system enables pri-miRNA
generation to be monitored in vivo. Lee et al. [11]
used the miRNA23a promoter to acquire images
that showed differences in the endogenous expression
of pri-miRNA23a in HeLa, 293 and P19 cells. Ko
et al. [12] also monitored the neuronal-specific
pri-miRNA9 during neurogenesis by using its upstream
region.
Second, to acquire the images of a pre-miRNA that
was cleaved from pri-miRNA by Drosha and DGCR8,
Lee et al. [11] designed sense and antisense oligonucle-
otides of the pri-miRNA23a that were annealed and
cloned between the cytomegalovirus (CMV) promoter
and the start codon of the Gaussia luciferase gene
of the optical reporter gene vector (Fig. 1C). The
Bioimaging of miRNA biogenesis and regulation S. Kim et al.
2166 FEBS Journal 276 (2009) 2165–2174 ª 2009 The Authors Journal compilation ª 2009 FEBS

Fig. 1. Schematic illustration of detection systems for imaging miRNA biogenesis and regulation. (A) Steps of miRNA processing. Precursor
miRNA is generated from longer primary transcripts by reaction with Drosha RNase III and the duplex miRNA form is produced by Dicer ribo-
nuclease enzyme in the cytoplasm, followed by the generation of mature miRNA after interaction with RNA-induced silencing complex
(RISC). (B) Design for imaging pri-miRNAs. The 5¢-regulatory upstream sequence of pri-miRNAs can be identified by the general database
program (UCSC) and be split into different size segments of the chosen upstream region of miRNAs for acquisition of a strong optical signal
[12]. The upstream region of pri-miRNAs can be fused into an imaging reporter gene to examine pri-miRNA expression [11–13]. The miRNA
promoter-restricting reporter gene is transfected into several cell lines, and the harvested cells are implanted into a mouse. The optical biolu-
minescence image can be acquired in a time-dependent manner. (C) Imaging strategy for pre-miRNAs. The generation of pre-miRNAs can
be detected by the signal activity of the reporter gene when it is cleaved by the Drosha enzyme [11]. (D) Molecular beacon for imaging a
partially double-stranded miRNA complex (miRNA–miRNA*), which is released from pre-miRNA by Dicer. The synthetic duplex form of pre-
miRNAs carrying quencher and organic dye at each strand can be designed. In the presence of the Dicer enzyme, the quencher molecule
and fluorophore dye are separated from each other after interaction of Dicer at the Dicer recognition site present on the pre-miRNAs, and
the fluorescent signals are thereby released [19]. (E) Schematic strategy for the reporter gene imaging of mature miRNAs. Perfectly matched
complementary sequence of mature miRNAs can be designed and cloned downstream of the reporter system under the control of the cyto-
megalovirus (CMV) promoter [11,20]. In the presence of the mature miRNA, bioluminescent signals are reduced by the miRNA function,
mRNA destabilization. (F) Reporter-gene frame imaging of miRNA targets. The 3¢-UTR of an miRNA target containing the seed region of
miRNA can be isolated and transferred into a downstream region of the reporter gene that is regulated by a constitutive promoter such as
CMV. When the imaging reporter gene of the miRNA target interacts with mature miRNAs in the cells, the activity of the reporter gene is
turned off [24–28].
S. Kim et al. Bioimaging of miRNA biogenesis and regulation
FEBS Journal 276 (2009) 2165–2174 ª 2009 The Authors Journal compilation ª 2009 FEBS 2167
pre-miRNA imaging system showed an increase in
luciferase activity when the pri-miRNA23a was cleaved
by Drosha and DGCR8.
Third, to monitor a partial double-stranded miRNA
complex, the functional imaging of Dicer is critical
(Dicer is the ribonuclease III enzyme that plays an
essential role in the production of mature miRNA by
cleaving the pre-miRNA). Several reports have sup-
ported the importance of Dicer, showing that deletion

of the Dicer enzyme causes phenotypic defects during
development, which results from the generation of
abnormal miRNA maturation [17,18]. One study
reported assaying the cleavage of pre-miRNA by the
Dicer enzyme using quencher-based pre-miRNA to
detect endogenous or exogenous Dicer enzyme; the
results showed intense fluorescence signals with dis-
placement of the fluorescence dye from the quenching
molecule by the Dicer enzyme, which cleaved the syn-
thetic let-7 precursor miRNA (Fig. 1D) [19]. In the
absence of the Dicer protein, the emission energy of
the fluorescence dye attached to the end of one strand
of the pre-miRNA (as shown in Fig. 1D) is absorbed
by the quenching molecule, showing the quenched flu-
orescence signal. By contrast, with Dicer present there
is cleavage of the end of the pre-miRNA, and acti-
vated fluorescence signals are observed, which implies
that the fluorescence beacon system is useful for the
detection of the functional action of Dicer in the intra-
cellular space (Fig. 1D).
Fourth, imaging of mature miRNA has been per-
formed with the optical reporter gene system, using
green fluorescent protein and luciferase, which has
enabled the endogenous production pattern of mature
miRNA to be monitored [11–13,20]. This imaging
method focuses on the concept that the binding or
function of the mature miRNAs is based on nucleotide
sequence homology. A perfectly matched complemen-
tary sequence of mature miRNA was cloned immedi-
ately after the stop codon of a reporter gene (Fig. 1E).

The reporter gene activities were decreased by the
mRNA destabilization of the reporter gene following
interaction of the imaging system with the mature
miRNA in cells (Fig. 1E).
Giraldez et al. presented a good method for evaluat-
ing the existence of mature miRNAs using a single
optical reporter imaging vector containing perfect
target sequences of miRNAs. The green fluorescent
protein reporter imaging vector system was used to
examine the production pattern of mature miRNAs by
generation of Dicer mutants in zebrafish, elucidating
the vital role of miRNAs associated in morphogenesis
[20]. However, this method was used only for the
detection of mature miRNAs.
Generally, miRNAs are produced via complex pro-
cesses controlled by a variety of proteins, including
Drosha and Dicer. The production of mature miRNA
is dependent on the functional action of many pro-
teins that control the generation of mature miRNAs.
Tomson et al. [21] reported that several miRNAs,
including let-7, showed an unbalanced pattern of pro-
duction during biogenesis, of a large amount of pri-
miRNA and a low level of mature miRNA, without
being processed by the RNase III enzyme Drosha,
and that no correlation between the production of
pri-miRNAs and mature miRNAswas observed in
cancer samples, compared with the correlation in nor-
mal cells. This information indicates that the matura-
tion process is controlled by an unknown regulatory
factor, and thus different levels of primary transcript

and mature miRNA were shown to occur simulta-
neously in living cells. Therefore, the simultaneous
monitoring of production of both pri-miRNA and
mature miRNA is required using different optical
reporter genes.
The bioluminescent imaging proteins, firefly lucifer-
ase and Gaussia luciferase, have their own sub-
strates, d-luciferin and coelenterazaine, respectively,
with their unique peak light emissions, at 480 and
560 nm, respectively. These proteins can thus be used
to image two different molecular actions simulta-
neously. Lee et al. [11] reported that the two differ-
ent reporter gene systems (firefly luciferase and
Gaussia luciferase, described above) could simulta-
neously image primary and mature forms of
miRNA23a in cancer cells (Fig. 2). The in vivo bio-
luminescence signals from pri-miR23a and mature
miR23a were observed in cancer-grafted nude mice.
Firefly luciferase reporters revealed the primary tran-
script activity of miRNA23 using the miRNA23
promoter (miR23P639 ⁄ Fluc)-controlled firefly lucifer-
ase reporter system. Conversely, the production of
mature miR23a was detected using the Gaussia lucif-
erase reporter system, which contains three copies of
the miR23a-binding sequence (CMV ⁄ Gaussia lucifer-
ase ⁄ 3 · PT_miR23a). Interestingly, in this report,
unlike the results on bioluminescence studies carried
out on HeLa and P19 cells that showed increased
firefly luciferase signals for pri-miRNA23a and the
resultant highly reduced Gaussia luciferase activities

for mature miRNA23a, 293 cells showed a slow
turnover from pri-miR23a to mature miRNA23a by
unknown mechanisms, implying the capability of the
reporter gene system to assess and visualize the post-
transcriptional regulation of miRNA [11]. Therefore,
a variety of imaging strategies, showing each step in
the generation of miRNAs, will provide critical
Bioimaging of miRNA biogenesis and regulation S. Kim et al.
2168 FEBS Journal 276 (2009) 2165–2174 ª 2009 The Authors Journal compilation ª 2009 FEBS
information for understanding the miRNA biogenesis
related to human diseases.
Imaging miRNA targets
While hundreds of miRNAs are known, identification
of their target genes has been a considerable challenge.
The results of bioinformatics and microarray experi-
ments have shown that miRNAs can target hundreds
of transcripts, both directly and indirectly [13,22]. Gen-
eral methodologies by which the functional regulation
of endogenous miRNA is investigated utilize a bioin-
formatically analyzed seed region that contains seven
nucleotides of a miRNA response sequence in the
3¢-UTR of target genes [23]. This 3¢-UTR-based lucif-
erase system, containing the seed region, has been
widely used for target identification and for elucidating
the functional relationship between miRNA and its
targets (Fig. 1F) [13,24–28]. Computer database
programs, such as the PicTar database, offer an easy
approach with which to identify the 3¢-UTR of the
miRNA target candidates by scoring the dependent
target prediction. Many studies have reported a variety

of target genes that were significantly down-regulated
by specific miRNAs. These targets were determined
in vitro using a reporter gene system containing the
3¢-UTR of the targets located immediately after the
stop codon of the luciferase reporter gene [13,29]. From
this reporter system, partial interaction of miRNAs
with a 3¢-UTR region in reporter gene constructs
resulted in reduction of the reporter signal in cells.
Using the 3¢-UTR-based Renilla luciferase reporter
gene system, Yan et al. [22] obtained quantitative
in vitro images of nine transcripts that were predicted
to be miRNA9 targets using the ensemble machine-
learning algorithm. However, in vivo imaging of
miRNA regulation has not been reported, with the
exception of a report by Kim et al. [13], in which
homeobox B5 (which was verified by microarray exper-
iments) was identified to be one of the endogenous
targets of miRNA221 that is highly expressed in
papillary thyroid carcinoma. The in vivo biolumines-
cent signals from the 3¢-UTR region of homeobox B5
containing the miRNA221 seed region fused into the
Gaussia reporter gene were significantly decreased by
mature miRNA221 during the development of
Fig. 2. Noninvasive bioimaging of miRNA23 biogenesis to examine the production of both the pri-miR23a transcript and the mature miR23a
in nude mice. MiR23P639 ⁄ Fluc (firefly luciferase reporter system regulated by miR23a promoter) and CMV ⁄ Gluc ⁄ 3 · PT_miR23a (Gaussia
luciferase reporter system containing three copies of an miR23a-binding sequence that is completely complementary to mature miR23a)
were cotransfected with 1 · 10
7
HeLa, 293 and P19 cells using the lipofection method (implantation into the right thigh of nude mice).
pGL3 ⁄ basic (promoterless-based firefly luciferase reporter system)-transfected and cytomegalovirus ⁄ Gluc (Gaussia luciferase vector driven

by the cytomegalovirus promoter)-transfected cell lines were also implanted into the left thigh of mice as controls. The bioluminescence
activity of three cell lines transfected with the firefly luciferase vector, controlled by the miRNA23 promoter, was greater than for cells trans-
fected with promoterless vector controls and represents the degree of miRNA23 production in HeLa cells (A, upper), 293 cells (B, upper)
and P19 embryonic carcinoma cells (C, upper). Simultaneous monitoring of the bioluminescence signals in the same mouse was detected
using the Gaussia luciferase reporter system. Production of in vivo mature miRNA23 was detected in three cell lines using the Gaussia lucif-
erase reporter vector containing three tandem repeat regions of miRNA23 binding sequence (reprinted with permission [11]).
S. Kim et al. Bioimaging of miRNA biogenesis and regulation
FEBS Journal 276 (2009) 2165–2174 ª 2009 The Authors Journal compilation ª 2009 FEBS 2169
papillary thyroid carcinoma [13]. These results were
obtained from directly monitoring the cells using the
3¢-UTR-based Gaussia luciferase reporter gene system.
One limitation of the 3¢-UTR-based reporter gene sys-
tem for imaging endogenous targets of miRNA was
focused on the targets containing the seed region,
which resulted in exclusion of targets regulated by
other miRNA mechanisms, such as genes regulated by
the translational inhibition of miRNA.
Unlike reporter imaging systems based on perfect
target sequences, as shown in Fig 1E, the 3¢-UTR-
based reporter system can be used to elucidate the real
function of miRNAs (Fig. 1F). In general, the func-
tional actions of miRNAs in mammalian cells, unlike
plant cells, are associated with the translational inhibi-
tion of target mRNAs by binding miRNA to the seed
region of the target mRNA with noncomplementarity
between the miRNA and its target. Therefore, the
3¢-UTR-based imaging reporter system reflects the real
action of mature miRNA in mammalian cells, com-
pared with reporter-imaging systems that use perfect
target sequences from miRNAs.

In vivo imaging of miRNA in cancers
and neuronal development
miRNAs have been associated with developmental
processes in many types of cells, including embryonic,
neuronal, muscular and lymphatic cells. Abnormal cel-
lular development, as occurs with cancer, has also been
associated with miRNAs. Indeed, a wide range of
miRNAs, including miRNA221, 21 and 142, are
involved in multiple oncogene targets in cancer cells,
which are thought to originate from two main basic
mechanisms, that is, functioning as either a tumor sup-
pressor gene or as an oncogenic gene [13,30–32]. First,
the widespread down-regulated form of miRNA that
regulates multiple oncogenes causes tumorigenesis,
resulting from abnormalities occurring during each of
the steps of miRNA biogenesis. Second, the oncogenic
characteristics of the miRNAs regulate the progression
of cancer by preventing the tumor suppressor genes
from producing tumor suppressor protein [33]. These
contrasting roles of cancer-related miRNAs have been
considered for the development of therapeutic tools to
be used against cancer cells. Recent evidence has indi-
cated that the use of anti-miRNA296 showed a signifi-
cant therapeutic effect by targeting miRNA296, which
is highly expressed in endothelial tumor cells [34]. The
therapeutic effect of anti-miRNA296 for reduction of
tumor angiogenesis was evaluated in tumor-xenograft-
ed mice by measuring the decrease of optical signal
in vivo [34]. Also, miRNA21 is a potential therapeutic
target for cancer treatment, as overexpression of anti-

miRNA21 in hepatocellular carcinoma and glioblas-
toma cells was shown to down-regulate the oncogenic
miRNA21 [35]. Based on the use of anti-miRNA21 as
the therapeutic agent, the therapeutic effects on the
tumor using anti-miRNA21, which is an inhibitor of
miRNA21, have been reported for the first time; these
findings indicated that the real-time tumor regression
could be clearly visualized in luciferase-expressing
tumor-bearing mice [36]. Corsten et al. [36] showed sig-
nificant cytoreduction effects in tumors using a dual
therapeutic method with locked nucleic acid-modified
anti-miRNA21 and neural precursor cells expressing
Secretable form of tumor necrosis factor-related apop-
tosis inducing ligand (S-TRAIL) (Fig. 3). However,
this study used in vitro transfection of anti-miRNA21
for in vivo cancer therapy, indicating that the in vivo
delivery of miRNAs or anti-miRNAs remains to be
demonstrated. Recently, our group developed an
in vivo system for monitoring miRNA221, which is
known to be overexpressed in papillary thyroid carci-
noma cells. Our system can be used to image the
production of miRNA221 in thyroid cancers. With
chemically modified anti-miRNA221 it can also be
used to examine therapeutic effects [13].
Previous studies have suggested that MYC and RAS
are putative target genes for the let-7 miRNA family
in lung cancer [37,38], which can show potential effects
on the treatment of cancer using let-7 as the therapeu-
tic agent. Therefore, the therapeutic imaging approach
using anti-oncogenic miRNAs, such as let-7, should be

further explored to validate its therapeutic efficacy. In
this manner, noninvasive cancer-associated miRNA-
imaging systems can be developed to monitor the
therapeutic effects, not only by blocking the oncogenic
miRNA participating in suppressing the tumor-
suppressor genes, but also by the over production of
miRNAs that can repress several oncogenes.
miRNAs play an important role in determining the
specification and fate of cells by regulating a number
of genes responsible for governing cellular develop-
ment. The tissue-specific or cell-specific distribution of
miRNAs might be a good target for monitoring the
tissue-restricted production of miRNAs [30]. It has
been shown that some miRNAs, including miR-
NA124a, 9, 9*, 124, 128 and 132, are primarily
enriched in brain tissue, where they are implicated in
neuronal development. Indeed, the production of these
miRNAs increased as neuronal lineages progressed
[39–41]. Ko et al. [12] reported that in vivo imaging of
miRNA9 and miRNA9* (the opposite sequence of
miRNA9), generated from the same pre-miRNA9,
showed distinctive and unbalanced patterns of expres-
Bioimaging of miRNA biogenesis and regulation S. Kim et al.
2170 FEBS Journal 276 (2009) 2165–2174 ª 2009 The Authors Journal compilation ª 2009 FEBS
sion during neurogenesis. This illustrates that the
imaging of neuron-specific miRNA9 and miRNA9*
can be important for understanding the functional role
of miRNA during neurogenesis, and for investigating
differences between miRNA and miRNA*. Generally,
antisense miRNAs (miRNA*), namely complementary

sequences of sense miRNAs that participate in the real
functions of miRNAs, are known for their low produc-
tion and fast degradation of miRNA* by endonucleases,
compared with sense miRNAs [42]. However, for
several miRNAs, including miRNA9, it has been
shown that both sense and antisense strands can
regulate target mRNA [39,43]. A noninvasive system
for imaging the production of neuron-specific miRNA9
and miRNA9* would facilitate a better understanding
of the unique molecular mechanisms associated with
the biogenesis of miRNA during neuronal develop-
ment in living subjects.
In this context, noninvasive bioimaging systems using
neuron-specific miRNAs could be applied not only to
monitor the functional roles and production profiles of
neuron-related miRNAs, but also as imaging tools to
track neuronal differentiation imaging based on mole-
cular imaging techniques. The development of imaging
strategies related to miRNAs will continue to be
applied as imaging tools for detecting the progression
of neuronal development, as well as for cancer diagnos-
tics and therapeutic application in human medicine.
Conclusion
Recently, the increased interest in miRNAs, and con-
cerns that miRNAs are known to play important roles
in clinical diseases, have attracted many molecular
researchers to study miRNAs associated with the bio-
genesis and functional role of miRNAs. To study a
variety of biological phenomena related to miRNAs,
noninvasive miRNA imaging techniques have been

developed to track the generation and function of
miRNAs by monitoring their targets regulating cellular
and molecular events, as well as the expression levels
of pri-miRNA and mature miRNA. To visualize the
generation of miRNAs using reporter imaging systems,
optical imaging strategies using the promoter region of
the miRNAs or miRNA responsive elements provides
non-invasive molecular imaging information regarding
how and when they are transcribed by transcription
factors and where they originate, in the case of a
miRNA located on multiple chromosomal loci. In
addition, these techniques show the extent to which
mature miRNAs are formed during cellular develop-
ment. Optical reporter genes, such as firefly or Gaussia
luciferase genes, are well suited for monitoring miRNA
biogenesis in small animals because they have the
advantages of low background noise and high sensitiv-
ity. However, these optical systems remain a distant
goal for clinical application because of the optical
signal attenuation by a tissue depth. Therefore, further
investigation of miRNAs and development of target
detection methods based on radionuclide imaging
modalities, such as the sodium iodide symporter and
Fig. 3. Bioluminescence imaging system created to evaluate the
therapeutic potential of anti-miRNA21. (A, B) After firefly luciferase-
expressing U87 glioblastoma cells were treated with locked nucleic
acid anti-miRNA21, the U87 cells collected were intracranially
injected into the brains of mice. The bioluminescence signals from
implanted U87 cells expressing luciferase dramatically reduced with
time, showing a therapeutic effect of anti-miRNA. This effect was

supported by the results of quantitative region of interest analysis
(reprinted with permission [36]).
S. Kim et al. Bioimaging of miRNA biogenesis and regulation
FEBS Journal 276 (2009) 2165–2174 ª 2009 The Authors Journal compilation ª 2009 FEBS 2171
the MR imaging reporter gene (such as the transferrin
receptor) are required to further the advancement
towards application to human medicine [44]. However,
the optical signals from the mature miRNA-based
reporter gene are basically a ‘turn-off’ system as a
result of the function of the miRNAs; this might intro-
duce ambiguity with regard to the interpretation of
whether the observed signaling-off data results from
the production of numerous mature miRNAs or only
from cell death.
An alternative strategy for imaging mature miRNAs
that overcomes the limitations of the signaling-off prob-
lem in the reporter gene system is a tunable fluorescence
system that uses a quenching molecule-based molecular
beacon. The molecular beacon is a fluorescence detec-
tion system composed of a single-stranded and stem–
loop nucleotide that is complementary to the target
DNA sequence or structurally bound to the protein of
the target. This has been exploited as a powerful imag-
ing probe for the detection of intracellular targets
[45,46]. The molecular beacon approach can be applied
to the investigation of endogenous miRNA production
profiles as a result of miRNAs being bound to their
targets based on sequence homology. The signal-on
imaging strategy, using the molecular beacon to image
mature miRNAs, is as follows. In the absence of mature

miRNA molecules, the fluorescence energy from the
fluorophore dye is absorbed by quencher molecules,
resulting in no fluorescence signal. By contrast, by
displacing the quencher from the organic fluorophore
dye, the presence of mature miRNA causes the mole-
cular beacon light-on result. The molecular beacon
approach used for the detection of miRNAs will pro-
vide more accurate imaging information about miRNA
biogenesis in cellular developments and disease.
In summary, molecular imaging techniques can be
used to monitor the in vivo dynamics of miRNAs non-
invasively, including their production profiles and regu-
lation. First, the miRNA-based optical system, as
proposed in several previous studies, can be used for
the in vitro and in vivo investigations of miRNA
production patterns. Second, noninvasive monitoring
of miRNAs provides useful information of the possible
role of miRNAs involving cellular developments.
Third, the in vivo molecular reporter system might be
used to monitor repetitively, with precise measurement,
the therapeutic effect using miRNAs relevant to cancer
in living animals. A study of the expression patterns of
miRNA, providing fine tuning of gene regulation, will
help to understand the interplay of complex gene regu-
latory networks during developmental stages and the
changes of these networks in disease and after applica-
tion of a variety of therapeutic strategies. Critically,
the noninvasive imaging approach of miRNA genera-
tion and its activity will improve our ability to diag-
nose and treat human disease.

Acknowledgements
This work was supported by the Nano Bio Rege-
nomics Project and by the Nuclear R&D program
through the Korea Science and Engineering Founda-
tion funded by the Ministry of Education, Science and
Technology (M20704000039-08M0400-03910) and by
the Seoul R & BD program (10550) and National
R&D Program for Cancer Control of the Ministry of
Health & Welfare (0820320).
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