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MINIREVIEWA study of microRNAs in silico and in vivo: bioimaging ofmicroRNA biogenesis and regulationSoonhag Kim1,2,*, Do W. Hwang2,3and Dong S. Lee2,3,*1 Medical Research Center, Seoul National University College of Medicine, Korea2 Department of Nuclear Medicine, Seoul National University College of Medicine, Korea3 Programs in Neuroscience, Seoul National University, KoreaMicroRNA (miRNA) has been recognized as a criticalregulatory gene involved in various biologicalprocesses, such as development, cellular proliferationand differentiation, in mammalian cells. Recently, theprogress in miRNA research has accelerated the paceof molecular diagnostics and therapeutics for clinicalapplication [1–3].To date, the detection and analysis of endogenousmiRNA production has been conducted with micro-arrays and ﬂuorescence in situ hybridization using opti-cal probes [4–6]. However, these techniques require theﬁxation or lysis of cells and thus cannot be used tostudy miRNA production in living cells. A noninvasivemonitoring method capable of real-time image acquisi-tion is needed to assess the miRNA production patternin vivo. Remarkable advances in molecular imagingtechniques have resulted in the ability to not onlyprovide noninvasive information and repetitive imageKeywordsbioimaging; microRNA; primary RNA;luciferaseCorrespondenceS. Kim, Department of Nuclear Medicine,Medical Research Center, Seoul NationalUniversity College of Medicine, 28Yongon-dong, Jongno-gu, Seoul 110 744,KoreaFax: +82 (2) 3668 7090Tel: +82 (2) 3668 7028E-mail: email@example.comD. S. Lee, Department of Nuclear Medicine,Seoul National University College ofMedicine, 28 Yongon-dong, Jongno-gu,Seoul 110 744, KoreaFax: +82 (2) 3668 7090Tel: 82 (2) 2072 2501E-mail: firstname.lastname@example.org*These authors contributed equally to thiswork(Received 25 August 2008, revised 8December 2008, accepted 21 January 2009)doi:10.1111/j.1742-4658.2009.06935.xMany recent studies have reported that microRNA (miRNA) biogenesisand function are related to the molecular mechanisms of various clinicaldiseases. 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-productionproﬁle in a noninvasive manner is demanded for the application ofmiRNAs to human medicine. Here, we describe a noninvasive system formonitoring miRNA biogenesis, from the stage of primary transcripts tothat of mature miRNA regulation. We review the optical methods thathave been developed to image miRNA production at each step of themiRNA-processing pathway in living subjects. We propose that an opticalmiRNA-imaging strategy, based on molecular imaging, can be used asan miRNA imaging detector to monitor various miRNAs, by using differ-ent reporters, simultaneously, for high-throughput screening, and willprovide potential application for the diagnosis and therapeutics of multiplediseases.AbbreviationsCMV, cytomegalovirus; DGCR8, DiGeorge syndrome critical region gene 8; miR, microRNA; pre-miR, precursor microRNA; pri-miR, primarymicroRNA.FEBS Journal 276 (2009) 2165–2174 ª 2009 The Authors Journal compilation ª 2009 FEBS 2165aquisition, but also to carry out imaging over anextended period of time without having to kill experi-mental subjects. Several reporter-based imagingprobes, including luciferase for optical imaging andsodium iodide symporter and herpes simplex virus1-thymidine kinase for radionuclide imaging, havebeen widely used to track the distribution of implantedstem cells and to evaluate endogenous gene expression[7–10]. The luciferase optical reporter genes, whichinclude ﬁreﬂy, Renilla and Gaussia luciferases, havebeen widely used to visualize bioluminescence signalsin living animals. While the ﬁreﬂy luciferase generatesbioluminescence energy (emission wavelength: 480 nm)by catalyzing the oxidation of d-luciferin to oxylucifer-in, Gaussia luciferase catalyzes the oxidation of itssubstrate, coelenterazine, to produce bioluminescencelight (emission wavelength: 560 nm).Although newly identiﬁed miRNAs in mammaliancells have been intensively studied to establish theirrole in human disease, including cancers, there hasbeen limited research on miRNA imaging. Therefore,active investigation of the production pattern andfunctional action of miRNAs is needed. The severalavailable imaging strategies used to detect endogenousmiRNA production can be used to monitor both theprimary transcript and the mature form of miRNA(Fig. 1) [11–13].As miRNAs are essential in all biological areas, anoninvasive technique for monitoring miRNA biogene-sis would help to eluciate the versatile functions andproduction patterns of miRNAs relative to geneticmodulation, cell development and multiple diseases,in vivo. In addition, the bioimaging techniques formiRNAs based on molecular imaging methods couldbe applied as target imaging indicators to help under-stand the developmental process, in the developmentof cancer biomarkers and to evaluate the therapeuticeffects, 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 ofthe production and function of miRNA in vivo as wellas for monitoring the therapeutic potential of miRNAsin cancer.Bioimaging of microRNA biogenesisThe molecular mechanisms involved in miRNA gener-ation are complex, and at least several processing stepsin the nucleus and cytoplasm should be monitored, byimaging, as follows (Fig. 1A): (a) imaging of a primarymiRNA (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) thatis cleaved from pri-miRNA by Drosha and DiGeorgesyndrome critical region gene 8 (DGCR8) (Fig. 1C),(c) imaging a partially double-stranded miRNAcomplex (miRNA–miRNA*) that is released from thepre-miRNA by Dicer (Fig. 1D), (d) imaging a single-stranded mature miRNA (Fig. 1E) and (e) imagingmiRNA function that either destabilizes mRNA orinhibits the translation of target genes by binding totarget genes (Fig. 1F).First, imaging of the generation of pri-miRNArevealed that the 5¢ upstream region of genomicmiRNA controls the long primary transcripts ofmiRNA that shape a single or a large family ofmiRNA gene clusters. Interestingly, several miRNAs,including miRNA9 and miRNA124, are located atmultiple loci, each of which can produce pri-miRNA,pre-miRNA and mature miRNA ; this implies thatthe 5¢ upstream region from a few different loci shouldbe investigated concurrently to provide an accuratereﬂection of the generation of a pri-miRNA. Likeother eukaryotic mRNAs, the primary transcript ofmiRNA is controlled by RNA polymerase II, and sev-eral transcription factors, including Oct4, c-Myc andNanog, regulate the expression of primary miRNAs bybinding them to the 5¢ terminal regulatory region ofthe miRNAs that participate in critical molecularand ⁄ or cellular processes during the developmentalstage [14–16]. Similarly to the reporter gene assay ofthe eukaryotic promoter, the 5¢ upstream region of amiRNA that proportionally reﬂects the endogenousexpression level of a pri-miRNA can be fused into thecassette of a promoterless optical reporter gene vector(Fig. 1B). The cloned miRNA-speciﬁc reporter-imagingvector can be transfected into cultured cells, and thecell lines can be collected and implanted (e.g. into thethighs of a mouse). The expression level of the pri-miRNA transcript can be obtained from living animalsby imaging the in vivo bioluminescence signals.The optical reporter gene system enables pri-miRNAgeneration to be monitored in vivo. Lee et al. used the miRNA23a promoter to acquire imagesthat showed differences in the endogenous expressionof pri-miRNA23a in HeLa, 293 and P19 cells. Koet al.  also monitored the neuronal-speciﬁcpri-miRNA9 during neurogenesis by using its upstreamregion.Second, to acquire the images of a pre-miRNA thatwas cleaved from pri-miRNA by Drosha and DGCR8,Lee et al.  designed sense and antisense oligonucle-otides of the pri-miRNA23a that were annealed andcloned between the cytomegalovirus (CMV) promoterand the start codon of the Gaussia luciferase geneof the optical reporter gene vector (Fig. 1C). TheBioimaging of miRNA biogenesis and regulation S. Kim et al.2166 FEBS Journal 276 (2009) 2165–2174 ª 2009 The Authors Journal compilation ª 2009 FEBSFig. 1. Schematic illustration of detection systems for imaging miRNA biogenesis and regulation. (A) Steps of miRNA processing. PrecursormiRNA 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 databaseprogram (UCSC) and be split into different size segments of the chosen upstream region of miRNAs for acquisition of a strong optical signal. The upstream region of pri-miRNAs can be fused into an imaging reporter gene to examine pri-miRNA expression [11–13]. The miRNApromoter-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 canbe detected by the signal activity of the reporter gene when it is cleaved by the Drosha enzyme . (D) Molecular beacon for imaging apartially 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 moleculeand ﬂuorophore dye are separated from each other after interaction of Dicer at the Dicer recognition site present on the pre-miRNAs, andthe ﬂuorescent signals are thereby released . (E) Schematic strategy for the reporter gene imaging of mature miRNAs. Perfectly matchedcomplementary 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 ofmiRNA can be isolated and transferred into a downstream region of the reporter gene that is regulated by a constitutive promoter such asCMV. When the imaging reporter gene of the miRNA target interacts with mature miRNAs in the cells, the activity of the reporter gene isturned off [24–28].S. Kim et al. Bioimaging of miRNA biogenesis and regulationFEBS Journal 276 (2009) 2165–2174 ª 2009 The Authors Journal compilation ª 2009 FEBS 2167pre-miRNA imaging system showed an increase inluciferase activity when the pri-miRNA23a was cleavedby Drosha and DGCR8.Third, to monitor a partial double-stranded miRNAcomplex, the functional imaging of Dicer is critical(Dicer is the ribonuclease III enzyme that plays anessential role in the production of mature miRNA bycleaving the pre-miRNA). Several reports have sup-ported the importance of Dicer, showing that deletionof the Dicer enzyme causes phenotypic defects duringdevelopment, which results from the generation ofabnormal miRNA maturation [17,18]. One studyreported assaying the cleavage of pre-miRNA by theDicer enzyme using quencher-based pre-miRNA todetect endogenous or exogenous Dicer enzyme; theresults showed intense ﬂuorescence signals with dis-placement of the ﬂuorescence dye from the quenchingmolecule by the Dicer enzyme, which cleaved the syn-thetic let-7 precursor miRNA (Fig. 1D) . In theabsence of the Dicer protein, the emission energy ofthe ﬂuorescence dye attached to the end of one strandof the pre-miRNA (as shown in Fig. 1D) is absorbedby the quenching molecule, showing the quenched ﬂu-orescence signal. By contrast, with Dicer present thereis cleavage of the end of the pre-miRNA, and acti-vated ﬂuorescence signals are observed, which impliesthat the ﬂuorescence beacon system is useful for thedetection 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, usinggreen ﬂuorescent protein and luciferase, which hasenabled the endogenous production pattern of maturemiRNA to be monitored [11–13,20]. This imagingmethod focuses on the concept that the binding orfunction of the mature miRNAs is based on nucleotidesequence 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 themRNA destabilization of the reporter gene followinginteraction of the imaging system with the maturemiRNA in cells (Fig. 1E).Giraldez et al. presented a good method for evaluat-ing the existence of mature miRNAs using a singleoptical reporter imaging vector containing perfecttarget sequences of miRNAs. The green ﬂuorescentprotein reporter imaging vector system was used toexamine the production pattern of mature miRNAs bygeneration of Dicer mutants in zebraﬁsh, elucidatingthe vital role of miRNAs associated in morphogenesis. However, this method was used only for thedetection of mature miRNAs.Generally, miRNAs are produced via complex pro-cesses controlled by a variety of proteins, includingDrosha and Dicer. The production of mature miRNAis dependent on the functional action of many pro-teins that control the generation of mature miRNAs.Tomson et al.  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, withoutbeing processed by the RNase III enzyme Drosha,and that no correlation between the production ofpri-miRNAs and mature miRNAswas observed incancer samples, compared with the correlation in nor-mal cells. This information indicates that the matura-tion process is controlled by an unknown regulatoryfactor, and thus different levels of primary transcriptand mature miRNA were shown to occur simulta-neously in living cells. Therefore, the simultaneousmonitoring of production of both pri-miRNA andmature miRNA is required using different opticalreporter 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 and560 nm, respectively. These proteins can thus be usedto image two different molecular actions simulta-neously. Lee et al.  reported that the two differ-ent reporter gene systems (ﬁreﬂy luciferase andGaussia luciferase, described above) could simulta-neously image primary and mature forms ofmiRNA23a in cancer cells (Fig. 2). The in vivo bio-luminescence signals from pri-miR23a and maturemiR23a were observed in cancer-grafted nude mice.Fireﬂy luciferase reporters revealed the primary tran-script activity of miRNA23 using the miRNA23promoter (miR23P639 ⁄ Fluc)-controlled ﬁreﬂy lucifer-ase reporter system. Conversely, the production ofmature miR23a was detected using the Gaussia lucif-erase reporter system, which contains three copies ofthe miR23a-binding sequence (CMV ⁄ Gaussia lucifer-ase ⁄ 3 · PT_miR23a). Interestingly, in this report,unlike the results on bioluminescence studies carriedout on HeLa and P19 cells that showed increasedﬁreﬂy luciferase signals for pri-miRNA23a and theresultant highly reduced Gaussia luciferase activitiesfor mature miRNA23a, 293 cells showed a slowturnover from pri-miR23a to mature miRNA23a byunknown mechanisms, implying the capability of thereporter gene system to assess and visualize the post-transcriptional regulation of miRNA . Therefore,a variety of imaging strategies, showing each step inthe generation of miRNAs, will provide criticalBioimaging of miRNA biogenesis and regulation S. Kim et al.2168 FEBS Journal 276 (2009) 2165–2174 ª 2009 The Authors Journal compilation ª 2009 FEBSinformation for understanding the miRNA biogenesisrelated to human diseases.Imaging miRNA targetsWhile hundreds of miRNAs are known, identiﬁcationof their target genes has been a considerable challenge.The results of bioinformatics and microarray experi-ments have shown that miRNAs can target hundredsof transcripts, both directly and indirectly [13,22]. Gen-eral methodologies by which the functional regulationof endogenous miRNA is investigated utilize a bioin-formatically analyzed seed region that contains sevennucleotides of a miRNA response sequence in the3¢-UTR of target genes . This 3¢-UTR-based lucif-erase system, containing the seed region, has beenwidely used for target identiﬁcation and for elucidatingthe functional relationship between miRNA and itstargets (Fig. 1F) [13,24–28]. Computer databaseprograms, such as the PicTar database, offer an easyapproach with which to identify the 3¢-UTR of themiRNA target candidates by scoring the dependenttarget prediction. Many studies have reported a varietyof target genes that were signiﬁcantly down-regulatedby speciﬁc miRNAs. These targets were determinedin vitro using a reporter gene system containing the3¢-UTR of the targets located immediately after thestop codon of the luciferase reporter gene [13,29]. Fromthis reporter system, partial interaction of miRNAswith a 3¢-UTR region in reporter gene constructsresulted in reduction of the reporter signal in cells.Using the 3¢-UTR-based Renilla luciferase reportergene system, Yan et al.  obtained quantitativein vitro images of nine transcripts that were predictedto be miRNA9 targets using the ensemble machine-learning algorithm. However, in vivo imaging ofmiRNA regulation has not been reported, with theexception of a report by Kim et al. , in whichhomeobox B5 (which was veriﬁed by microarray exper-iments) was identiﬁed to be one of the endogenoustargets of miRNA221 that is highly expressed inpapillary thyroid carcinoma. The in vivo biolumines-cent signals from the 3¢-UTR region of homeobox B5containing the miRNA221 seed region fused into theGaussia reporter gene were signiﬁcantly decreased bymature miRNA221 during the development ofFig. 2. Noninvasive bioimaging of miRNA23 biogenesis to examine the production of both the pri-miR23a transcript and the mature miR23ain nude mice. MiR23P639 ⁄ Fluc (ﬁreﬂy luciferase reporter system regulated by miR23a promoter) and CMV ⁄ Gluc ⁄ 3 · PT_miR23a (Gaussialuciferase reporter system containing three copies of an miR23a-binding sequence that is completely complementary to mature miR23a)were cotransfected with 1 · 107HeLa, 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 drivenby the cytomegalovirus promoter)-transfected cell lines were also implanted into the left thigh of mice as controls. The bioluminescenceactivity 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 detectedusing 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 ).S. Kim et al. Bioimaging of miRNA biogenesis and regulationFEBS Journal 276 (2009) 2165–2174 ª 2009 The Authors Journal compilation ª 2009 FEBS 2169papillary thyroid carcinoma . These results wereobtained from directly monitoring the cells using the3¢-UTR-based Gaussia luciferase reporter gene system.One limitation of the 3¢-UTR-based reporter gene sys-tem for imaging endogenous targets of miRNA wasfocused on the targets containing the seed region,which resulted in exclusion of targets regulated byother miRNA mechanisms, such as genes regulated bythe translational inhibition of miRNA.Unlike reporter imaging systems based on perfecttarget sequences, as shown in Fig 1E, the 3¢-UTR-based reporter system can be used to elucidate the realfunction of miRNAs (Fig. 1F). In general, the func-tional actions of miRNAs in mammalian cells, unlikeplant cells, are associated with the translational inhibi-tion of target mRNAs by binding miRNA to the seedregion of the target mRNA with noncomplementaritybetween the miRNA and its target. Therefore, the3¢-UTR-based imaging reporter system reﬂects the realaction of mature miRNA in mammalian cells, com-pared with reporter-imaging systems that use perfecttarget sequences from miRNAs.In vivo imaging of miRNA in cancersand neuronal developmentmiRNAs have been associated with developmentalprocesses in many types of cells, including embryonic,neuronal, muscular and lymphatic cells. Abnormal cel-lular development, as occurs with cancer, has also beenassociated with miRNAs. Indeed, a wide range ofmiRNAs, including miRNA221, 21 and 142, areinvolved in multiple oncogene targets in cancer cells,which are thought to originate from two main basicmechanisms, 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 thatregulates multiple oncogenes causes tumorigenesis,resulting from abnormalities occurring during each ofthe steps of miRNA biogenesis. Second, the oncogeniccharacteristics of the miRNAs regulate the progressionof cancer by preventing the tumor suppressor genesfrom producing tumor suppressor protein . Thesecontrasting roles of cancer-related miRNAs have beenconsidered for the development of therapeutic tools tobe used against cancer cells. Recent evidence has indi-cated that the use of anti-miRNA296 showed a signiﬁ-cant therapeutic effect by targeting miRNA296, whichis highly expressed in endothelial tumor cells . Thetherapeutic effect of anti-miRNA296 for reduction oftumor angiogenesis was evaluated in tumor-xenograft-ed mice by measuring the decrease of optical signalin vivo . Also, miRNA21 is a potential therapeutictarget for cancer treatment, as overexpression of anti-miRNA21 in hepatocellular carcinoma and glioblas-toma cells was shown to down-regulate the oncogenicmiRNA21 . Based on the use of anti-miRNA21 asthe therapeutic agent, the therapeutic effects on thetumor using anti-miRNA21, which is an inhibitor ofmiRNA21, have been reported for the ﬁrst time; theseﬁndings indicated that the real-time tumor regressioncould be clearly visualized in luciferase-expressingtumor-bearing mice . Corsten et al.  showed sig-niﬁcant cytoreduction effects in tumors using a dualtherapeutic method with locked nucleic acid-modiﬁedanti-miRNA21 and neural precursor cells expressingSecretable form of tumor necrosis factor-related apop-tosis inducing ligand (S-TRAIL) (Fig. 3). However,this study used in vitro transfection of anti-miRNA21for in vivo cancer therapy, indicating that the in vivodelivery of miRNAs or anti-miRNAs remains to bedemonstrated. Recently, our group developed anin vivo system for monitoring miRNA221, which isknown to be overexpressed in papillary thyroid carci-noma cells. Our system can be used to image theproduction of miRNA221 in thyroid cancers. Withchemically modiﬁed anti-miRNA221 it can also beused to examine therapeutic effects .Previous studies have suggested that MYC and RASare putative target genes for the let-7 miRNA familyin lung cancer [37,38], which can show potential effectson the treatment of cancer using let-7 as the therapeu-tic agent. Therefore, the therapeutic imaging approachusing anti-oncogenic miRNAs, such as let-7, should befurther explored to validate its therapeutic efﬁcacy. Inthis manner, noninvasive cancer-associated miRNA-imaging systems can be developed to monitor thetherapeutic effects, not only by blocking the oncogenicmiRNA participating in suppressing the tumor-suppressor genes, but also by the over production ofmiRNAs that can repress several oncogenes.miRNAs play an important role in determining thespeciﬁcation and fate of cells by regulating a numberof genes responsible for governing cellular develop-ment. The tissue-speciﬁc or cell-speciﬁc distribution ofmiRNAs might be a good target for monitoring thetissue-restricted production of miRNAs . It hasbeen shown that some miRNAs, including miR-NA124a, 9, 9*, 124, 128 and 132, are primarilyenriched in brain tissue, where they are implicated inneuronal development. Indeed, the production of thesemiRNAs increased as neuronal lineages progressed[39–41]. Ko et al.  reported that in vivo imaging ofmiRNA9 and miRNA9* (the opposite sequence ofmiRNA9), 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 FEBSsion during neurogenesis. This illustrates that theimaging of neuron-speciﬁc miRNA9 and miRNA9*can be important for understanding the functional roleof miRNA during neurogenesis, and for investigatingdifferences between miRNA and miRNA*. Generally,antisense miRNAs (miRNA*), namely complementarysequences of sense miRNAs that participate in the realfunctions of miRNAs, are known for their low produc-tion and fast degradation of miRNA* by endonucleases,compared with sense miRNAs . However, forseveral miRNAs, including miRNA9, it has beenshown that both sense and antisense strands canregulate target mRNA [39,43]. A noninvasive systemfor imaging the production of neuron-speciﬁc miRNA9and miRNA9* would facilitate a better understandingof the unique molecular mechanisms associated withthe biogenesis of miRNA during neuronal develop-ment in living subjects.In this context, noninvasive bioimaging systems usingneuron-speciﬁc miRNAs could be applied not only tomonitor the functional roles and production proﬁles ofneuron-related miRNAs, but also as imaging tools totrack neuronal differentiation imaging based on mole-cular imaging techniques. The development of imagingstrategies related to miRNAs will continue to beapplied as imaging tools for detecting the progressionof neuronal development, as well as for cancer diagnos-tics and therapeutic application in human medicine.ConclusionRecently, the increased interest in miRNAs, and con-cerns that miRNAs are known to play important rolesin clinical diseases, have attracted many molecularresearchers to study miRNAs associated with the bio-genesis and functional role of miRNAs. To study avariety of biological phenomena related to miRNAs,noninvasive miRNA imaging techniques have beendeveloped to track the generation and function ofmiRNAs by monitoring their targets regulating cellularand molecular events, as well as the expression levelsof pri-miRNA and mature miRNA. To visualize thegeneration of miRNAs using reporter imaging systems,optical imaging strategies using the promoter region ofthe miRNAs or miRNA responsive elements providesnon-invasive molecular imaging information regardinghow and when they are transcribed by transcriptionfactors and where they originate, in the case of amiRNA located on multiple chromosomal loci. Inaddition, these techniques show the extent to whichmature miRNAs are formed during cellular develop-ment. Optical reporter genes, such as ﬁreﬂy or Gaussialuciferase genes, are well suited for monitoring miRNAbiogenesis in small animals because they have theadvantages of low background noise and high sensitiv-ity. However, these optical systems remain a distantgoal for clinical application because of the opticalsignal attenuation by a tissue depth. Therefore, furtherinvestigation of miRNAs and development of targetdetection methods based on radionuclide imagingmodalities, such as the sodium iodide symporter andFig. 3. Bioluminescence imaging system created to evaluate thetherapeutic potential of anti-miRNA21. (A, B) After ﬁreﬂy luciferase-expressing U87 glioblastoma cells were treated with locked nucleicacid anti-miRNA21, the U87 cells collected were intracraniallyinjected into the brains of mice. The bioluminescence signals fromimplanted U87 cells expressing luciferase dramatically reduced withtime, showing a therapeutic effect of anti-miRNA. This effect wassupported by the results of quantitative region of interest analysis(reprinted with permission ).S. Kim et al. Bioimaging of miRNA biogenesis and regulationFEBS Journal 276 (2009) 2165–2174 ª 2009 The Authors Journal compilation ª 2009 FEBS 2171the MR imaging reporter gene (such as the transferrinreceptor) are required to further the advancementtowards application to human medicine . However,the optical signals from the mature miRNA-basedreporter gene are basically a ‘turn-off’ system as aresult of the function of the miRNAs; this might intro-duce ambiguity with regard to the interpretation ofwhether the observed signaling-off data results fromthe production of numerous mature miRNAs or onlyfrom cell death.An alternative strategy for imaging mature miRNAsthat overcomes the limitations of the signaling-off prob-lem in the reporter gene system is a tunable ﬂuorescencesystem that uses a quenching molecule-based molecularbeacon. The molecular beacon is a ﬂuorescence detec-tion system composed of a single-stranded and stem–loop nucleotide that is complementary to the targetDNA sequence or structurally bound to the protein ofthe 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 appliedto the investigation of endogenous miRNA productionproﬁles as a result of miRNAs being bound to theirtargets based on sequence homology. The signal-onimaging strategy, using the molecular beacon to imagemature miRNAs, is as follows. In the absence of maturemiRNA molecules, the ﬂuorescence energy from theﬂuorophore dye is absorbed by quencher molecules,resulting in no ﬂuorescence signal. By contrast, bydisplacing the quencher from the organic ﬂuorophoredye, the presence of mature miRNA causes the mole-cular beacon light-on result. The molecular beaconapproach used for the detection of miRNAs will pro-vide more accurate imaging information about miRNAbiogenesis in cellular developments and disease.In summary, molecular imaging techniques can beused to monitor the in vivo dynamics of miRNAs non-invasively, including their production proﬁles and regu-lation. First, the miRNA-based optical system, asproposed in several previous studies, can be used forthe in vitro and in vivo investigations of miRNAproduction patterns. Second, noninvasive monitoringof miRNAs provides useful information of the possiblerole of miRNAs involving cellular developments.Third, the in vivo molecular reporter system might beused to monitor repetitively, with precise measurement,the therapeutic effect using miRNAs relevant to cancerin living animals. A study of the expression patterns ofmiRNA, providing ﬁne tuning of gene regulation, willhelp to understand the interplay of complex gene regu-latory networks during developmental stages and thechanges 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.AcknowledgementsThis work was supported by the Nano Bio Rege-nomics Project and by the Nuclear R&D programthrough the Korea Science and Engineering Founda-tion funded by the Ministry of Education, Science andTechnology (M20704000039-08M0400-03910) and bythe Seoul R & BD program (10550) and NationalR&D Program for Cancer Control of the Ministry ofHealth & Welfare (0820320).References1 Negrini M, Ferracin M, Sabbioni S & Croce CM (2007)MicroRNAs in human cancer: from research to ther-apy. J Cell Sci 120, 1833–1840.2 Blenkiron C & Miska EA (2007) miRNAs in cancer:approaches, aetiology, diagnostics and therapy. HumMol Genet 16, R106–R113.3 Van Rooij E & Olson EN (2007) MicroRNAs: powerfulnew regulators of heart disease and provocative thera-peutic targets. 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