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: kimsoonhag@empal.com 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: dsl@plaza.snu.ac.kr *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. 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