Bioimaging of the unbalanced expression of microRNA9 and microRNA9* during the neuronal differentiation of P19 cells Mee Hyang Ko 1,2,3 , Soonhag Kim 2,4, *, Do Won Hwang 1,2,3 , Hae Young Ko 2,3 , Young Ha Kim 2,3 and Dong Soo Lee 2,4, * 1 Programs in Neuroscience, Seoul National University, Korea 2 Department of Nuclear Medicine, Seoul National University College of Medicine, Korea 3 Laboratory of Molecular Imaging and Therapy of Cancer Research Institute, Seoul National University College of Medicine, Korea 4 Medical Research Center, Seoul National University College of Medicine, Korea MicroRNAs (miRs) are a class of small non-coding RNA molecules, encoded as short inverted repeats in the genomes of plants and animals. miRs are believed to modulate the post-transcriptional regulations of their targets in diverse biological regulatory systems including cellular development [1,2], cell differentiation [3], fat metabolism [4], cell proliferation and cell death [5]. Hundreds of miRs have been isolated from mam- malian species and a dozen of these, including miR124a, miR9, miR128, miR131, miR178 and Keywords bioimaging; Luciferase; microRNA; microRNA9 and microRNA9*; neurogenesis Correspondence S. Kim, Department of Nuclear Medicine, Medical Research Center, Seoul National University College of Medicine, 28 Yongon- dong, Jongno-gu, Seoul 110 744, Korea Fax: +82 2 3668 7090 Tel: +82 2 3668 7028 E-mail: kimsoonhag@empal.com D. S. Lee, Department of Nuclear Medicine, Medical Research Center, Seoul National University College of Medicine, 28 Yongon- dong, Jongno-gu, Seoul 110 744, Korea Fax: +82 2 3668 7090 Tel: +82 2 2072 2501 E-mail: dsl@plaza.snu.ac.kr *These authors contributed equally to this work (Received 18 January 2008, revised 10 March 2008, accepted 17 March 2008) doi:10.1111/j.1742-4658.2008.06408.x Generally, the 3¢-end of the duplex microRNA (miR) precursor (pre-miR) is known to be stable in vivo and serve as a mature form of miR. However, both the 3¢-end (miR9) and 5¢-end (miR9*) of a brain-specific miR9 have been shown to function biologically in brain development. In this study, real-time PCR analysis and in vitro ⁄ in vivo bioluminescent imaging demon- strated that the upstream region of a primary miR9-1 (pri-miR9-1) can be used to monitor the highly expressed pattern of endogenous pri-miR9-1 during neurogenesis, and that the Luciferase reporter gene can image the unequal expression patterns of miR9 and miR9* seen during the neuronal differentiation of P19 cells. This demonstrates that our bioimaging system can be used to study the participation of miRs in the regulation of neur- onal differentiation. Abbreviations Dicer, RNase III endonuclease; FLuc, Firefly Luciferase; GLuc, Gaussia Luciferase; miR, microRNA; miR9*, microRNA9*, 5¢-end of pre-miR9; miR9, microRNA9, 3¢-end of miR9 precursor; piRNA, Piwi-interacting RNA; pre-miR, precursor microRNA; pre-miR9, miR9 precursor or precursor miR9; pri-miR9-1, primary miR9-1; ROI, region of interest analysis. FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS 2605 miR125b, have been found to be associated with polyr- ibosomes in primary neurons [6,7]. These studies have shown that both microRNA9 [miR9, 3¢-end of miR9 precursor (pre-miR9)] and microRNA9* (miR9*, 5¢-end of pre-miR9) originate from the hairpin-loop structure of the same pre-miR9, and are highly co-expressed and neuron-specific during brain devel- opment. In general, intergenic or intragenic miRs are tran- scribed into primary miRNA (pri-miR) by RNA polymerase II and processed into a 70-nucleotide hair- pin-structured pre-miR in the nucleus by Drosha [8–10]. Pre-miRs are transported to the cytoplasm by exportin-5 (a member of the Ran transport receptor family) and another factor Ran [11]. Pre-miR hairpin is further processed into a 19- to 23-nucleotide single- stranded mature miR by RNase III endonuclease (Dicer) [12]. During Dicer cleavage, duplex pre-miRs are uncoiled by helicase into two single strands, mature miR (from the 3¢-end of duplex pre-miR) or miR* (opposite strand, from the 5¢-end of duplex pre-miR), although miR* is generally rapidly degraded by an unknown enzyme nuclease [13]. Mature miRs are then incorporated into the RNA- induced silencing complex and bound to the 3¢-UTR of its target mRNA to induce either mRNA degrada- tion or translational inhibition [2,14,15]. Interestingly, unlike most miRs, which have a single mature miR, miR cloning and sequencing from the human and miRNAMap database (http://mirnamap.mbc.nctu. edu.tw) showed that a few miRs, including miR302b, miR302c, miR373 and miR9, have two types of mature form, miR and miR* [16,17]. This is similar to the final functional forms of Piwi-interacting RNA (piRNA), which are also small RNA molecules although distinct in size from miR. Even though 25- to 31-nucleotide long piRNAs are not generated by Dicer, both sense and antisense strands of the piRNA hairpin are involved in formation of the piR- NA-interacting complex (piRC) and function in the transcriptional gene silencing of retrotransposons and genetic elements in germline cells [18]. Investigations into the gene expression of endoge- nous miR in cells or tissues are useful for understand- ing cellular metabolism, disease diagnosis and the effects of therapies related to miR. However, current methods of monitoring endogenous miR levels, such as northern blotting, RT-PCR, and microarrays are time- consuming, laborious, and non-reproducible. In a pre- vious study, we successfully imaged miR23a biogenesis in small animals to noninvasively monitor the expres- sion patterns of endogenous miR23a in different cells [19]. This type of bioluminescence imaging technology may be clinically relevant and could be applied to the real-time analysis of miR biogenesis in living animals. Currently, the most widely used bioluminescent pro- teins in living animals are Gaussia Luciferase (GLuc) and Firefly Luciferase (FLuc). GLuc emits light at a 480 nm by oxidizing its substrate coelenterazine [20], FLuc emits at 562 nm when it oxidizes d-luciferine [21]. We cloned the upstream region of miR9 and studied the expression pattern of endogenous pri-miR9-1 to try to understand miR9 biogenesis during the neuronal differentiation of P19 cells using RT-PCR and biolu- minescent imaging. The unbalanced biogenesis of mature miR9 and miR9* during neurogenesis was monitored by real-time PCR and in vitro and in vivo Luciferase reporter gene systems. Results Detection of the endogenous level of the pri-miR9s To study miR9 biogenesis during neurogenesis, we first investigated the primary transcript level of miR9 in P19 cells that had differentiated into neuronal cells following retinoic acid treatment. Mouse and human genomes from the UCSC database showed three dif- ferent loci that can be processed into mature miR9 and ⁄ or miR9*. Three different primary transcripts of miR9 in mouse are located at chromosome 3 (pri-miR9-1), chromosome 13 (pri-miR9-2), and chro- mosome 7 (pri-miR9-3; Fig. 1A). The gene-expression levels of the primary transcripts of miR9 were moni- tored by sequence-specific RT-PCR analysis using total RNA from P19 cells induced to differentiate by retinoic acid. PCR primers were designed by aligning the sequences of three different pri-miR9s to match their unique pri-miR9s, but not to amplify alternative sequences (Fig. 1B). Gene-expression analysis of P19 cells treated with retinoic acid for 6 days showed a gradual increase in MAP2 transcript levels, a neuro- nal marker gene, which was expected to occur during neuronal differentiation (Fig. 1C). The gene expres- sions of the three different pri-miR9s exhibited vari- ous transcript patterns during neuronal differentiation of P19 cells. Pri-miR9-1 showed a dramatic change in gene expression immediately after treatment with reti- noic acid, i.e. a gradual increase in primary transcript level until the third day, followed by a sudden decrease. By contrast, pri-miR9-3 was relatively highly expressed even before neuronal differentiation, increasing gradually during neurogenesis until the fourth day and then completely disappeared. Unlike Bioimaging of miR9 and miR9* during neurogenesis M. H. Ko et al. 2606 FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS the other two pri-miR9s, pri-miR9-2 expression was barely detectable in undifferentiated and P19 cells differentiated by retinoic acid. This suggests that the transcript level of pri-miR9-1 is a good bioindicator for the neuronal differentiation of P19 cells treated with retinoic acid. Bioimaging of the highly expressed pri-miR9-1 during neurogenesis To monitor the endogenous expression of pri-miR9s using the bioluminescent Luciferase reporter gene system during neuronal differentiation in P19 cells, A B C Fig. 1. Detecting the gene-expression pat- terns of the three different pri-miR9s during neurogenesis. (A) Chromosomal locations of the three different pri-miR9s in mice from the UCSC database. (B) Primer positions and sequences used to amplify pri-miR9-1, 9-2 and 9-3. Arrows indicate the position and orientation of the primers. Bold font represents the sequence of mature miR9* and italic fonts the sequence of mature miR9. (C) RT-PCR analysis of pri-miR9s dur- ing the neuronal differentiation of P19 cells treated with retinoic acid (RA). pri-miR9-1 and -9-3 were gradually increased during neuronal differentiation, whereas the pri- miR9-2 transcript was barely detected. MAP2, a neuronal marker gene, showed that retinoic acid treatment efficiently induced neuronal differentiation of P19 cells. B-Actin was used as an internal control. M. H. Ko et al. Bioimaging of miR9 and miR9* during neurogenesis FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS 2607 1343 bp of the upstream region of the pri-miR9-1 from human genomic DNA was cloned and fused into a pro- moterless reporter vector, pGL3_Basic, which contained the ORF of the FLuc reporter gene (Fig. 2A). FLuc activity was measured to determine the promoter activ- ity of pri-miR9-1 during retinoic acid-induced neuronal differentiation. The upstream region of the pri-miR9-1 was randomly split into five different segments by PCR, )1387 to )44 bp (miR9-1PN1_Fluc), )846 to )44 bp (miR9-1PN2_Fluc), )530 to )44 bp (miR9-1PN3_Fluc), )236 to )44 bp (miR9-1PN4_Fluc), and )135 to )44 bp (miR9-1PN5_Fluc; Fig. 2B). These five different con- structs were then transfected into P19 cells and their pro- moter activities monitored using an in vitro Luciferase assay over 2 days following treatment of P19 cells with retinoic acid (Fig. 2C). Most of the constructs from P19 cells treated or not with retinoic acid had equal or lower promoter activities than did the pGL3_Basic vector used as a negative control. However, miR9-1PN3_Fluc showed a relatively stronger FLuc signal and a higher expression level after neuronal differentiation than the other segments, which indicated an increased endoge- nous level of pri-miR9-1 during neurogenesis. This indi- cates that negative promoter elements of pri-miR9-1 transcription may be involved in the upstream region between -846 and -531 bp and that positive elements may be involved between -530 and -237 bp. These find- ings indicate that the miR9-1PN3_Fluc construct could be used for in vivo imaging of gene expression of endoge- nous pri-miR9 during neurogenesis. To image in vivo the endogenous expression of the pri-miR9 in small animals, 2.5 · 10 6 of P19 cells bear- ing the miR9-1PN3_Fluc construct were subcutane- ously implanted into mice and region of interest (ROI) analysis was performed on the basis of the resultant bioluminescent signals obtained 2 days after inducing neuronal differentiation with retinoic acid (Fig. 2D). All the Luciferase signals of the CMV_Fluc, a positive control, from the right shoul- der, showed constant and high FLuc expression at 0, 18, 24, and 48 h after retinoic acid treatment. The negative control, pGL3_Basic, in left shoulders, was found to show weak or undetectable FLuc expression throughout the investigation. FLuc intensities of miR9-1PN3_Fluc (right thighs; normalized versus CMV_Fluc) showed a gradual increase in the presence of retinoic acid compared with left thighs which were not treated with retinoic acid. ROI analysis showed that miR9-1PN3_Fluc showed an almost fivefold increase in FLuc activity 1 day after retinoic acid treatment. The findings of our in vitro and in vivo Luciferase assays showed that miR9-1PN3_Fluc biolu- minescence reflects elevated endogenous pri-miR9-1 levels during the neuronal differentiation of P19 cells treated with retinoic acid. Mature miR9 was relatively higher expressed than mature miR9* during neurogenesis Mature miR9 and miR9*, which may be processed from the same pre-miR9, are known to be highly expressed at the same time during neuronal develop- ment [17]. To quantify their relative expression levels during neurogenesis, we conducted real-time PCR using small RNAs extracted from the neuronal differ- entiation of P19 cells at 0, 1, 2, 3, 4, 5 and 6 days after treatment with retinoic acid. The amplicons produced using pairs of specific primers for mature miR9 and miR9* were quantified and normalized using U6 small RNA. Endogenous mature miR9 and miR9* were barely detectable prior to the neuronal differentiation of P19 cells (Fig. 3A). However, these mature miRs showed a similar and gradually increased expression pattern during neuronal differentiation of P19 cells. Interestingly, mature miR9 was consistently expressed at a  40% higher level than mature miR9* during differentiation. To image the endogenously unequal expressions of mature miR9 and miR9* during neurogenesis, a GLuc reporter gene vector was first designed containing the following components in order: a CMV promoter, an ORF of GLuc, three copies of perfectly complemen- tary sequences of mature miR9 (designated as CMV ⁄ Gluc ⁄ 3xPT_mir9) or miR9* (designated as CMV ⁄ Gluc ⁄ 3xPT_mir9*) (Fig. 3B). When mature miR9 or miR9* is present in cells, the GLuc activities of CMV ⁄ Gluc ⁄ 3xPT_mir9 andCMV ⁄ Gluc ⁄ 3xPT_mir9* are repressed by cognate mature miR9 and miR9*, respectively. To demonstrate the specificity of the bio- luminescent reporter system to monitor both mature miR9 and miR9*, CMV ⁄ Gluc ⁄ 3xPT_mir9 or CMV ⁄ Gluc ⁄ 3xPT_mir9* with a negative control vector, CMV_Gluc, were transfected into HeLa cells which do not express mature miR (Fig. 3C). The CMV_Gluc construct, which was not repressed by exogenous pre- miR was used to normalize the GLuc activities of the CMV ⁄ Gluc ⁄ 3xPT_mir9 and CMV ⁄ Gluc ⁄ 3xPT_mir9* in HeLa cells treated with various concentrations (0, 2.5, 5, 10, 20 nm) of exogenously derived pre-miR9 or pre-miR9*. The GLuc expressions of both CMV ⁄ Gluc ⁄ 3xPT_mir9 and CMV ⁄ Gluc ⁄ 3xPT_mir9* showed a dramatic decrease in response to exogenous pre-miR9 and pre-miR9*, respectively. The CMV ⁄ Gluc ⁄ 3xPT_mir23a vector, which has previously been reported to monitor mature miR23a [19], was transfected into HeLa cells and found not to change Bioimaging of miR9 and miR9* during neurogenesis M. H. Ko et al. 2608 FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS A B C D Fig. 2. In vitro and in vivo GLuc expression of pri-miR9-1 during neuronal differentiation. (A) The chromosomal region of has-pri- miR9-1. (B) Schematic diagram of the upstream region of pri-miR9-1 fused to a promoterless FLuc reporter vector. The pro- moter sizes of the construct are indicated by numbers in parentheses. )44 means located 44 bp upstream of the first base pair at the 5¢-end of pre-miR-9-1 defined as +1. FLuc is the ORF of the Firefly Luciferase reporter gene. (C) In vitro bioluminescent assay of pri-miR9-1 during neuronal differen- tiation of P19 cells. Five different upstream regions of pri-miR9-1 were transfected into P19 cells, miR9-1PN3_Fluc produced a strong FLuc signal after neuronal differentia- tion of P19 cells, pGL3_Basic vector was used to normalize the FLuc activities obtained from the five different constructs. Transfections were performed in triplicate and results are expressed as mean ± SD. (D) Bioluminescence image of pri-miR9-1 expression in nude mice. P19 cells (2.5 · 10 6 ) were transiently transfected with miR9-1PN3_Fluc and injected into nude mice. In right thighs, neuronal differentiation was induced by retinoic acid, whereas left thighs were not treated with retinoic acid. The pGL3_Basic vector in the right shoul- ders was used as a negative control and CMV_Fluc in right shoulders as a positive control, and were used to normalize FLuc activities acquired on each day. Biolumines- cence intensities in right thighs, expressing pri-miR9-1 were increased during neuronal differentiation of P19 cells compared with the left thigh (n = 3 mice ⁄ group). The lower panel shows ROI analysis of the biolumines- cence image. Fold ratios of FLuc activities were normalized versus FLuc intensity on day 0. Experiments were performed in tripli- cate and results are expressed as mean ± SD. M. H. Ko et al. Bioimaging of miR9 and miR9* during neurogenesis FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS 2609 GLuc expression significantly after treatment with exogenous pre-miR9 or pre-miR9*. Both CMV ⁄ Gluc ⁄ 3xPT_mir9 and CMV ⁄ Gluc ⁄ 3xPT_mir9* reporter sys- tems demonstrated a great specificity of monitoring its cognate mature miR9 and miR9*, respectively. In vitro bioluminescent Luciferase assays of the unequally expressed mature miR9 and miR9* during neurogenesis were conducted in P19 cells treated with retinoic acid for 4 days. The CMV ⁄ Gluc ⁄ 3xPT_mir9 or CMV ⁄ Gluc ⁄ 3xPT_mir9* construct was transfected into P19 cells and GLuc activities, representing the endogenous levels of mature miR9 or miR9*, were measured and normalized versus CMV_Gluc (Fig. 3D). As observed for mature miR9 or miR9* during the neuronal differentiation of P19 cells by real-time PCR, GLuc expressions of CMV ⁄ Gluc ⁄ 3xPT_ mir9 and CMV ⁄ Gluc ⁄ 3xPT_mir9* were both significantly lower in neuronally differentiated than in undifferentiated P19 cells and were observed to gradu- ally decreased during the neuronal differentiation. In addition, the GLuc signal of CMV ⁄ Gluc ⁄ 3xPT_mir9 was relatively smaller than that of CMV⁄ Gluc ⁄ 3xPT_ mir9* throughout the investigation, which implies a A B C D Fig. 3. Gene expressions of mature miR9 and miR9* during neuro- nal differentiation. (A) Using real-time PCR. The gene expressions of endogenous mature miR9 and miR9* during the neuronal differ- entiation of P19 cells treated with retinoic acid for 6 days were determined. The expressions of mature miR9 and miR9* gradually increased during neurogenesis, but mature miR9 was found to be expressed 1.4-fold more than mature miR9*. The line with a trian- gular head represents the fold ratio of miR9 to miR9* expression values (right y-axis). The left y-axis shows the real-time PCR intensi- ties of each mature miRNAs normalized versus U6 snRNA (C T =C T-before –C T-day, C T =C T-miRNA –C T-U6RNA ). Experiments were performed in triplicate and results are expressed as mean ± SD. (B) Schematic diagram of the reporter genes used to monitor mature miR9 and miR9*. The black and gray boxes indicated three copies of perfectly complementary sequences of mature miR9 and miR9*, respectively. These three copies were located between the stop codon and the polyadenylation sequence of the GLuc gene. These systems were designed to repress GLuc expression when mature miR9 and miR9* were present. (C) Specification of the recombination constructs CMV ⁄ Gluc ⁄ 3xPT_mir9 and CMV ⁄ Gluc ⁄ 3xPT_mir9* regulated by miR9 and miR9*, respectively. The GLuc activities of CMV ⁄ Gluc ⁄ 3xPT_mir9, CMV ⁄ Gluc ⁄ 3xPT_mir9*, or CMV ⁄ Gluc ⁄ 3xPT_mir23a constructs were normalized using CMV_Gluc vector and determined at five different concentrations (n M on the x-axis) of exogenous pre-miR9 or pre-miR9* in HeLa cells. Experiments were performed in triplicate and results are shown as means ± SDs. (D) In vitro Luciferase assay of endoge- nous mature miR9 and miR9* during the neuronal differentiation of P19 cells. CMV ⁄ Gluc ⁄ 3xPT_mir9 or CMV ⁄ Gluc ⁄ 3xPT_mir9* were transfected and endogenous levels of mature miR9 and miR9* in P19 cells treated with retinoic acid were determined. The y-axis represents the fold ratio of GLuc expression during neuronal differ- entiation versus GLuc expression from undifferentiated P19 cells; the white bar represents mature miR9 and the black bar mature miR9*, and the line with a triangular head indicates the ratio (numerical values shown on the top of bar) of mature miR9 to miR9*. Experiments were performed in triplicate and results are expressed as mean ± SD. Bioimaging of miR9 and miR9* during neurogenesis M. H. Ko et al. 2610 FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS higher endogenous level of mature miR9 than of mature miR9*. In vivo visualization of unbalanced expressions of mature miR9 and miR9* during neurogenesis To monitor in vivo the endogenously unequal expres- sion of mature miR9 and miR9* during neuronal dif- ferentiation in P19 cells, CMV ⁄ Gluc ⁄ 3xPT_mir9, CMV ⁄ Gluc ⁄ 3xPT_mir9* or CMV_Gluc (a negative control), were transfected into 2.5 · 10 6 of P19 cells and subcutaneously implanted into nude mice in the presence or absence of retinoic acid (Fig. 4A). In addi- tion to in vivo imaging of endogenous mature miR9 or miR9* during neurogenesis, CMV_Fluc vector, which expressed constant FLuc activity regardless of the pres- ence of mature miR9 or miR9* or retinoic acid, was cotransfected with CMV ⁄ Gluc ⁄ 3xPT_mir9 or CMV ⁄ Gluc ⁄ 3xPT_mir9* into P19 cells as an internal control. FLuc activities of left thighs not treated with retinoic acid and of treated right thighs showed no significant change after CMV ⁄ Gluc ⁄ 3xPT_mir9 or CMV ⁄ Gluc ⁄ 3xPT_mir9* transfection (Fig. 4B,C, lower). GLuc signals, as determined by ROI analysis, from A B C Fig. 4. In vivo Luciferase imaging of the mature miR9 and miR9* during the neuronal differentiation. (A) The strategy used to implant P19 cells into mice to image mature miR9 and miR9* during neurogenesis. CMV_Gluc tranfected into right shoulders was used to normalize GLuc signals and CMV_Fluc transfected into left and right thighs was used as an internal control. (B,C) In vivo imaging of mature miR9 and miR9* during the neuronal differentiation of P19 cells treated with retinoic acid. FLuc signals of CMV_Fluc in the lower panel showed similar strong expressions in the presence (right thigh) or absence (left thigh) of retinoic acid. ROI analysis results in the right panel and bioluminescence imaging results in the left upper panel show that the GLuc activi- ties of CMV ⁄ Gluc ⁄ 3xPT_mir9 in right thighs (B) and left shoulders (C) were more repressed during the neuronal differentiation of P19 cells than CMV ⁄ Gluc ⁄ 3xPT_mir9* in left shoulders (B) and right thighs (C). ROI fold ratios were normalized versus GLuc intensity at 0 h. Experiments were per- formed in triplicate and results are expressed as mean ± SD. M. H. Ko et al. Bioimaging of miR9 and miR9* during neurogenesis FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS 2611 CMV ⁄ Gluc ⁄ 3xPT_mir9 with retinoic acid in right thighs were dramatically reduced compared with CMV ⁄ Gluc ⁄ 3xPT_mir9 without retinoic acid in left thighs, and had almost disappeared 2 days after neuro- nal differentiation (Fig. 4B,C, upper). Similarly, CMV ⁄ Gluc ⁄ 3xPT_mir9* in right thighs showed signifi- cant GLuc repression during the neuronal differentia- tion of P19 cells treated with retinoic acid compared with left thighs not treated with retinoic acid. Fold ratios of ROI of CMV⁄ Gluc ⁄ 3xPT_mir9 and CMV ⁄ Gluc ⁄ 3xPT_mir9* on day 1 between pre- and post differentiation were 6- and 14-fold, respectively. The bioluminescent signals and ROI analysis in Fig. 4B,C showed that CMV ⁄ Gluc ⁄ 3xPT_mir9 had higher repression of the GLuc intensity during neuro- genesis than CMV ⁄ Gluc ⁄ 3xPT_mir9*, indicating that mature miR9 are relatively more expressed than miR9* during neuronal differentiation of P19 cells treated with retinoic acid. Discussion Thousands of miRs proven by the cloning of hundreds of miRs from various species have been identified by bioinformatics analysis [22] and tens of miRs have been reported to be related to specific tissue develop- ment, cellular differentiation, proliferation, apoptosis, and various diseases including cancers, cardiovascular diseases, neurological diseases and metabolic disorders [4,5]. Even though the regulation and functions of miRs are unclear, the basic molecular mechanisms of miR biogenesis in cells have been shown to be pro- cessed into the primary, precursor, and mature form of miRs by RNA polymerase II, Drosha, exportin-5, Dicer and RNA-induced silencing complex [1,11,12]. However, cellular gene-expression analysis of miRs has been restricted to laborious and irreproducible meth- ods like in situ hybridization and northern blotting. Moreover, these methods have been used to detect only endogenous mature miRs in cells [17,23]. Few studies have examined miRs to determine initial gene expression associated with miR biogenesis using the upstream region of miRs, which is considered a pro- moter. Using the developed bioluminescent imaging system to monitor pri-miR9-1 we found that pri-miR9- 1 is highly and specifically expressed in neurons during the retinoic acid-induced differentiation of P19 cells. The upstream region of the pri-miR9-1 from )530 to )44 bp was found to show substantial promoter activ- ity during neurogeneis, whereas other constructs with longer or shorter fragments were not found to be effec- tive enough to monitor differences in endogenous pri-miR9 expression during the neuronal differentia- tion of P19 cells. Recently, a number of important transcription factors, such as repressor element silenc- ing transcription factor, cAMP response element-bind- ing protwin, Nanog, and Octamer4 have been suggested to be involved in the transcriptional regula- tion of neuronal miRs [24–26]. Unfortunately, the upstream region of the pri-miR9-1 does not have any homologue-binding sequence for the transcription factors that are required to maintain the neuronal differentiation of stem cells. Moreover, the molecular mechanisms of the bioge- neses of a number of miRs are equivocal. miR23a, which was previously reported upon by our laboratory, showed unbalanced biogenesis in pri-miR23a and resultant mature 23a in HEK293 cells, but not in HeLa cells and P19 cells [19]. Highly expressed pri- miR23a produced a relatively low endogenous level of mature miR23a in HEK293 cells, indicating a slow turnover from pri-miR23a to mature miR23a. In this study, real-time PCR and in vitro and in vivo biolumi- nescent imaging demonstrated relatively higher expres- sion levels of mature miR9 than of miR9* during the neuronal differentiation of P19 cells treated with reti- noic acid, even though both mature miRs probably originated from the hairpin sequences of the same pre- miR. For strand selection from the secondary structure of pre-miRs to be a single-stranded mature miR, ther- modynamic profiling of duplex pre-miR hairpin showed that in general, the 5¢ terminal sequence of pre-miR hairpin has less internal stability than the 3¢ terminal sequences of the pre-miRs, which implies that the mature miRs prefer miRs to miR*s [13,27]. How- ever, this hypothesis is not applicable to several miRs cloned from several species. miR18, miR106, miR16 and miR105 have a 5¢-end of precursor form in the mature form and miR142, miR17, miR302, miR373 and miR9 have both ends in mature forms [7,16,17,24]. Even though the molecular mechanism of miR biogenesis is still unclear, interestingly, northern blotting and microarray analysis using human brain tissues also demonstrated that miR9 is more highly expressed than miR9* [6,17]. Our previously reported dual Luciferase system will provide clearer and simul- taneous imaging of this phenomenon during miR bio- genesis [19]. The endogenous expression of mature miR9 has been recently reported to contribute to the develop- mental shift from neuron generation to glial cell gener- ation, and to be related to the expression of granuphilin ⁄ slp4 in insulin-producing cells [28]. More- over, miR9 and other neuronal miRs including miR125b and miR128, are involved in the Alzheimer’ disease [29]. Bioimaging of miR9 and miR9* during neurogenesis M. H. Ko et al. 2612 FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS Our noninvasive bioluminescent imaging systems devised to monitor miR9 can also be usefully applied to study and monitor the biogenesis of other miRs related to neuronal development, differentiation, and neuronal diseases. In addition, the in vitro and in vivo imaging systems will undoubtedly provide information about the molecular patterns and mechanisms of miR biogenesis in various heterogeneous cells. Experimental procedures Recombinant constructions of reporter gene to monitor primary and mature form of miR9 To detect the transcript level of pri-miR9, the upstream region of pri-miR9-1 was isolated from the genomic DNA of HeLa cells and cloned into a promoterless vector, pGL3_Basic vector (Promega, Madison, WI) containing the ORF of FLuc. Five different fragments, which were fused into the HindIII site of the reporter gene and designated miR9-1PN1_Fluc, miR9-1PN2_Fluc, miR9-1PN3_Fluc, miR9-1PN4_Fluc, and miR9-1PN5_Fluc, were amplified using the primer pairs listed in Table 1, and then sequenced to determine the orientation of the fragments in the repor- ter vector. These constructs were then transfected into P19 cells by liposome-mediated transfection using a Lipofectin reagent kit (Invitrogen, Grand Island, NY, USA) and FLuc activity was monitored during the neuronal differentiation of P19 cells treated with retinoic acid (retinoic acid). To study the mature forms miR9 and miR9*, mature sequences of has-miR-9 and mature has-miR-9* were obtained from the MirnaMap database (http://mirna- map.mbc.nctu.edu.tw) and oligonucleotides containing three copies of a perfectly complementary sequence of mature miR9 or miR9* were synthesized (Table 1). Each pair of sense and antisense oligos was annealed in annealing buffer (·1 TE buffer + 50 mm NaCl) for 10 min at 60 °C and ligated into the XhoI and XbaI sites of CMV_Gluc vec- tor (Targeting Systems, San Diego, CA, USA) to create CMV ⁄ Gluc ⁄ 3xPT_mir9 and CMV ⁄ Gluc ⁄ 3xPT_mir9*. Cell culture and neuronal induction of P19 cells P19 (a mouse embryonic carcinoma cell line) was purchased from the American Type Culture Collection (Manassa, VA, USA). P19 cells were grown in a-MEM (Gibco, Grand Island, NY, USA) supplemented with 2.5% fetal bovine serum (Cellgro, Herndon, VA, USA), 7.5% bovine calf serum (Gibco), and 1% antibiotics–antimycotic (Cellgro) [30]. To induce neuronal differentiation, P19 cells were cul- tured under serum-free conditions in Dulbecco’s modified Eagle’s medium ⁄ 12(1 : 1) media (Gibco) supplemented with insulin, transferring, and selenium (ITS; Gibco) and then treated with 5 · 10 )7 m all-trans retinoic acid (Sigma, St Louis, MO, USA) for 3 days. HeLa cells (an adenocarci- noma cell line) were cultured routinely in RPMI (Jeil Bio- techservices Inc, Daegu, Korea) containing 10% fetal bovine serum and 1% antibiotics–antimycotic. Transfection of CMV ⁄ Gluc ⁄ 3xPT_mir9 & CMV ⁄ Gluc ⁄ 3xPT_mir9* and precursor miR9 & miR9* P19 cells were seeded at 0.6 · 10 5 (undifferentiated), 0.8 · 10 5 (1 day), 0.6 · 10 5 (2 days), 0.4 · 10 5 (3 days), and 0.2 · 10 5 (4 days) in a six-well plate 24 h prior to Table 1. Primers used to amplify pri-miR9-1 promoter and clone the perfect target sequence of mature miR9 and miR9*. Name of primer Sequence (5¢-to3¢) Primer applications miR9-1PN1 GCA CAA GCT TGG AGT GTG AAA GGA TGA Amplify 1343mer promoter fragment miR9-1PN2 GCA CAA GCT TCT TTC CTC CCC TCC GCC CCT CTC AT Amplify 802mer promoter fragment miR9-1PN3 GCA CAA GCT TCA CCG CGG CTC CCC ATT TCC ATC Amplify 486mer promoter fragment miR9-1PN4 GCA CAA GCT TGG TCA TCG CGT CCT TTC CAC GCC Amplify 192mer promoter fragment miR9-1PN5 GCA CAA GCT TTC ACC CTC CCC CTC AAC TCC ACT AG Amplify 91mer promoter fragment miR9-1P reverse GCA CAA GCT TGC CGC CGC CGC CAG CAC CTC Amplify N1–N5 promoter fragments Perfect target seq. of miR9 sense TCGAGAATCTAGT TCA TAC AGC TAG ATA ACC AAA GA TAGTA TCA TAC AGC TAG ATA ACC AAA GA TAGTA TCA TAC AGC TAG ATA ACC AAA GAT Construction of CMV ⁄ Gluc ⁄ 3xPT_mir9 Perfect target seq. of miR9 anti-sense CTAGA TCT TTG GTT ATC TAG CTG TAT GA TACTA TCT TTG GTT ATC TAG CTG TAT GA TACTA TCT TTG GTT ATC TAG CTG TAT GA ACTAGATTC Construction of CMV ⁄ Gluc ⁄ 3xPT_mir9 Perfect target seq. of miR9* sense TCGAGAATC TAG TAC TTT CGG TTA TCT AGC TTT A TAGTA ACT TTC GGT TAT CTA GCT TTA TAGTA ACT TTC GGT TAT CTA GCT TTAT Construction of CMV ⁄ Gluc ⁄ 3xPT_mir9* Perfect target seq. of miR9* anti-sense CTA GAT AAA GCT AGA TAA TTG AAA GT TACTA TAA AGC TAG ATA ACC GAA AGT TACTA TAA ACG TAC ATA ACC GAA AGT ACTAGATTC Construction of CMV ⁄ Gluc ⁄ 3xPT_mir9* M. H. Ko et al. Bioimaging of miR9 and miR9* during neurogenesis FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS 2613 tranfection. Transient transfections were performed using 1 lg of DNA using lipofectamine (Invitrogen). HeLa (a miR9 non-producing cell line) was seeded at 1 · 10 5 cells to determine the expressions of miR9 and miR9*. Luciferase assays for FLuc and GLuc activities P19 and HeLa cells were washed with NaCl ⁄ P i and treated with lysis buffer (200 lLÆwell )1 ) for Luciferase assays. Lysed cells were transferred to a 96-well white microplate and Luciferase activities were measured using luminometer (TR717; Applied Biosystems, Foster City, CA, USA) and an exposed time of 1s. All data are presented as means ± SD calculated from triplicate wells. RT-PCR analysis in undifferentiated and differentiated P19 cells Total RNA was isolated from cultured cells using Tri- zol reagent (Invitrogen). Reverse transcription to synthesize first-strand cDNA was carried out using random-hexamer primer and SuperScript II reverse transcriptase (Invitrogen), according to the manufacturer’s instructions, and used as a template for PCR amplification. PCR amplifications of MAP2 and b-actin cDNA were performed using i-Taq DNA polymerase (Table 2) (iNtRON; Korea). PCR prod- ucts were loaded on agarose gels containing ethidium bromide, and bands were revealed under UV. For pri- miR9-1, pri-miR9-2 and pri-miR9-3, first-strand cDNA synthesis was carried out using random-hexamer primer and promoter primers (Table 2). Quantitative RT-PCR of mature miR9 and miR9* Small RNA was isolated from cultured cells using mirVana miRNA isolation kits (Ambion, Austin, TX, USA), and qRT-PCR was performed using mirVana TM qRT-PCR miR detection kits (Ambion) using a has-miR-9 or a has-miR-9* primer set (Ambion) according to the manufacturer’s instructions. To normalize experimental samples for RNA content, the U6 snRNA primer set (Ambion) was used as a control. In vivo visualization of primary miR9 expression or mature miR9 and miR9* expressions in undifferentiated and differentiated P19 cells The miR9-1PN3_Fluc construct was transfected into P19 cells, which were divided into retinoic acid-treated and non- retinoic acid treated groups for in vivo imaging. At 48 h after transfection, 1 · 10 6 P19 cells were harvested with 100 lL NaCl ⁄ P i , and resuspended with retinoic acid for the neuronal differentiation group. P19 cells were then subcuta- neously injected into each thigh of 6-week-old male Balb ⁄ c nude mice, 3 mg of d-luciferin was administered intraperi- toneally [31]. This study was approved by the IACUC (Institutional Animal Care and Use Committee) of Clinical Research Institute, Seoul National University Hospital (AAALAC accredited faculty). Bioluminescence images were acquired using an IVIS100 (In vivo Imaging System; Xenogen, Alameda, CA, USA) with the integration time of 5 min. For in vivo GLuc imaging, nude mice were imaged using the IVIS100 system after direct administering 50 lg of coelenterazine. Acknowledgements This study was supported by Nano Bio Regenomics Project of Korean Science and Engineering Founda- tion and by Innovation Cluster for Advanced Medical Imaging Technology. This study was made easier using KREONET, Korean Research Network, a nationwide giga-bps network system. References 1 Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR & Ruvkun G (2000) The 21-nucleotide let-7 RNA regulates Table 2. Primers used in real-time polymerase chain reaction (RT-PCR). Name of primer Sequence (5¢-to3¢) Primer applications Mouse 9-1 upper TCA TAA AGC TAG ATA ACC GAA GAT RT-PCR for expression of pri-miR9-1 Mouse 9-1 lower TCC AGA GGC GAC CCC AGA GC RT-PCR for expression of pri-miR9-1 Mouse 9-2 upper TTG GTT ATC TAG CTG TAT GAG TGT RT-PCR for expression of pri-miR9-2 Mouse 9-2 lower AGC CTG GCC CCT TTA GAT TTC RT-PCR for expression of pri-miR9-2 Mouse9-3 upper GAG TGC CAC AGA GCC GTC ATA AA RT-PCR for expression of pri-miR9-3 Mouse9-3 lower CTG GGT GGC GTG GGC TTC TCT GG RT-PCR for expression of pri-miR9-3 MAP2 forward CCC AAG AAC CAA CAA GAT GAA RT-PCR for neuronal differentiation MAP2 reverse AAT CAA GGC AAG ACA TAG CGA RT-PCR for neuronal differentiation b-actin forward TGA CGG GGT CAC CCA ACT GTG CCC ATC TA Control b-actin reverse CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG Control Bioimaging of miR9 and miR9* during neurogenesis M. H. Ko et al. 2614 FEBS Journal 275 (2008) 2605–2616 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... 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