[1] 16w Bioimaging of the unbalanced expression of microRNA9 and microRNA9* during the neuronal differentiation of P19 cells

[1] 75w 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 mammalian species and a dozen of these, including miR124a, miR9, miR128, miR131, miR178 and

[1] 274w 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 different 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 chromosome 7 (pri-miR9-3; Fig. 1A). The gene-expression levels of the primary transcripts of miR9 were monitored 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 neuronal marker gene, which was expected to occur during neuronal differentiation (Fig. 1C). The gene expressions of the three different pri-miR9s exhibited various transcript patterns during neuronal differentiation of P19 cells. Pri-miR9-1 showed a dramatic change in gene expression immediately after treatment with retinoic 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 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.

[2] 261w To monitor the endogenous expression of pri-miR9s using the bioluminescent Luciferase reporter gene system during neuronal differentiation in P19 cells, 1343 bp of the upstream region of the pri-miR9-1 from human genomic DNA was cloned and fused into a promoterless reporter vector, pGL3_Basic, which contained the ORF of the FLuc reporter gene (Fig. 2A). FLuc activity was measured to determine the promoter activity 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 constructs were then transfected into P19 cells and their promoter 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 endogenous level of pri-miR9-1 during neurogenesis. This indicates 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 findings indicate that the miR9-1PN3_Fluc construct could be used for in vivo imaging of gene expression of endogenous pri-miR9 during neurogenesis.

[3] 187w To image in vivo the endogenous expression of the pri-miR9 in small animals, 2.5 • 10 6 of P19 cells bearing the miR9-1PN3_Fluc construct were subcutaneously 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 shoulder, 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 bioluminescence reflects elevated endogenous pri-miR9-1 levels during the neuronal differentiation of P19 cells treated with retinoic acid.

[4] 137w 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 development [17]. To quantify their relative expression levels during neurogenesis, we conducted real-time PCR using small RNAs extracted from the neuronal differentiation 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.

[5] 280w 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 complementary 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 bioluminescent 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 GLuc expression significantly after treatment with exogenous pre-miR9 or pre-miR9*. Both CMV ⁄ Gluc ⁄ 3xPT_mir9 and CMV ⁄ Gluc ⁄ 3xPT_mir9* reporter systems demonstrated a great specificity of monitoring its cognate mature miR9 and miR9*, respectively.

[6] 158w 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 gradually 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 higher endogenous level of mature miR9 than of mature miR9*.

[7] 298w To monitor in vivo the endogenously unequal expression of mature miR9 and miR9* during neuronal differentiation 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 addition to in vivo imaging of endogenous mature miR9 or miR9* during neurogenesis, CMV_Fluc vector, which expressed constant FLuc activity regardless of the presence 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 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 neuronal differentiation (Fig. 4B,C, upper). Similarly, CMV ⁄ Gluc ⁄ 3xPT_mir9* in right thighs showed significant GLuc repression during the neuronal differentiation 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 neurogenesis 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.

[1] 299w 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 development, 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 processed 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 methods 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 promoter. 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 activity during neurogeneis, whereas other constructs with longer or shorter fragments were not found to be effective 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 silencing transcription factor, cAMP response element-binding protwin, Nanog, and Octamer4 have been suggested to be involved in the transcriptional regulation of neuronal miRs [24][25][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.

[2] 266w Moreover, the molecular mechanisms of the biogeneses 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 bioluminescent imaging demonstrated relatively higher expression levels of mature miR9 than of miR9* during the neuronal differentiation of P19 cells treated with retinoic 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, thermodynamic 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]. However, 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 simultaneous imaging of this phenomenon during miR biogenesis [19].

[3] 55w The endogenous expression of mature miR9 has been recently reported to contribute to the developmental shift from neuron generation to glial cell generation, and to be related to the expression of granuphilin ⁄ slp4 in insulin-producing cells [28]. Moreover, miR9 and other neuronal miRs including miR125b and miR128, are involved in the Alzheimer' disease [29].

[4] 58w 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.

[1] 13w Recombinant constructions of reporter gene to monitor primary and mature form of miR9

[2] 122w 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 reporter 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).

[3] 96w 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 vector (Targeting Systems, San Diego, CA, USA) to create CMV ⁄ Gluc ⁄ 3xPT_mir9 and CMV ⁄ Gluc ⁄ 3xPT_mir9*.

[4] 166w 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 cultured 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 adenocarcinoma cell line) were cultured routinely in RPMI (Jeil Biotechservices Inc, Daegu, Korea) containing 10% fetal bovine serum and 1% antibiotics-antimycotic. 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*.

[5] 94w Total RNA was isolated from cultured cells using Trizol 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 products 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).

[6] 7w Quantitative RT-PCR of mature miR9 and miR9*

[7] 60w 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.

[8] 19w In vivo visualization of primary miR9 expression or mature miR9 and miR9* expressions in undifferentiated and differentiated P19 cells

[9] 145w The miR9-1PN3_Fluc construct was transfected into P19 cells, which were divided into retinoic acid-treated and nonretinoic 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 subcutaneously injected into each thigh of 6-week-old male Balb ⁄ c nude mice, 3 mg of d-luciferin was administered intraperitoneally [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.

[1] 179w 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 demonstrated 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 neuronal differentiation. miR125b, have been found to be associated with polyribosomes 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 development.

[2] 251w In general, intergenic or intragenic miRs are transcribed into primary miRNA (pri-miR) by RNA polymerase II and processed into a 70-nucleotide hairpin-structured pre-miR in the nucleus by Drosha [8][9][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 singlestranded 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 RNAinduced silencing complex and bound to the 3¢-UTR of its target mRNA to induce either mRNA degradation 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].

[3] 138w Investigations into the gene expression of endogenous miR in cells or tissues are useful for understanding 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 timeconsuming, laborious, and non-reproducible. In a previous study, we successfully imaged miR23a biogenesis in small animals to noninvasively monitor the expression 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 proteins 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].

[4] 58w 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 bioluminescent 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.

[5] 64w P19 and HeLa cells were washed with NaCl ⁄ P i and treated with lysis buffer (200 lLAEwell )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.