MicroRNAs (miRNAs) are a class of small noncoding RNAs that are processed by Dicer from precursors with a characteristic hairpin secondary structure (Ambros et al., 2003). Hundreds of miRNAs have been identified from plants, animals, and viruses (miRBase; http://microrna.sanger.ac.uk/sequences/). miRNAs are implicated in various cellular processes, such as cell fate determination, cell death, and tumorigenesis (for review see Bartel, 2004). Many miRNAs are expressed in a tissue-specific manner (Lagos-Quintana et al., 2002; Babak et al., 2004; Barad et al., 2004; Liu et al., 2004; Sempere et al., 2004; Thomson et al., 2004; Baskerville and Bartel, 2005; Wienholds et al., 2005), suggesting a role of the miRNAs in the specification of the tissue during differentiation.

Among the hundreds of miRNAs, only a small fraction have assigned target mRNAs or an established role. Valid target prediction is a major problem in the study of miRNAs. Although several algorithms for target prediction have been based on sequence similarity between targets and miRNAs (Bentwich, 2005), the small size of the miRNAs and the tolerance for mismatches and bulges in the recognition sequence result in most of these algorithms' predicting too many targets.

The mode of action of miRNAs on their targets is controversial. Classic results from lin-4 miRNAs suggested that the miRNAs bind to their targets with imperfect complementarity and decrease the levels of encoded proteins without decreasing the target mRNA (Olsen and Ambros, 1999; Seggerson et al., 2002). In contrast, target mRNA is cleaved specifically at the recognition site by siRNA (Elbashir et al., 2001b), many plant miRNAs (for reviews see Kidner and Martienssen, 2005; Millar and Waterhouse, 2005), and at least one animal miRNA (Yekta et al., 2004). In all cases where the target mRNA is cleaved, the interaction between the small RNA and the target mRNA is nearly perfect. Therefore, the degree of complementarity has been thought to be a major determinant in dictating whether a miRNA promotes mRNA degradation or inhibits protein synthesis. Although this hypothesis is supported by mutation analyses of miRNAs and their target mRNAs (Doench et al., 2003; Saxena et al., 2003), a recent report demonstrated that a miRNA can regulate the levels of several target mRNAs despite mismatches and bulges between the miRNA and the targets (Lim et al., 2005). This was shown true for even lin-4 and let-7 miRNAs (Bagga et al., 2005), which had been thought to block only the translational step.

Differentiation down a specific lineage is characterized by the activation of tissue-specific transcription factors and modulation of gene expression. To study the role of miRNA in such a process and begin the process of identifying potential targets, we studied muscle differentiation using the C2C12 myoblast (MB) cell line as a model system (Yaffe and Saxel, 1977; Andres and Walsh, 1996). Upon serum depletion, muscle-specific transcription factors such as myogenin are induced and many muscle genes are turned on. Subsequently, cells become elongated and fused to each other to form multinucleate myotubes (MTs). Another critical event during the differentiation process is a decrease in DNA synthesis and cell cycle arrest. We show here that miRNAs miR-206, -1, and -133 on their own change the gene expression profile of C2C12 toward the differentiated state and that miR-206 induces many of the markers of differentiation. The miRNAs regulate many target mRNAs as revealed by microarray screening. Antisense oligonucleotides to these miRNAs inhibit muscle differentiation and entry into cell quiescence. We predicted the putative direct targets of miR-206 by intersecting the mRNAs down-regulated in the microarray data with the computational prediction of targets based on sequence match to the miRNA. As an example of the utility of this approach, we identify four mRNAs, including that of the largest subunit (p180 subunit; Pola1) of DNA polymerase α (DNA pol α) as being directly regulated by miR-206. Two of the targets, including DNA pol α, are cleaved at multiple sites by the miRNA. The effect of miR-206 on DNA pol α, thereby DNA synthesis, is a new example of miRNA function connecting the cell quiescence event with the differentiation process. In addition, inhibitors of myogenic transcription factors are indirectly down-regulated by miR-206, thereby further promoting the differentiation process. Recently, another group reported a critical role of miR-1 and -133 in C2C12 differentiation (Chen et al., 2006). Together, we can conclude that all three miRNAs induced during C2C12 differentiation are very important for myogenesis.

Figure 1. The muscle-specific expression of the miRNAs miR-1, -133, and -206. (A) RNase protection assays were performed with RNA from various tissues to detect miR-1, -206, and -133. 5S ribosomal RNA (rRNA) was used as a control. The different panels have different (more ...)

C2C12 mouse MBs can be induced to differentiate into MTs by serum depletion, as indicated here by the induction of myogenin, cell cycle inhibitor p21, and myosin heavy chain (MHC) with a constant level of Cdk4 as a loading control (Fig. 1 B). miR-1, -133, and -206 were not expressed in undifferentiated C2C12 but were induced during muscle differentiation (Fig. 1 B; Chen et al., 2006), whereas two other miRNAs (let-7 and miR-125b) were expressed constantly throughout the differentiation process. Using this in vitro differentiation of C2C12, we studied whether the miRNAs play an active role during skeletal myogenesis.

Figure 2. The muscle-specific miRNAs induce the differentiation of MBs by regulating many genes. (A) Synthetic oligonucleotides complementary to miR-1, -133, and -206 were inserted downstream of Renilla luciferase ORF in pRL-CMV(MCS) to generate pRL1, pRL133, and (more ...)

We began our studies with miR-206 because of the skeletal muscle–specific expression of miR-206 (Fig. 1 A) and because of its similarity to miR-1. miR-206 transfection advanced MHC expression after changing to differentiation medium (DM; Fig. 2 C), whereas miR-133 did not (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200603008/DC1) under the same experimental condition.

Upon transfection in the continued presence of serum, miR-206 markedly up-regulated the percentage of cells expressing MHC and the muscle-specific transcription factor myogenin, relative to cells at comparable density transfected with GL2 control (Fig. 2, D and E; and Fig. S1, B and C). In addition, 28% of MHC-positive cells are multinucleated after miR-206 treatment, whereas no multinucleated cells were detected in GL2 control (Fig. 2 D and not depicted). Therefore, physiological levels of miR-206 induce skeletal myogenesis from C2C12 even without serum depletion.

To measure the extent of differentiation and to extend the studies to the other muscle-specific miRNAs, microarray experiments were used to measure changes in global gene expression profile after muscle differentiation by serum deprivation (DM). The microarray profile after differentiation was compared with that obtained after miRNA transfection in the presence of serum (growth medium [GM]). We focused our analysis on the ~100 genes that were induced or repressed the most after C2C12 differentiation to MTs by serum depletion (Fig. 2 F). Genes up- or down-regulated after muscle differentiation were changed similarly by transfection of the miRNAs (in GM), with miR-206 and -1 having similar effects and miR-133 affecting a slightly different subset of genes. Given the importance of the first eight nucleotides of miRNA in target interaction (Doench and Sharp, 2004), the identical seed sequences between miR-1 and -206 explain why the two miRNAs had almost identical changes in gene expression. These data suggest that the individual miRNAs change the repertoire of expressed genes in a direction mimicking that seen during muscle differentiation. Further analysis of the microarray data will be addressed in the next section.

To determine whether the miRNAs were essential for differentiation, C2C12 cells were treated with 2′-O-methyl antisense oligonucleotides against the miRNAs (Hutvagner et al., 2004; Meister et al., 2004) and then induced to differentiate by serum depletion. Inhibition of the miRNAs reduced the number of MHC-positive cells (Fig. 3, A and B) and decreased MHC levels in an immunoblot (Fig. 3 C), with little effect when only miR-133 was inhibited. BrdU immunostaining shows that a significant fraction of the cells remain in active DNA synthesis when the miRNAs are inhibited (42% for the anti-miR mix relative to 26% for the anti-GL2 control; P = 0.0071; Fig. 3, A and B). Inhibition of the miRNAs prevented the elongation of MTs seen during differentiation (Fig. 3 D). The cells remained relatively short and thick even after expression of MHC: 86% of MHC-positive cells in the anti-GL2 control sample were elongated, as opposed to only 25% in the anti-miR mix sample. Although inhibition of miR-1 and -206 (without inhibition of miR-133) was sufficient to decrease MHC expression and derepress DNA synthesis (Fig. 3 B), it was not sufficient to inhibit cell elongation (not depicted), suggesting that miR-133 might have specific targets relevant to MT elongation. Collectively, the results suggest that the three miRNAs are required for complete differentiation to muscle, with miR-206 (and miR-1) being particularly important for induction of cell quiescence.

Figure 3. Inhibition of the miRNAs by 2′-O-methyl antisense oligonucleotides inhibits differentiation. (A) C2C12 cells transfected with antisense oligonucleotide against GL2 or a mixture of antisense oligonucleotides against miR-1, -133, and -206 (anti-mix). (more ...)

In contrast, the list of down-regulated mRNAs (Fig. 2 F and Table S2, available at http://www.jcb.org/cgi/content/full/jcb.200603008/DC1) might include direct targets of the miRNAs if, as in plants, the miRNAs promote target RNA cleavage. To identify such direct targets, we intersected the list of genes down-regulated in the microarrays with the list of predicted genes with miRNA target sites (generated using the miRanda program [Enright et al., 2003]). We focused our analysis on miR-206 because of its similarity with miR-1 and its pronounced effect on muscle differentiation and cell proliferation (Figs. 2 and 3). On this intersection list, we were pleased to find the largest subunit of DNA pol α (Pola1; available from GenBank/EMBL/DDBJ under accession no. NM_008892.1), the replicative polymerase expected to be very important for cell proliferation.

miR-206 alone is sufficient to decrease Pola1 at the mRNA and protein levels as early as 24 h after transfection (Fig. 4 A). During differentiation of MBs to MTs, Pola1 mRNA and protein were also decreased (Fig. 4 A). The Pola1 promoter requires the activity of E2F, a transcription factor that is repressed by pRb during myogenesis (Kalma et al., 2001; Kitzmann and Fernandez, 2001), so we wondered how much of the down-regulation of Pola1 mRNA during differentiation was dependent on miR-206. Antisense to miR-206 significantly delayed the down-regulation of Pola1 mRNA (Fig. 4 D). This result is in agreement with the increase of BrdU-positive cells and decrease of MHC-positive cells when miR-206 was inhibited by antisense oligonucleotide (Fig. 3). The antisense to miR-206 does not, however, completely block differentiation (Fig. 3 B). Therefore, residual inhibition of Cdk2 kinase, hypophosphorylation of Rb, and repression of E2F probably account for the eventual repression of Pola1 in the presence of antisense to miR-206, albeit with delayed kinetics. Together, these results suggest that miR-206 is important for the early down-regulation of Pola1 during differentiation.

Figure 4. miR-206 directly down-regulates DNA pol α during differentiation. (A) Northern hybridization (top) and immunoblots (bottom) for the largest subunit of DNA pol α (Pola1) at 24 and 96 h after transfection of the indicated duplexes into C2C12 (more ...)

Pola1 has two potential target sites of miR-206 (Fig. 4 B; Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200603008/DC1; and see Fig. 7 A) in the 4991–5345 segment of the 3′UTR. Fusion of a luciferase reporter to this segment rendered luciferase repressible by miR-206 (Fig. 4 C). The level of repression by cotransfected miRNAs is comparable to the two- to threefold repression by miRNAs in all published experiments where a reporter gene is fused to naturally occurring target 3′UTRs (Krek et al., 2005; Lim et al., 2005; Yu et al., 2005). As a negative control, no repression was seen upon insertion of another part of the 3′UTR of Pola1 without any predicted target sites for miR-206 (DNA pol α 3985–4657). Point mutations in the target sites in pol α 4991–5345 revealed that the one encompassing nucleotides 5007–5029 (M1; Fig. 4, B and C; Fig. S5; and see Fig. 7 A) is necessary for the repression by miR-206, whereas the second site at 5090–5114 (M2) is not required. Therefore, miR-206 directly down-regulates Pola1 mRNA, a down-regulation that probably contributes to the prompt suppression of cell proliferation seen during differentiation. Pola1 is similarly down-regulated by miR-1 but not as much by miR-133 (Fig. 4 A and Fig. S2). However, miR-206 is likely to be more important than miR-1 for this function during C2C12 differentiation because it is present at a much higher level than miR-1 (Fig. 1 B).

Figure 7. The down-regulation of direct targets by miR-206 is posttranscriptional, dependent on target sites, and occurs through mRNA cleavage. (A) The schematic of B-ind1 mRNA (top) and 3′UTR of Pola1 mRNA (bottom). Vertical bars show potential binding (more ...)

Figure 5. Inhibition of DNA synthesis by the down-regulation of DNA pol α promotes withdrawal from the cell cycle in the continued presence of serum. (A) DNA synthesis (top) and cell growth (bottom) were measured after the transfection of GL2 (white bar) (more ...)

Next, we tested whether cells synchronized in the cell cycle responded to miR-206 with an arrest in G1. C2C12 cells were synchronized at the G1/S boundary by thymidine aphidicolin block (Fig. 5 D, middle). During the synchronization, we introduced miR-206 or GL2. Upon removal of aphidicolin, GL2-treated cells were released from G1 through the subsequent stages of the first cell cycle (2–8 h) and proceeded through the second cell cycle (8 h and later; Fig. 5 E). miR-206–treated cells were released into S phase with delayed kinetics (2–8 h), and the delay was even more marked in the second cell cycle (Fig. 5, D [bottom] and E [>8 h]). The time of second G1 (Fig. 5 E, 10–12 h) corresponds to 34–36 h after transfection. Because the kinetics of target repression after miR-206 transfection is likely to be slow, it is entirely expected that the cell cycle effect is more marked as the cell proceeds through successive cell cycles.

Figure 6. Examination of down-regulated genes from microarray to determine whether they are direct targets of miR-206. (A and C) Northern blots for MyoD inhibitors or down-regulated genes containing putative miRNA targets, as described in Fig. 4 A. (B and D) The (more ...)

The second approach to finding direct targets was to focus on genes that were decreased in the microarrays after miRNA introduction and contained putative target sites in the 3′ UTR. These include a Rac1 interacting protein named butyrate-induced transcript 1 (B-ind1), brain-derived neurotrophic factor, the gap-junction protein connexin43 (Cx43), nuclear receptor coactivator 5, AMP activated protein kinase, Hhip, junction adhesion molecule 4, and an adiponectin-related protein expressed in microglia called monocyte-to-macrophage differentiation-associated protein (Mmd). All of these mRNAs were decreased by normal differentiation and by transfection of the miRNA (Fig. 6 C). Although all of them have predicted target sites (Table S3), fusion to luciferase reporter reveals that only B-ind1, Cx43, and Mmd are direct targets of miR-206 like DNA pol α (Fig. 6 D).

Of the four direct targets, Pola1 has already been shown to be important for cell quiescence. B-ind1 might influence differentiation through its interaction with the G protein Rac-1. Down-regulation of B-ind1 mRNA was inversely related to the up-regulation of miR-206 during differentiation and was abrogated when miR-206 was inhibited by anti–miR-206 (Fig. 6 E). We do not have any evidence, however, that B-ind1 down-regulation is critical for differentiation. The known functions of Cx43 and Mmd do not suggest that their repression is critical for differentiation.

Nuclear run-on experiments were performed to determine whether there is any transcriptional repression of these direct target genes induced during differentiation or after introduction of miRNAs miR-1 or -206. Transcriptional activity of Pola1 was too low to be measured by nuclear run-on (unpublished data), but that of B-ind1 was measured and found to not decrease after C2C12 differentiation or after the introduction of miR-1 and -206 (Fig. 7 D). The Northern blots that accompanied this experiment show that the steady-state mRNA level of B-ind1 is repressed by at least fivefold (Fig. 7 D), suggesting that the miRNAs repress B-ind1 at the posttranscriptional level.

To test if the posttranscriptional reduction is due to the cleavage of mRNA by the miRNAs, we performed modified rapid amplification of cDNA ends (RACE)–PCR (Llave et al., 2002) to map the 5′ ends of potential cleavage fragments of B-ind1 and Pola1. Differentiated MTs (not depicted) and C2C12 MBs transfected with miR-206 and -1 (Fig. 7, E and F) generated more cleavage products from the B-ind1 transcript compared with the MBs or cells transfected with either GL2 control or miR-133. Similar results were obtained from Pola1 (unpublished data), supporting the hypothesis that the mRNAs are cleaved in the presence of the miRNAs. The sizes of RACE-PCR products derived from the B-ind1 (Fig. 7 E) or Pola1 (not depicted) after transfection of miR-206 are distinct from those obtained after transfection of miR-1 (Fig. 7 E and Fig. S3). As miR-1 and -206 recognize similar target sites and similarly decreased B-ind1 (Fig. 7 D), the different cleavage sites between the two miRNAs suggest that the miRNAs do not select the cleavage sites. To support this idea, sequencing of the RACE-PCR products revealed that the 5′ ends of cleavage fragments mapped at multiple sites in the B-ind1 mRNA (Fig. 7 A and Fig. S3) and were not confined to putative target sites of miR-206. B-ind1 mRNA was cleaved similarly during differentiation (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200603008/DC1). DNA pol α mRNA was cleaved by transfection of miR-206, and these cleavage sites also did not match with putative target sites (Fig. 7 A and Fig. S5). In contrast, siRNAs cleave the target mRNA at the center of RNA duplex by RISC (RNA-induced silencing complex; Elbashir et al., 2001b; Yekta et al., 2004). A control experiment with miR-206 siRNA against a perfectly matching site in luciferase 3′UTR showed that cleavage in this case was exactly at the siRNA target site (unpublished data). Thus, although the down-regulation of B-ind1 and Pola1 mRNA depends on miR-206 target sites with multiple mismatches and bulges, and although the target mRNA is cleaved, the 5′ ends of the cleavage fragments are distributed and do not match the target sites.

We show that miRNAs not only promote differentiation of MBs in vitro but are required for differentiation. Individual miRNAs like miR-206 produce large changes in gene expression at the mRNA level that mimic changes seen upon differentiation, and the down-regulation of a direct target of miR-206, Pola1, might contribute to the cell cycle quiescence upon differentiation. In addition, several inhibitors of myogenic transcription factors are repressed after miR-206 transfection, leading to further amplification of the prodifferentiation function. At least two of the direct targets are cleaved at the posttranscriptional level, but the 5′ ends of the cleavage fragments suggest the involvement of exonucleases, a finding that has implications for the proposed involvement of processing bodies (P-bodies) in miRNA function. Finally, we describe an efficient strategy for identifying direct targets of miRNAs by intersecting mRNAs down-regulated by miR-206 in microarray screens with computationally predicted targets and then checking the intersection list by transient transfection and luciferase fusion assays.

Transfection of miR-1 in a nonmuscle cell has been reported to alter the gene expression profile toward that of muscle (Lim et al., 2005), but there was no indication of the magnitude of the change or the importance of the miRNAs in the physiology of muscle differentiation. Our results suggest that muscle-specific miRNAs promote skeletal myogenesis through direct down-regulation of several target genes and are required for normal differentiation. The microarray result indicates that the gene expression changes effected by miR-206 or -1 are closer to that of normal differentiation compared with that effected by miR-133. Because miR-206 is specifically expressed in skeletal muscle, whereas miR-1 is present in both skeletal and cardiac muscle, it would be interesting to compare miR-206 and -1 in greater detail to identify overlapping or unique roles in myogenesis.

A block to DNA synthesis through the direct down-regulation of DNA pol α precedes the cell cycle withdrawal and is the first demonstration of miRNAs directly affecting DNA replication. This result is supported by the recent observation that overexpression of miR-1 decreased the pool of proliferating ventricular cardiomyocytes in transgenic mice (Zhao et al., 2005). It is not clear yet whether down-regulation of the three other targets identified, B-ind1, Cx43, and Mmd, contributes in any way to the muscle differentiation program. To fully understand the role of miR-206 in skeletal myogenesis, we expect to uncover more targets of the miRNA in the future. Combinatorial down-regulation of multiple targets may be necessary to fully mimic the effect of miR-206 transfection into MBs.

miR-135b, -338 (Wienholds et al., 2005), and -181 (Naguibneva et al., 2006) are also muscle specific, suggesting that there may be additional miRNA involved in the regulation of myogenesis. miR-181 (Naguibneva et al., 2006), -1, and -133 (Chen et al., 2006) have already been shown to play a role in muscle differentiation by repressing their cognate targets, and our results add miR-206 to the list. We expect that extensive cooperation between several miRNAs and several transcription factors is necessary to effect the complete differentiation program.

The mode of action of miRNAs is still being debated. The multiple mismatches with target sites are believed to prevent cleavage of the target by a siRNA-like mechanism. Our results indicate, however, that at least for four endogenous targets, the repression by a miRNA is accompanied by a decrease in mRNA levels. For one of these direct targets (B-ind1), we rule out any transcriptional component, and for two of them (B-ind1 and Pola1), we show that the repression is accompanied by generation of mRNA cleavage fragments with multiple 5′ ends that do not map to the essential target sites matching the miRNA sequence. The exact 5′ ends vary and do not overlap when B-ind1 is down-regulated by the transfection of miR-206 or -1 or serum deprivation. This variability of the 5′ ends of the cleavage fragments suggests that they may be created by exonuclease activity after an initial endonucleolytic cleavage. RNA P-bodies have recently been proposed as sites where miRNAs sequester their target mRNAs from the protein-synthesis machinery (Liu et al., 2005; Sen and Blau, 2005). P-bodies contain exonucleases, so that the degradation of target mRNAs observed in this paper could be the end stage of the sequestration of targets in P-bodies. By serving as a common platform for both processes, P-bodies might explain how miRNAs can in some cases repress protein levels but not mRNAs and in other cases promote the degradation of mRNAs. An important point is that changes in mRNA profile during differentiation, thought to be mainly the province of transcription factors, can also be dictated by miRNA-mediated posttranscriptional cleavage of mRNAs.

The approach described here of intersecting mRNAs discovered to be down-regulated in microarray screens after miRNA transfection with mRNAs that have computationally predicted matches to miRNAs provides a high yield of potential targets. Fusion of candidate 3′UTRs to luciferase can then be used to quickly narrow down the list of targets to those that are directly down-regulated by miRNAs. Combining this strategy with in vitro differentiation systems is likely to yield many miRNA targets critical for differentiation down specific lineages. We are aware, of course, that this strategy might overlook targets that are exclusively down-regulated at the protein level, such as HoxA11 by miR-181 (Naguibneva et al., 2006) and histone deacetylase by miR-1 (Chen et al., 2006). If, however, down-regulation of protein synthesis by miRNAs involves sequestration of the target mRNAs to P-bodies, we suspect that many direct targets of miRNAs will eventually be down-regulated at the mRNA level and thus be revealed in the microarray screens. Because different computational algorithms predict different sets of targets for a given miRNA, a future extension of this work will be to intersect the microarray data with the output from other target prediction programs.

The ability of miRNAs to affect many mRNAs is similar to the ability of transcription factors to regulate many promoters simultaneously. Thus, just like transcription factors, we predict that miRNAs will induce complex changes in rate-limiting steps of cell metabolism and thus have a profound effect on the differentiation program. An additional point of interest is the interaction between these miRNAs and transcription factors known to be involved in muscle differentiation. For example, MyoD has already been suggested to be involved in the induction of miR-1 in cardiac myogenesis (Zhao et al., 2005). We suggest here that one of the muscle-specific miRNAs, miR-206, indirectly down-regulates Id1-3 and MyoR, inhibitors of myogenic transcription factors like MyoD. Such a positive feedback loop between the myogenic transcription factors and the muscle-specific miRNAs will be expected to push the equilibrium toward differentiation and could be very significant for holding muscle cells in a permanently differentiated state.

2′-O-methyl antisense oligonucleotides against miR-1, -133, and -206 and GL2 were synthesized by Dharmacon RNA Technologies. Transfection into C2C12 was performed with Lipofectamine 2000 reagent (Invitrogen), combined with 267 nM of each siRNA duplex or with 133 nM of 2′-O-methyl antisense oligonucleotide.

RNase protection assay was performed with miRNA detection kit (Ambion) with minor modifications. 20 μl of reaction contained the indicated amount of RNA, 0.6 μg of yeast tRNA, and the probe RNA, which was in vitro transcribed, labeled with α-[³²P]UTP using miRNA probe construction kit (Ambion), and purified from polyacrylamide gel. Digestion products were resolved by electrophoresis in an 18% polyacrylamide gel with 7 M urea.

At 20 h after transfection of the miRNAs and the luciferase plasmids into C2C12 cells, luciferase assays were performed with Dual-luciferase reporter assay system (Promega) per the manufacturer's instructions. Luminescent signal was quantified by luminometer (Monolight 3020; BD Biosciences). Each value from Renilla luciferase construct (Rr) was first normalized to the firefly (Photinus pyralis) luciferase assay value (Pp) from the cotransfected pGL3-control vector (Promega). Each Rr/Pp value was again normalized to the Rr/Pp value from miR-125b as a control. Each value is a mean of three transfections.

The luciferase constructs containing various putative target genes include B-ind1 (1248–2334 of accession no. NM_021345.1, available from GenBank/EMBL/DDBJ), brain-derived neurotrophic factor (700–1756 of accession no. NM_007540.3), Cx43 (82–2700 of accession no. BC006894), Hhip (800–2982 of accession no. NM_020259.3), junction adhesion molecule 4 (91–1491 of accession no. NM_028078.1), and Mmd (301–2510 of accession no. NM_026178.2). The indicated regions were PCR amplified and inserted into pRL-CMV.

This work was supported by grant RO1 CA89406 (to A. Dutta) from the National Institutes of Health.