To understand the potential involvement of miRNAs in skeletal muscle proliferation and differentiation, we analyzed the expression of miRNAs during skeletal muscle differentiation using an established microarray analysis9
. We used C2C12 myoblasts because these cells faithfully mimic skeletal muscle differentiation in vitro
, as shown by their induction to terminally differentiated myotubes when serum is withdrawn from the culture medium10–12
. We found that the expression of a few of the miRNAs examined was upregulated in differentiated C2C12 myoblasts or myotubes ( and Supplementary Fig. 1
online). This increase in expression of miR-1 and miR-133 in differentiated myoblasts was confirmed by RNA blot analysis ( and Supplementary Fig. 2
Figure 1 Expression of miR-1 and miR-133 in cardiac and skeletal muscle during development. (a) miRNA array expression data from C2C12 myoblasts cultured in growth medium (GM) or in differentiation medium (DM) for 0, 1, 3 or 5 d. Normalized log2 data were hierarchically (more ...)
miR-1 and miR-133 are specifically expressed in adult cardiac and skeletal muscle tissues, but not in other tissues tested13–15
( and Supplementary Fig. 3
online). Little is known, however, about the spatio-temporal distribution of specific miRNAs during mammalian development. We therefore examined the expression of miR-1 and miR-133 in mouse embryos and neonates. miR-1 and miR-133 are expressed in very small amounts in the developing hearts and skeletal muscle of embryonic day 13.5 (E13.5) and E16.5 mice ( and Supplementary Fig. 3
). Increasing expression of miR-1 and miR-133 was found in neonatal hearts and skeletal muscle, although it was still substantially lower than that in adult tissues ( and Supplementary Fig. 3
). These data are consistent with findings in zebrafish showing that most miRNAs are expressed relatively late during embryogenesis16
miR-1 and miR-133 are clustered together both on mouse chromosome 2, where they are separated by 9.3 kb, and on mouse chromosome 18, where they are separated by 2.5 kb (Supplementary Fig. 4
online and ref. 14
). We carried out northern blot analysis using genomic probes of ~300 bp including the miR-1 or miR-133 sequence (Supplementary Fig. 4
). The probes for miR-1 and miR-133 on chromosome 18 detected a single primary transcript of ~6 kb in total RNAs isolated from heart and skeletal muscle (Supplementary Fig. 4
), indicating that miR-1 and miR-133 are indeed transcribed together. Whereas the probes for miR-1 and miR-133 on chromosome 2 both detected a transcript of ~10 kb in the heart and skeletal muscle, the miR-133 probe hybridized to two additional transcripts of ~4.5 kb and ~2.2 kb, and the miR-1 probe detected an additional major transcript of ~6 kb (Supplementary Fig. 4
), suggesting that post-transcriptional processing may be involved in the production of miR-1 and miR-133. Together, our data indicate that cardiac-specific and skeletal muscle–specific expression of miR-1 and miR-133 is dictated at the primary transcription step.
We reasoned that the regulatory elements that control the transcription of both the chromosome 2 and the chromosome 18 miR-1 and miR-133 clusters are probably conserved. We therefore used sequence analysis to identify a highly conserved region (~2 kb) that lies about 50 kb upstream of the miR-1–miR-133 clusters on both chromosome 2 and chromosome 18 (Supplementary Fig. 5 online). When this genomic fragment from chromosome 2 was used to drive the expression of a dsRed reporter gene in transgenic X. laevis, we found cardiac-specific and skeletal muscle–specific expression of the transgene (Supplementary Fig. 5).
To assess the function of miR-1 and miR-133 in skeletal muscle, we first attempted to overexpress miR-1 and miR-133 in mammalian cells. We tested and validated the expression and activity of both miRNAs by RNA blot analysis, as well as by using miR-1 and miR-133 ‘sensors’17
, in which the complementary sequences of miR-1 and miR-133 are cloned downstream of a dsRed coding sequence (Supplementary Fig. 6
online and data not shown). We transfected C2C12 myoblasts with miR-1 or miR-133 and then either maintained cells in growth medium or transferred them to differentiation medium after transfection. miR-1 strongly enhanced myogenesis, as indicated by an increase in expression of the respective early and late myogenic markers myogenin and myosin heavy chain (MHC), as well as other myogenic markers including MyoD, MEF2 and skeletal α-actin ( and ). miR-1 induced the expression of myogenic marker genes in cells maintained in the log-phase growth condition () and in the differentiation condition ().
Figure 2 Regulation of myoblast proliferation and differentiation by miR-1 and miR-133. C2C12 myoblasts cultured in growth medium were electroporated with double-stranded miR-1, miR-133 or control miGFP. (a,b) Cells were continuously cultured in growth medium (more ...)
Effect on myogenic proliferation and differentiation of miR-1 and miR-133 overexpression and knock down
Accelerated myogenic differentiation induced by miR-1 was accompanied by a decrease in cell proliferation, as marked by a significant decrease in expression of phosphorylated histone H3 (phospho–histone H3; and ). We found that miR-1-induced myogenesis is specific, because overexpression of a green fluorescent protein control RNA duplex (miGFP) or miR-208, which is not endogenously expressed in skeletal muscle cells, showed no effect (). In addition, mutations introduced into miR-1 ‘seed’ sequences abolished its ability to activate myogenic gene expression (). By contrast, overexpression of miR-133 repressed the expression of myogenin and MHC ( and ) and promoted myoblast proliferation ( and ). Again, we found that the effect of miR-133 on myoblasts proliferation is specific, because controls showed no effect and the mutation introduced abolished the function of miR-133 ().
We carried out a reciprocal experiment wherein we transfected C2C12 myoblasts with 2′-O
-methyl antisense inhibitory oligoribonucleotides, which have been shown to inhibit the function of miRNAs18,19
, targeted towards miR-1 or miR-133 (or control miGFP and miR-208). Cells transfected with the miR-1 inhibitor showed inhibition of myogenesis and promotion of myoblast proliferation, as indicated by a decrease in myogenic markers and an increase in phospho–histone H3 ( and ). Consistent with the role of miR-133 in promoting myoblast proliferation and repressing differentiation, inhibition of miR-133 caused an opposing effect, whereby myogenesis was enhanced and cell proliferation was repressed ( and ). By contrast, the control 2′-O
-methyl inhibitors showed no effects (). We conclude that miR-1 and miR-133 have distinct roles in skeletal muscle proliferation and differentiation: miR-1 promotes myoblast differentiation, whereas miR-133 stimulates myoblast proliferation.
Both miR-1 and miR-133 have been found in most animal species, from Drosophila to human, suggesting that they are evolutionary conserved. To test the effects of miR-1 and miR-133 on skeletal muscle and heart development in vivo, we identified copies of miR-1 and miR-133 in X. laevis and tested their function through misexpression. Introduction of miR-1 at the one-cell stage led to a markedly shortened axis with an accompanying reduction in anterior structures and an increase in body size along the dorsal-ventral axis, as compared with either uninjected or miGFP-injected controls (n > 50, two independent experiments; ). Although somites formed in embryos injected with miR-1 (), whole-mount antibody staining and serial sectioning showed that the tissue was highly disorganized and did not develop into segmented structures (). Cardiac tissue was completely absent, as judged by histology, staining for tropomyosin (,j) and staining for cardiac actin (data not shown). In addition to these defects, there was a marked decrease in phospho–histone H3 staining (), consistent with the notion that miR-1 is essential in regulating muscle cell proliferation and differentiation.
Figure 3 Control of cardiac and skeletal muscle development by miR-1 and miR-133 in vivo. (a–h) Images of uninjected (a,b), control miGFP-injected (c,d), miR-1-injected (e,f) and miR-133-injected (g,h) X. laevis embryos stained with anti-tropomyosin and (more ...)
Misexpression of miR-133 also led to a reduction in anterior structures and defects in somite development but, in contrast to misexpression of miR-1, there was only a modest reduction in anterior-posterior length and somitic defects were most severe in the more anterior or posterior aspects of the embryo where somites failed to form (). In addition, cardiac tissue frequently formed in miR-133-injected embryos, although it was highly disorganized and did not undergo cardiac looping or chamber formation ( and data not shown). Collectively, these data suggest that correct temporal expression and amounts of both miR-1 and miR-133 are required for proper skeletal muscle and heart development.
To identify target genes that might mediate the observed effects of miR-1 and miR-133 on skeletal muscle proliferation and differentiation, we next examined potential targets of these two miRNAs. Many computational and/or bioinformatics-based approaches have been used to predict numerous potential targets of miRNAs20–22
. Strikingly, many transcription factors have been suggested to be targets of miRNAs, raising the possibility that miRNAs might be involved in transcriptional regulation of gene expression. Among the predicted targets of miR-1, HDAC4 has been shown to inhibit muscle differentiation and skeletal muscle gene expression, mainly by repressing MEF2C, an essential muscle-related transcription factor12,23
. HDAC4 contains two naturally occurring putative miR-1-binding sites at its 3′ UTR, which are evolutionarily conserved among vertebrate species (Supplementary Fig. 7
online). Similarly, two conserved miR-133-binding sites are found in the 3′ UTR of the mammalian gene encoding SRF (Supplementary Fig. 7
), which has been shown to be important in muscle proliferation and differentiation in vitro
and in vivo11,24,25
We fused the 3′ UTRs of mouse SRF and HDAC4 to a luciferase reporter gene and transfected these constructs, along with transfection controls, into mammalian cells. Ectopic overexpression of miR-1 strongly repressed the HDAC4 3′ UTR luciferase reporter gene, whereas over-expression of miR-133 inhibited the SRF 3′ UTR luciferase reporter gene (). By contrast, mutations introduced into miR-1 or miR-133 seed sequences abolished this repression, indicating the specificity of the action ().
Figure 4 Identification of miR-1 and miR-133 target genes in skeletal muscle. (a) Repression of SRF and HDAC4 3′ UTRs by miR-133 and miR-1. Luciferase reporters containing either miR-133 complementary sites from mouse SRF 3′ UTR (SRF-3′-UTR), (more ...)
When the above reporters were transfected into C2C12 myoblasts and luciferase activity was measured before and after the induction of cell differentiation, reporter gene activity was markedly repressed in differentiated cells (), indicating that the increase in endogenous miR-1 and miR-133 inhibited the reporter gene. The effects and specificity of endogenous miR-1 and miR-133 were monitored by the miRNA sensor (Supplementary Fig. 6). By contrast, the activity of a luciferase reporter gene for MCK, an indicator of muscle differentiation, was increased in differentiated muscle cells (). In addition, overexpression of miR-1 led to the downregulation of endogenous HDAC4 protein in C2C12 cells in both the growth condition () and the differentiation condition (), whereas overexpression of miR-133 repressed the expression of endogenous SRF proteins (). By contrast, the mRNA levels of SRF and HDAC4 were not altered by these miRNAs (), supporting the notion that miRNAs repress the function of their target genes mainly by inhibiting translation. The application of 2′-O-methyl-antisense oligoribonucleotides targeted towards miR-1 or miR-133 relieved the repression of HDAC4 or SRF protein, respectively (), but had no effect on their mRNA levels ().
To verify that HDAC4 and SRF are cognate targets of miR-1 and miR-133 in regulating skeletal muscle gene expression, we tested whether cotransfecting expression plasmids encoding SRF and HDAC4 could ‘suppress’ miRNA-mediated myogenesis. Indeed, over-expression of SRF partially reversed the myogenic gene repression induced by miR-133 (), whereas overexpression of HDAC4 counteracted the effects of miR-1 on skeletal muscle gene expression (). Consistent with the potential involvement of HDAC4 and SRF in miR-1- and miR-133-dependent skeletal muscle proliferation and differentiation, endogenous HDAC4 and SRF protein was down-regulated in differentiated C2C12 cells, coupled with a concomitant increase in expression of myogenic differentiation markers and a decrease in expression of the mitotic index marker phospho–histone H3 ( and Supplementary Fig. 2 online). The decrease in expression of SRF and HDAC4 protein was accompanied by an increase in expression of miR-1 and miR-133 (compare with ). Together, these data show that miR-1 and miR-133 specifically repress HDAC4 and SRF protein, respectively, which in turn contributes to (at least in part) the regulatory effects of these miRNAs on myoblast proliferation and differentiation.
In summary, we have characterized cardiac-specific and skeletal muscle–specific miR-1 and miR-133 and have shown their essential functions in controlling skeletal muscle proliferation and differentiation. Notably, we have shown that miR-1 and miR-133, which are clustered on the same chromosomal loci and transcribed together as a single transcript, become two independent, mature miRNAs with distinct biological functions achieved by inhibiting different target genes. This finding implicates miRNAs in complex molecular mechanisms. Although the tissue-specific expression of miR-1 and miR-133 is controlled by MyoD and SRF8
, expression of SRF is repressed by miR-133. Thus, these findings identify a negative regulatory loop in which miRNAs participate to control cellular proliferation and differentiation (). In the future, it will be interesting to determine whether miR-1 and miR-133 are involved in cardiac-related and skeletal muscle–related human diseases.
Figure 5 Model of miR-1 and miR-133-mediated regulation of skeletal muscle proliferation and differentiation. Tissue-specific expression of miR-1 and miR-133 clusters is regulated by SRF and myogenic transcription factor MyoD. miR-1 and miR-133 modulate muscle (more ...)