The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation

Abstract

Understanding the molecular mechanisms that regulate cellular proliferation and differentiation is a central theme of developmental biology. MicroRNAs (miRNAs) are a class of regulatory RNAs of approximately 22 nucleotides that post-transcriptionally regulate gene expression. Increasing evidence points to the potential role of miRNAs in various biological processes. Here we show that miRNA-1 (miR-1) and miRNA-133 (miR-133), which are clustered on the same chromosomal loci, are transcribed together in a tissue-specific manner during development. miR-1 and miR-133 have distinct roles in modulating skeletal muscle proliferation and differentiation in cultured myoblasts in vitro and in Xenopus laevis embryos in vivo. miR-1 promotes myogenesis by targeting histone deacetylase 4 (HDAC4), a transcriptional repressor of muscle gene expression. By contrast, miR-133 enhances myoblast proliferation by repressing serum response factor (SRF). Our results show that two mature miRNAs, derived from the same miRNA polycistron and transcribed together, can carry out distinct biological functions. Together, our studies suggest a molecular mechanism in which miRNAs participate in transcriptional circuits that control skeletal muscle gene expression and embryonic development.

Figures

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 clustered by gene and are plotted as a heat map. The signal ranged from a fourfold decrease to a fourfold increase. Yellow denotes high expression and blue denotes low expression relative to the median; only the miRNA nodes that were upregulated in differentiation medium are shown. (b) Northern blot analysis of miR-1 and miR-133 expression using total RNA isolated from C2C12 myoblasts cultured in growth medium or in differentiation medium DM for 0, 1, 3 or 5 d. tRNAs were used as a loading control. (c) Northern blot analysis of miR-1 and miR-133 expression in adult mouse tissues. (d) Northern blot analysis of miR-1 and miR-133 expression in E13.5 and E16.5 mouse tissues. (e) Northern blot analysis of miR-1 and miR-133 expression in neonatal mouse tissues. The same amount of total RNAs from adult heart and skeletal muscle was loaded on the blots to compare embryonic and neonate RNA (d,e).

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 for 24 h after transfection and then transferred to differentiation medium for either 12 h before immunostaining for myogenin (a) or 36 h before immunostaining for MHC (b). (c–e) C2C12 myoblasts cultured in GM were electroporated with double-stranded miR-1, miR-133 (or their mutants as indicated) or control miR-208 or miGFP and cultured for 24 h before being either subjected to immunoblotting with the indicated antibodies (c), or transferred to differentiation medium for 24 h and subjected to RT-PCR for the indicated genes (d) or to immunoblotting with the indicated antibodies (e). (f–h) C2C12 myoblasts cultured in GM were electroporated with a 2′-O-Methyl antisense oligonucleotide inhibitors of miR-1, miR-133, miR-208 or miGFP as controls. Cells were cultured in growth medium for 24 h after transfection and then transferred into differentiation medium for 12 h before immunostaining for phospho–histone H3 (f), 24 h before RT-PCR for the indicated genes (g) or 24 h before immunoblotting with the indicated antibodies (h). (i,j) C2C12 myoblasts cultured in growth medium were electroporated with miRNA duplexes or with 2′-O-Methyl antisense oligonucleotide inhibitors as indicated. Cells were cultured in growth medium for 24 h after transfection and then transferred into DM for 12 h before immunostaining for myogenin (i) or phospho–histone H3 (j). Positive stained cells were counted and data are presented as the expression level relative to a miGFP control (100%).

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 shown at stage 32 under bright-field (a,c,e,g) or fluorescence (b,d,f,h) microscopy. Note the lack of staining for heart tissue (H, arrows) and the disruption of segmented somites (S, arrows) in f and h. (i–k) Transverse sections corresponding to the position of the heart at stage 32 in uninjected (i), miR-1-injected (j) and miR-133-injected (k) X. laevis embryos stained with anti-tropomyosin to visualize somites (S, arrows) and cardiac tissue (H, arrows), and antibody to phospho–histone H3 (red) to visualize cells in S phase. Each set of injections was conducted at least twice independently, and the phenotype was observed in at least 90% of a minimum of 50 embryos scored by whole-mount immunostaining.

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), miR-1 complementary sites from mouse HDAC4 3′ UTR (HDAC4-3′-UTR) or the perfect antisense sequences of miR-133 (miR-133-luc) or miR-1 (miR-1-luc) were cotransfected with the indicated miRNA expression vectors. Luciferase activity was determined 48 h after transfection. Data represent the mean ± s.d. from at least three independent experiments done in duplicate (*P < 0.05). (b) SRF-3′-UTR, HDAC4-3′-UTR and MCK-luc luciferase reporters were transfected into C2C12 myoblasts. Cells were maintained in growth medium for 24 h (GM) or transferred into differentiation medium for 1 d (DM1) or 3 d (DM3) before luciferase activity was determined. (c–e) C2C12 myoblasts cultured in growth medium were electroporated with the indicated miRNA duplexes (or their mutants), or miR-208 and miGFP as controls. Cells were cultured in growth medium for 24 h after transfection before being either subjected to immunoblotting with anti-SRF and anti-HDAC4 antibodies (c), or transferred into differentiation medium for 24 h and subjected to RT-PCR for the indicated genes (d) or to immunoblotting with the indicated antibodies (e). (f,g) C2C12 myoblasts cultured in growth medium were electroporated with the indicated 2′-O-Methyl antisense oligonucleotide inhibitors. Cells were cultured in growth medium for 24 h after transfection and transferred into differentiation medium for 24 h before being subjected to RT-PCR for the indicated genes (f) or to immunoblotting with indicated antibodies (g). (h) C2C12 myoblasts cultured in growth medium were electroporated with the indicated miRNA duplexes and/or expression plasmids for SRF or HDAC4, as indicated. Cells were cultured in growth medium for 24 h after transfection. Immunoblotting with the indicated antibodies was done 24 h after transfer into differentiation medium. (i) C2C12 myoblasts were cultured in growth medium or differentiation medium for 0, 1, 3 or 5 d and subjected to immunoblotting with the indicated antibodies.

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 proliferation and differentiation, in part, by targeting HDAC4 and SRF, respectively.