miR-186 inhibits muscle cell differentiation through myogenin regulation

Abstract

The complex process of skeletal muscle differentiation is organized by the myogenic regulatory factors (MRFs), Myf5, MyoD, Myf6, and myogenin, where myogenin plays a critical role in the regulation of the final stage of muscle differentiation. In an effort to investigate the role microRNAs (miRNAs) play in regulating myogenin, a bioinformatics approach was used and six miRNAs (miR-182, miR-186, miR-135, miR-491, miR-329, and miR-96) were predicted to bind the myogenin 3'-untranslated region (UTR). However, luciferase assays showed only miR-186 inhibited translation and 3'-UTR mutagenesis analysis confirmed this interaction was specific. Interestingly, the expression of miR-186 mirrored that of its host gene, ZRANB2, during development. Functional studies demonstrated that miR-186 overexpression inhibited the differentiation of C2C12 and primary muscle cells. Our findings therefore identify miR-186 as a novel regulator of myogenic differentiation.

Keywords: Differentiation; Gene Regulation; MicroRNA; Muscle; Myogenesis; Myogenin.

FIGURE 1.

Identification of miRNAs for myogenin 3′-UTR. A, following transfections of miRNAs into HeLa cells, only miR-186 caused a significant decrease in luciferase/mouse myogenin 3′-UTR activity, as compared with plasmid only (Myog 3′-UTR) or control transfections with negative miRNA (Myog 3′-UTR + control miRNA). Normalized luciferase activities were shown as the percentage relative to the negative control which was set as 1. B, map of the mouse myogenin gene locus indicating the length of the 3′-UTR and binding position of the miR-186. C, seed matched sequences in the myogenin 3′-UTR (indicated in red) show full conservation between at least 4 species (indicated by asterisks) and partial conservation (indicated by a dash). D, following transfections of miRNAs, miR-186 and miR-182 caused a significant decrease in luciferase/human myogenin 3′-UTR activity, as compared with plasmid only (Myog 3′-UTR) or control transfections with negative miRNA (Myog 3′-UTR + control miRNA). Normalized luciferase activities were shown as the percentage relative to the negative control, which was set as 1. Experiments were performed in triplicate, and the data represent the mean ± S.D. * represents a significance of (p < 0.01) using Student's t test.

FIGURE 2.

Myogenin 3′-UTR is down-regulated by miR-186. A, Predicted binding of miR-186 to the 3′-UTR of mouse myogenin and introduction of an 8 bp mutation for ensuring specificity. B, following transfections into HeLa cells, the presence of mutations in the miR-186 binding site caused no decrease in luciferase activity, indicating specificity for miR-186. No reduction was observed either in cells transfected with miR-186 and amiR-186 together (Myog 3′-UTR + miR-186 + amiR-186), and plasmid only (Myog 3′-UTR) or control transfections with negative miRNA (Myog 3′-UTR + control miRNA). C, following transfection into C2C12 cells (left) and mouse primary cells (right), miR-186 caused in both cases a decrease in luciferase activity compared with the mutated myogenin 3′-UTR and the miR-186 mutant as well as the rest of the experimental control transfections. Normalized luciferase activities were shown as the percentage relative to the negative control, which was set as 1. D, miR-186 was found to down-regulate luciferase gene expression through RNA repression as compared with negative miRNA control following transfections in HeLa cells. GAPDH was used as an internal control. Normalized expression levels were shown as the percentage relative to the negative control which was set as 1. Experiments were performed in triplicate, and the data represent the mean ± S.D. * represents a significance of (p < 0.01) using Student's t test.

FIGURE 3.

The expression pattern of miR-186 is mirrored to its host gene ZRANB2. Ai, schematic representation of the ZRANB2 gene indicating exons numbered from 1–10. Positions of primers, used for genomic or cDNA amplification are shown along with distance in base pairs (bp) between the pair of primers. ii. amplification of cDNA shows detection of exonic sequence devoid of the intron, containing the miR-186. As a control, the same primers amplified the larger genomic area which also contains the intron. B–D, following differentiation of C2C12 myoblasts, RNA or protein was extracted from different time points before (with growth medium, GM) and during (with differentiation medium, DM) differentiation (Day −1 to Day 4). Real-time PCR and Western blot analysis showed the similarity in the expression profile between ZRANB2 and miR-186. Myogenin protein levels were found to be low at proliferation and gradually increased up until day 3 of differentiation. Myogenin protein levels then gradually declined in mature myotubes. SNO-135 and GAPDH were used as internal controls for real-time PCR and Western blot analysis respectively. Immunoblots were measured using image J software. Experiments were performed in triplicate, and the data represent the mean ± S.D. * represents a significance of (p < 0.01) using ANOVA between different days in GM or DM.

FIGURE 4.

miR-186 targets myogenin during differentiation but not during proliferation. A, miR-186 mimics or amiR-186 reduced or increased C2C12 endogenous myogenin levels, respectively, compared with the negative control (negative miRNA or anti-miR control accordingly) following transfection in differentiating cells. Similar transfections of amiR-186 in proliferating cells did not alter myogenin levels. Immunoblots were measured using image J software. Experiments were performed in triplicate, and the data represent the mean ± S.D. * represents a significance of (p < 0.01) using Student's t test. B, luciferase-myogenin-3′-UTR dose response assays on amiR-186 transfections in proliferating cells show reduction in endogenous miRNA activity above a certain threshold (5 ng plasmid), compared with the negative control (anti-miR control). Normalized activities were shown as the percentage relative to the negative control. Experiments were performed in triplicate, and the data represent the mean ± S.D. * represents a significance of (p < 0.01) using Student's t test between amiR-186 transfected and the negative control. Ci, following transfections of miR-186, no decrease was observed in luciferase-cyclin-D1–3′-UTR activity, as compared with plasmid only (cyclin 3′-UTR) or negative miRNA transfections. Normalized luciferase activities were shown as the percentage relative to the negative control which was set as 1. ii, amiR-186 transfections did not alter the endogenous C2C12 myogenin levels, compared with control miRNA transfections (anti-miR control) following transfection in proliferating cells. GAPDH was used as an internal control.

FIGURE 5.

miR-186 decreases C2C12 muscle cell differentiation. A, C2C12 cells were transfected with miR-186 mimic, siRNA against myogenin, amiR-186, mutant miR-186 along with negative controls (negative miRNA and anti-miR-control) during differentiation and left to differentiate for a further 48 h following transfection (Day 3). Myotubes were fixed and stained with MyHC and nucleus-directed Hoechst. miR-186 mimic and myogenin siRNA reduced differentiation and fusion index, whereas amiR-186 increased both parameters. Scale bar, 200 μm. B, MyoD, Myf5, and Myf6 protein levels following overexpression and inhibition of miR-186. Protein levels of MyoD, Myf5, and Myf6 were measured following transfection of miR-186 and amiR-186 along with their negative controls (negative miRNA or anti-miR control accordingly). GAPDH was used as an internal control. C, skeletal muscle markers following overexpression and inhibition of miR-186. Muscle markers myogenin, troponin, and skeletal actin protein levels were detected following transfections on Day 0 (Proliferating) and Day 3 (Differentiating) of miR-186 and amiR-186 along with negative miRNA and anti-miR control, respectively. GAPDH was used as an internal control.

FIGURE 6.

miR-186 decreases muscle cell differentiation in primary mouse muscle cells. A and B, (A) miR-186 mimic or (B) amiR-186 transfected primary mouse cells showed decrease and increase in the endogenous myogenin protein levels, respectively, by Western blotting, as compared with the negative control (negative miRNA) Experiments were performed in triplicate, and the data represent the mean ± S.D. * represents a significance of (p < 0.01) using Student's t test between either miR-186 or amiR-186 compared with their negative controls. C, primary muscle cells were transfected with miR-186 mimic or amiR-186 and left to differentiate for 3 days. Myotubes were fixed and stained with MyHC and nucleus-directed Hoechst. miR-186 mimic reduced differentiation and fusion index, whereas amiR-186 increased both parameters. Transfections using a negative control (negative miRNA) were carried out in parallel. Scale bar, 200 μm.

FIGURE 7.

Myogenin and miR-186 display opposing expression profiles during mouse muscle regeneration. A, H&E staining of mouse TA muscle following 3, 5, 14 days after cardiotoxin injection. Saline-injected mice were used as a control “Day 0” Scale bar, 50 μm. B, myogenin expression levels were maximum when muscle injury was also at maximum (Day 3 and Day 5) at the time points indicated. GAPDH was used as an internal control. C, real time PCR of miR-186 was dramatically decreased during maximum muscle injury (Day 3 and Day 5). SNO-135 was used as an internal control.