doi: 10.1371/journal.pone.0007607.
Microrna-221 and microrna-222 modulate differentiation and maturation of skeletal muscle cells
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- PMID:19859555
- PMCID: PMC2762614
- DOI: 10.1371/journal.pone.0007607
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Microrna-221 and microrna-222 modulate differentiation and maturation of skeletal muscle cells
Beatrice Cardinali et al. PLoS One..
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Abstract
Background: MicroRNAs (miRNAs) are a class of small non-coding RNAs that have recently emerged as important regulators of gene expression. They negatively regulate gene expression post-transcriptionally by translational repression and target mRNA degradation. miRNAs have been shown to play crucial roles in muscle development and in regulation of muscle cell proliferation and differentiation.
Methodology/principal findings: By comparing miRNA expression profiling of proliferating myoblasts versus differentiated myotubes, a number of modulated miRNAs, not previously implicated in regulation of myogenic differentiation, were identified. Among these, miR-221 and miR-222 were strongly down-regulated upon differentiation of both primary and established myogenic cells. Conversely, miR-221 and miR-222 expression was restored in post-mitotic, terminally differentiated myotubes subjected to Src tyrosine kinase activation. By the use of specific inhibitors we provide evidence that expression of miR-221 and miR-222 is under the control of the Ras-MAPK pathway. Both in myoblasts and in myotubes, levels of the cell cycle inhibitor p27 inversely correlated with miR-221 and miR-222 expression, and indeed we show that p27 mRNA is a direct target of these miRNAs in myogenic cells. Ectopic expression of miR-221 and miR-222 in myoblasts undergoing differentiation induced a delay in withdrawal from the cell cycle and in myogenin expression, followed by inhibition of sarcomeric protein accumulation. When miR-221 and miR-222 were expressed in myotubes undergoing maturation, a profound alteration of myofibrillar organization was observed.
Conclusions/significance: miR-221 and miR-222 have been found to be modulated during myogenesis and to play a role both in the progression from myoblasts to myocytes and in the achievement of the fully differentiated phenotype. Identification of miRNAs modulating muscle gene expression is crucial for the understanding of the circuits controlling skeletal muscle differentiation and maintenance.
Conflict of interest statement
Figures
miRNA expression was measured in QMb-ts, cultivated either at 35°C or 41°C for 48 hours and in QMb-ts maintained at 41°C for 30 hours and shifted to 35°C for additional 18 hours. Values in table were normalized according to median values and expressed as a function of values at 35°C DM using the comparative Ct method. Standard error (s.e.) and statistical significance (p, n = 6) are shown. P values in red are not statistically significant. Heat map representing miRNA modulation is expressed in a linear scale with a maximum value of 16 fold to highlight differences within this range. Green and red luts indicate down- and up-regulation, respectively.
Northern blot analysis showing expression of miR-221 and miR-222, compared to muscle-specific miR-133, in QMb-ts myoblasts at 35°C, myotubes at 41°C and myotubes shifted back to 35°C. Numbers represent the quantity of each miR expressed as fold decrease/increase relative to that measured in proliferating myoblasts (35°), taken as 1.0. U6 snRNA was used for normalization.
(A) QMb-ts myoblasts allowed to differentiate in DM upon temperature shift from 35°C to 41°C and (B) myotubes differentiated in DM for 40 hours at 41°C (41-T0) subjected to Src activation at 35°C were analysed at different time points for expression of p27 protein by Western blot (upper panels) and of p27 mRNA, miR-221 and miR-222 by real time RT-PCR. In B, 41-T24 represents control myotubes kept at 41°C for additional 24 hours. Accumulation levels of p27 protein and mRNA, miR-221 and miR-222, expressed relative to levels detected at 41°C, taken as 1.0, are shown in the histograms, normalized for p38 protein, GAPDH mRNA and miR-16 respectively. Error bars represent standard error (n = 3).
(A, B) Proliferating QMb-ts were treated in DM at 35°C for two days with DMSO (vehicle) or with the MEK inhibitor U0126 (U0126), or infected with adenoviruses expressing GFP (ad-GFP) or a dominant interfering Ras mutant (ad-RasN17). QMb-ts in DM at 41°C are shown for comparison. After 2 days, cells were analysed for (A) expression of miR-221, miR-222 and miR-1 by real time RT-PCR and (B) accumulation of p27 and muscle-specific proteins by western blot. (C) QMb-ts myotubes kept at 41° for 30 hours (41° (T0)) were treated with DMSO (vehicle) or U0126 (U0126) and shifted to 35° for 20 hours. Fold modulation of miRNA expression shown in (A) and (C), normalized for miR-16 expression, is expressed relative to cell extracts at 35°C (A) and myotubes differentiated for 30 hours at 41°C (C), taken as 1.0. Error bars represent standard error and the observed differences are statistically significant (p<0.05; n≥3).
Western blot analysis of p27 in myotubes derived from (A) QMb-ts and primary QMb and (B) C2C12, L6C5 and MSC myoblasts transfected with control siGFP (siGFP), miR-221 (221), miR-222 (222) or both (221+222) duplex RNAs and allowed to differentiate for two days. p27 expression in (C) QMb-ts myotubes transfected with antisense inhibitors to GFP (aGFP), used as control, miR-221 (a221), miR-222 (a222) or both (a221+a222) and shifted to 35°C for 24 hours and in (D) C2C12 myoblasts transfected with the same duplexes in GM and analysed 24 hours. Untransfected (n.t.) myoblasts in DM at 35°C or GM and (n.t.) myotubes in DM are shown as controls. Numbers represent levels of p27 expressed as fold decrease/increase relative to that measured in controls (siGFP), taken as 1.0. p38 protein levels were used for normalization.
Firefly luciferase activity was measured in QMb-ts after transfection of pGL3 reporter vectors carrying the entire (UTR) or a portion (s-UTR) of the quail p27-3′UTR containing the target sites for miR-221 and miR-222. (A) pGL3 reporter vectors were transfected and luciferase activities were measured in myoblasts (35°) and myotubes (41°) cultivated in DM; (B) pGL3 reporter vectors were cotransfected with antisense inhibitors to GFP (aGFP) and miR-221 and miR-222 (a221+a222) in myoblasts at 35°C in DM; (C) pGL3 reporter vectors were cotransfected with control (GFP) or miR-221 and miR-222 (221+222) duplexes in myoblasts at 35°C. In (C) also a human p27-3′UTR (h-UTR) is shown for comparison. The transfection efficiency was accounted for by cotransfection with the renilla luciferase reporter pRL. Activities of UTR, s-UTR and h-UTR were normalized on activity of pGL3 empty vector transfected in parallel plates, and expressed relative to activity at 35° (A), to activity of control aGFP (B) and control GFP (C), taken as 1.0. Error bars represent standard error and the observed differences are all statistically significant (p<0.05; n≥3).
QMb-ts were transfected with control (siGFP) or miR-221 and miR-222 duplexes (miR221+222) in GM and transferred to 41°C in DM 24 hours later. At different time points after temperature shift cells were pulse-labeled (for 2 hours) with BrdU, fixed and stained for immunofluorescence with antibodies to (A) BrdU and (B) myogenin. The percentage of positive cells relative to total cells is shown in the histograms. Error bars represent standard error; (*) = p<0.05; (**) = p<0.01; n≥3.
(A) QMb-ts transfected at 35°C with control siGFP, miR-221 (221), miR-222 (222) and both (221+222) duplex RNAs were shifted to 41°C in DM and allowed to differentiate for two days. Cell extracts were analyzed by immunoblotting with antibodies specific for the indicated proteins. (B, C) MSC myocytes kept in DM for 24 hours were transfected with control siGFP, miR-221 and miR-222 (221+222), and, after 2 days, were either analyzed by Western blot with antibodies to the proteins indicated in (B) or processed for immunofluorescence and scored for degree of cell fusion (C). Protein accumulation levels expressed as fold decrease/increase relative to those measured in control siGFP, taken as 1.0, are shown in (A, B). p38 and vinculin protein levels were used for normalization. (C) Histogram displaying the size distribution of MSC myotubes, labeled with antibody to myosin heavy chain and with the fluorescent dye Hoechst 33258, expressed as function of the number of nuclei per myotube. Error bars represent standard error.
(A) QMb-ts myoblasts were transfected with control duplexes (siGFP) or a mixture of miR-221 and miR-222 duplexes (221+222), induced to differentiate in DM at 41°C and, two days later, subjected to double immunofluorescence with actin-staining phalloidin and with antibodies specific for p27, as indicated. (B) QMb-ts myotubes, differentiated in DM at 41°C for 24 hours, were transfected with control duplexes (siGFP) or a mixture of miR-221 and miR-222 duplexes (221+222), maintained in DM at 41°C for further 30 hours and subjected to double immunofluorescence with antibodies specific for skeletal myosin and p27, as indicated. (C–D) MSC myocytes, differentiated in DM for 24 hours, were transfected with control duplexes (siGFP) or a mixture of miR-221 and miR-222 duplexes (221+222), maintained in DM for additional 2 days and subjected to immunofluorescence with antibodies specific for skeletal myosin. (A–D) Nuclei were counterstained with Hoechst dye. Note that in (B) and (D) high magnification micrographs are shown to allow visualization of sarcomeres. Scale bars: 40 µm (A, C); 10 µm (B–D).
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