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Transforming growth factor-beta-regulated miR-24 promotes skeletal muscle differentiation.

Sun Q, Zhang Y, Yang G, Chen X, Zhang Y, Cao G, Wang J, Sun Y, Zhang P, Fan M, Shao N, Yang X - Nucleic Acids Res. (2008)

Bottom Line: In this study, we found that miR-24 was upregulated during myoblast differentiation and could be inhibited by TGF-beta1.TGF-beta1 was unable to inhibit miR-24 expression in Smad3-deficient myoblasts, which exhibited accelerated myogenesis.This is the first study demonstrating a critical role for miRNAs in modulating TGF-beta-dependent inhibition of myogenesis, and provides a novel mechanism of the genetic regulation of TGF-beta signaling during skeletal muscle differentiation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Proteomics, Genetic Laboratory of Development and Diseases, Institute of Biotechnology, 20 Dongdajie, Beijing 100071, PR China.

ABSTRACT
MicroRNAs (miRNAs) have recently been proposed as a versatile class of molecules involved in regulation of a variety of biological processes. However, the role of miRNAs in TGF-beta-regulated biological processes is poorly addressed. In this study, we found that miR-24 was upregulated during myoblast differentiation and could be inhibited by TGF-beta1. Using both a reporter assay and Northern blot analysis, we showed that TGF-beta1 repressed miR-24 transcription which was dependent on the presence of Smad3 and a Smads binding site in the promoter region of miR-24. TGF-beta1 was unable to inhibit miR-24 expression in Smad3-deficient myoblasts, which exhibited accelerated myogenesis. Knockdown of miR-24 led to reduced expression of myogenic differentiation markers in C2C12 cells, while ectopic expression of miR-24 enhanced differentiation, and partially rescued inhibited myogenesis by TGF-beta1. This is the first study demonstrating a critical role for miRNAs in modulating TGF-beta-dependent inhibition of myogenesis, and provides a novel mechanism of the genetic regulation of TGF-beta signaling during skeletal muscle differentiation.

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Enhanced myogenesis in C2C12 cells ectopically expressing miR-24. (A) Schematic structures of the retroviral vector pINCO-miR24. (B) Northern blot analysis of miR-24 expression in C2C12 cells infected with pINCO-miR24. U6 RNAs detected by Northern blot were used as loading controls. (C) Activity detection of ectopically expressed miR-24. Reporter vector of miR-24 ‘sensor’, in which the complementary sequence of miR-24 was cloned downstream of luciferase coding sequences, was co-transfected with pINCO-miR24. pGL3-control vector was used as a negative control. Luciferase activity was measured 2 days after transfection. Two stars mean P < 0.01. (D) C2C12 cells infected with pINCO-miR24 or pINCO were transferred into DM with or without TGF-β1 (5 ng/ml) for 36 h. Immunofluorescence staining was performed to detect the expression of MHC. DAPI staining was done to visualize the nuclei. (E) The expression of MHC shown in (D) was quantified after standardization with the expression level of MHC in controls. Two stars mean P < 0.01. (F) and (G) Overexpression of miR-24 elevated the expression of myogenic factors, which was assessed by RT-PCR (F) and Western blot analyses (G).
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Figure 6: Enhanced myogenesis in C2C12 cells ectopically expressing miR-24. (A) Schematic structures of the retroviral vector pINCO-miR24. (B) Northern blot analysis of miR-24 expression in C2C12 cells infected with pINCO-miR24. U6 RNAs detected by Northern blot were used as loading controls. (C) Activity detection of ectopically expressed miR-24. Reporter vector of miR-24 ‘sensor’, in which the complementary sequence of miR-24 was cloned downstream of luciferase coding sequences, was co-transfected with pINCO-miR24. pGL3-control vector was used as a negative control. Luciferase activity was measured 2 days after transfection. Two stars mean P < 0.01. (D) C2C12 cells infected with pINCO-miR24 or pINCO were transferred into DM with or without TGF-β1 (5 ng/ml) for 36 h. Immunofluorescence staining was performed to detect the expression of MHC. DAPI staining was done to visualize the nuclei. (E) The expression of MHC shown in (D) was quantified after standardization with the expression level of MHC in controls. Two stars mean P < 0.01. (F) and (G) Overexpression of miR-24 elevated the expression of myogenic factors, which was assessed by RT-PCR (F) and Western blot analyses (G).

Mentions: To address the role of miR-24 during myogenesis, we transfected C2C12 myoblasts with 2′-O-methyl antisense inhibitory oligoribonucleotides, which have been shown to inhibit the function of miRNAs (48,49). Treatment with miR-24 antisense oligoribonucleotides dramatically inhibited C2C12 myoblast differentiation, as indicated by reduced myotube formation (Figure 5A and B). The reduced expression of miR-24 was confirmed by Northern blot (Figure 5C). A decrease in both early and late myogenic markers, Myogenin and MHC, as well as MEF2, α-actin and caveolin3 upon the treatment of miR-24 antisense oligoribonucleotides was also detected by real-time PCR (Figure 5D) and Western blot (Figure 5E). Synthetic control oligoribonucleotides did not significantly affect differentiation (Figure 5A–E). Furthermore, we overexpressed miR-24 using the retroviral vector pINCO (Figure 6A) (37) in C2C12 cells treated with miR-24 antisense oligoribonucleotides, and found that overexpression of miR-24 almost restored the expression of myogenic markers (Figure 5F–H). All these results suggested that miR-24 is a promoting factor of myogenesis.Figure 5.


Transforming growth factor-beta-regulated miR-24 promotes skeletal muscle differentiation.

Sun Q, Zhang Y, Yang G, Chen X, Zhang Y, Cao G, Wang J, Sun Y, Zhang P, Fan M, Shao N, Yang X - Nucleic Acids Res. (2008)

Enhanced myogenesis in C2C12 cells ectopically expressing miR-24. (A) Schematic structures of the retroviral vector pINCO-miR24. (B) Northern blot analysis of miR-24 expression in C2C12 cells infected with pINCO-miR24. U6 RNAs detected by Northern blot were used as loading controls. (C) Activity detection of ectopically expressed miR-24. Reporter vector of miR-24 ‘sensor’, in which the complementary sequence of miR-24 was cloned downstream of luciferase coding sequences, was co-transfected with pINCO-miR24. pGL3-control vector was used as a negative control. Luciferase activity was measured 2 days after transfection. Two stars mean P < 0.01. (D) C2C12 cells infected with pINCO-miR24 or pINCO were transferred into DM with or without TGF-β1 (5 ng/ml) for 36 h. Immunofluorescence staining was performed to detect the expression of MHC. DAPI staining was done to visualize the nuclei. (E) The expression of MHC shown in (D) was quantified after standardization with the expression level of MHC in controls. Two stars mean P < 0.01. (F) and (G) Overexpression of miR-24 elevated the expression of myogenic factors, which was assessed by RT-PCR (F) and Western blot analyses (G).
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Figure 6: Enhanced myogenesis in C2C12 cells ectopically expressing miR-24. (A) Schematic structures of the retroviral vector pINCO-miR24. (B) Northern blot analysis of miR-24 expression in C2C12 cells infected with pINCO-miR24. U6 RNAs detected by Northern blot were used as loading controls. (C) Activity detection of ectopically expressed miR-24. Reporter vector of miR-24 ‘sensor’, in which the complementary sequence of miR-24 was cloned downstream of luciferase coding sequences, was co-transfected with pINCO-miR24. pGL3-control vector was used as a negative control. Luciferase activity was measured 2 days after transfection. Two stars mean P < 0.01. (D) C2C12 cells infected with pINCO-miR24 or pINCO were transferred into DM with or without TGF-β1 (5 ng/ml) for 36 h. Immunofluorescence staining was performed to detect the expression of MHC. DAPI staining was done to visualize the nuclei. (E) The expression of MHC shown in (D) was quantified after standardization with the expression level of MHC in controls. Two stars mean P < 0.01. (F) and (G) Overexpression of miR-24 elevated the expression of myogenic factors, which was assessed by RT-PCR (F) and Western blot analyses (G).
Mentions: To address the role of miR-24 during myogenesis, we transfected C2C12 myoblasts with 2′-O-methyl antisense inhibitory oligoribonucleotides, which have been shown to inhibit the function of miRNAs (48,49). Treatment with miR-24 antisense oligoribonucleotides dramatically inhibited C2C12 myoblast differentiation, as indicated by reduced myotube formation (Figure 5A and B). The reduced expression of miR-24 was confirmed by Northern blot (Figure 5C). A decrease in both early and late myogenic markers, Myogenin and MHC, as well as MEF2, α-actin and caveolin3 upon the treatment of miR-24 antisense oligoribonucleotides was also detected by real-time PCR (Figure 5D) and Western blot (Figure 5E). Synthetic control oligoribonucleotides did not significantly affect differentiation (Figure 5A–E). Furthermore, we overexpressed miR-24 using the retroviral vector pINCO (Figure 6A) (37) in C2C12 cells treated with miR-24 antisense oligoribonucleotides, and found that overexpression of miR-24 almost restored the expression of myogenic markers (Figure 5F–H). All these results suggested that miR-24 is a promoting factor of myogenesis.Figure 5.

Bottom Line: In this study, we found that miR-24 was upregulated during myoblast differentiation and could be inhibited by TGF-beta1.TGF-beta1 was unable to inhibit miR-24 expression in Smad3-deficient myoblasts, which exhibited accelerated myogenesis.This is the first study demonstrating a critical role for miRNAs in modulating TGF-beta-dependent inhibition of myogenesis, and provides a novel mechanism of the genetic regulation of TGF-beta signaling during skeletal muscle differentiation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Proteomics, Genetic Laboratory of Development and Diseases, Institute of Biotechnology, 20 Dongdajie, Beijing 100071, PR China.

ABSTRACT
MicroRNAs (miRNAs) have recently been proposed as a versatile class of molecules involved in regulation of a variety of biological processes. However, the role of miRNAs in TGF-beta-regulated biological processes is poorly addressed. In this study, we found that miR-24 was upregulated during myoblast differentiation and could be inhibited by TGF-beta1. Using both a reporter assay and Northern blot analysis, we showed that TGF-beta1 repressed miR-24 transcription which was dependent on the presence of Smad3 and a Smads binding site in the promoter region of miR-24. TGF-beta1 was unable to inhibit miR-24 expression in Smad3-deficient myoblasts, which exhibited accelerated myogenesis. Knockdown of miR-24 led to reduced expression of myogenic differentiation markers in C2C12 cells, while ectopic expression of miR-24 enhanced differentiation, and partially rescued inhibited myogenesis by TGF-beta1. This is the first study demonstrating a critical role for miRNAs in modulating TGF-beta-dependent inhibition of myogenesis, and provides a novel mechanism of the genetic regulation of TGF-beta signaling during skeletal muscle differentiation.

Show MeSH
Related in: MedlinePlus