Korean J Physiol Pharmacol.  2023 May;27(3):277-287. 10.4196/kjpp.2023.27.3.277.

Saturated fatty acid-inducible miR-103-3p impairs the myogenic differentiation of progenitor cells by enhancing cell proliferation through Twinfilin-1/F-actin/YAP1 axis

• Nguyen MT
• Lee W

• KMID: 2541639
• DOI: http://doi.org/10.4196/kjpp.2023.27.3.277

Abstract

Actin dynamics play an essential role in myogenesis through multiple mechanisms, such as mechanotransduction, cell proliferation, and myogenic differentiation. Twinfilin-1 (TWF1), an actin-depolymerizing protein, is known to be required for the myogenic differentiation of progenitor cells. However, the mechanisms by which they epigenetically regulate TWF1 by microRNAs under muscle wasting conditions related to obesity are almost unknown. Here, we investigated the role of miR-103-3p in TWF1 expression, actin filament modulation, proliferation, and myogenic differentiation of progenitor cells. Palmitic acid, the most abundant saturated fatty acid (SFA) in the diet, reduced TWF1 expression and impeded myogenic differentiation of C2C12 myoblasts, while elevating miR-103-3p levels in myoblasts. Interestingly, miR-103-3p inhibited TWF1 expression by directly targeting its 3’UTR. Furthermore, ectopic expression of miR-103-3p reduced the expression of myogenic factors, i.e., MyoD and MyoG, and subsequently impaired myoblast differentiation. We demonstrated that miR-103-3p induction increased filamentous actin (F-actin) and facilitated the nuclear translocation of Yes-associated protein 1 (YAP1), thereby stimulating cell cycle progression and cell proliferation. Hence, this study suggests that epigenetic suppression of TWF1 by SFA-inducible miR-103-3p impairs myogenesis by enhancing the cell proliferation triggered by F-actin/YAP1.

Keyword

Actin cytoskeleton; miR-103-3p; Myogenesis; Twinfilin-1; YAP1

Figure

• 

  Fig. 1 Palmitic acid (PA) inhibits myoblast differentiation while upregulating miR-103-3p expression. C2C12 cells were pretreated with PA (100 µM) for 24 h before differentiation. (A) The cells were stained with an anti-MyHC antibody (green) and Hoechst 33342 nuclear dye (blue). Scale bar: 50 μm. (B) MyHC-positive areas, differentiation indices, and fusion indices were analyzed as described in the Materials and Methods. (C, D) Representative immunoblots of myogenic factors and TWF1 on day three of differentiation and analyzed densitometry. The immunoblot intensities were normalized to the amount of β-Actin. The values are shown as relative ratios, with the intensity of the normalized vehicle control set to one. (E) The expressions of miR-103-3p (miR-103) were determined after 24 h of PA treatment by qRT-PCR and normalized to the amount of U6. All data are shown as the mean ± SEM (n > 3), where the level of significance is represented as *p < 0.05; ***p < 0.001 vs. controls. MyHC, myosin heavy chain; TWF1, Twinfilin-1; qRT-PCR, real-time qualitative polymerase chain reaction.

• 

  Fig. 2 MiR-103-3p suppresses TWF1 expression by targeting TWF1 3’UTR. (A) The tentative miR-103-3p (miR-103) target site of the TWF1 3’UTR. (B) Schematic diagram of wild-type TWF1 3’UTR segment (TWF1wt) and mutant TWF1 3’UTR segment (TWF1mut) containing miR-103 target sites cloned in pmirGLO dual-luciferase reporter plasmids and the seed sequence of miR-103 target sites on the TWF1wt. (C) C2C12 cells were co-transfected with scRNA control or miR-103-3p mimic (miR-103) and either TWF1wt or TWF1mut. Luciferase activities were determined 24 h after transfection. (D) The protein levels of TWF1 in C2C12 cells were detected by immunoblotting after 24 h of transfection with scRNA control, miR-103-3p mimic (miR-103), or antimiR-103. The immunoblot intensities were normalized to the amount of β-Actin. The values are shown as relative ratios, with the intensity of the normalized scRNA control set to one. All data are shown as the mean ± SEM (n > 3), where the level of significance is represented as **p < 0.01; ***p < 0.001 vs. scRNA. TWF1, Twinfilin-1; CDS, coding sequence; ns, no significance.

• 

  Fig. 3 MiR-103-3p inhibits the expressions of myogenic factors. C2C12 cells were transfected with scRNA, siTWF1, miR-103-3p mimic (miR-103), or antimiR-103 and differentiated for three days. (A) Representative immunoblots from three independent experiments on day three of differentiation. (B) The immunoblot intensities were normalized to the amount of β-Actin. The values are shown as relative ratios, with the intensity of the normalized scRNA control set to one. All data are shown as the mean ± SEM (n > 3), where the level of significance is represented as **p < 0.01; ***p < 0.001 vs. scRNA. TWF1, Twinfilin-1; ns, no significance.

• 

  Fig. 4 MiR-103-3p impairs myogenic differentiation and myotube formation. C2C12 cells were transfected with scRNA, siTWF1, miR-103-3p mimic (miR-103), or antimiR-103 and differentiated for five days. (A) The cells were stained with an anti-MyHC antibody (green) and Hoechst 33342 nuclear dye (blue). Scale bar: 50 μm. (B) MyHC-positive areas, differentiation indices, fusion indices, and myotube widths were analyzed as described in the Materials and Methods. All data are shown as the mean ± SEM (n > 3), where the level of significance is represented as ***p < 0.001 vs. scRNA. MyHC, myosin heavy chain; TWF1, Twinfilin-1; ns, no significance.

• 

  Fig. 5 MiR-103-3p facilitates F-actin accumulation and nuclear YAP1 localization. C2C12 cells were transfected with scRNA, siTWF1, or miR-103-3p mimic (miR-103) and cultured for 24 h. (A) The transfected cells were labeled with FITC-phalloidin (green) for F-actin staining and Hoechst 33342 (blue) for nucleus staining. Representative images from three independent experiments are shown. Scale bar: 25 μm. (B) Phalloidin intensities were analyzed by the ImageJ program and presented as relative ratios, with the intensity of the scRNA control set to one. (C) Representative immunoblots of YAP1 and phosphorylated YAP1 (pYAP1) protein in the cytoplasmic and nuclear fractions are shown. α-Tubulin and Lamin B were used as the cytoplasm and nucleus markers, respectively. β-Actin was used for loading control. (D) The immunoblot intensities were normalized to the amount of β-Actin or α-Tubulin, and the values are shown as relative ratios, with the intensity of the normalized scRNA control set to one. All data are shown as the mean ± SEM (n > 3), where the level of significance is represented as **p < 0.01; ***p < 0.001 vs. scRNA. F-actin, filamentous actin; YAP1, Yes-associated protein 1; TWF1, Twinfilin-1; ns, no significance.

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  Fig. 6 MiR-103-3p enhances myoblasts proliferation. C2C12 cells were transfected with scRNA, siTWF1, miR-103-3p mimic (miR-103), or antimiR-103 and cultured for 24 h. (A) The cells undergoing DNA replication were labeled with EdU (green), and Hoechst 33342 (blue) was used for nuclei staining. Scale bar: 50 μm. (B) The proportion of EdU-positive cells was measured using ImageJ software. (C, D) Flow cytometry with a scatter plot of cell cycle analysis after transfection with scRNA or miR-103-3p mimic (miR-103). (E) Relative expression levels of PCNA, CCNB1, and CCND1 were analyzed by qRT-PCR and normalized to U6 levels. The values are shown as relative ratios, with the intensity of the scRNA control set to one. All data are shown as the mean ± SEM (n > 3), where the level of significance is represented as *p < 0.05; **p < 0.01; ***p < 0.001 vs. scRNA. TWF1, Twinfilin-1; EdU, ethynyl deoxyuridine; qRT-PCR, real-time qualitative polymerase chain reaction; ns, no significance.

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