MiR-148a-3p Regulates Skeletal Muscle Satellite Cell Differentiation and Apoptosis via the PI3K/AKT Signaling Pathway by Targeting Meox2

Abstract

As bioinformatic approaches have been developed, it has been demonstrated that microRNAs (miRNAs) are involved in the formation of muscles and play important roles in regulation of muscle cell proliferation and differentiation. Previously, it has been demonstrated that miR-148a-3p is one of the most abundant miRNAs in chicken skeletal muscle. Here, we build on that work and demonstrate that miR-148a-3p is important in the control of differentiation of chicken skeletal muscle satellite cells (SMSCs). Elevated expression of miR-148a-3p significantly promoted differentiation and inhibited apoptosis of SMSCs but did not affect proliferation. Furthermore, it was observed that the mesenchyme homeobox 2 (Meox2) is a target gene of miR-148a-3p and that miR-148a-3p can down-regulate expression of Meox2, which promote differentiation of SMSCs and suppress apoptosis. Furthermore, miR-148a-3p overexpression encouraged activation of the PI3K/AKT signaling pathway, which could be recovered by overexpression of Meox2. Overall, these findings suggest that microRNA-148a-3p is a potent promoter of myogenesis via direct targeting of Meox2 and increase of the PI3K/AKT signaling pathway in chicken SMSCs.

Keywords: Meox2; PI3K/AKT pathway; differentiation; miR-148a-3p; skeletal muscle satellite cell.

FIGURE 1

Expression of miR-148a-3p in SMSCs. (A) Expression of miR-148a-3p in tissues of chickens. (B) Expression of miR-148a-3p in chest muscle during embryo development of ROSS 308 (broiler) and White Leghorn (layer). (C) Expression profile over time of miR-148a-3p from 1 to 5 days of satellite cell culturing. (D) Morphological changes in chicken skeletal muscle satellite cells after 1, 3, and 5 days.

FIGURE 2

Expression of miR-148-3p as modulated by a miR-148-3p mimic or inhibitor. (A,B) Expression of miR-148-3p in SMSCs was monitored using qRT-PCR following transfection with a miR-148-3p inhibitor or mimic. Data are presented as mean ± SEM of three individuals. The Student’s t-test was used to compare expression levels among different groups. **P < 0.01 vs. NC.

FIGURE 3

Effect of miR-148a-3p on the proliferation of SMSCs. (A) Cell growth curves as measured using the CCK-8 assay following transfection with a miR-148a-3p mimic, inhibitor or negative control in SMSCs. (B) Results of Edu assay for SMSCs transfected with a miR-148a-3p mimic, inhibitor, or negative control, where EdU (red) fluorescence is used as an indicator of proliferation and nuclei are indicated by Hoechst (blue) fluorescence. Photomicrographs were taken using a 100× magnification. (C) mRNA expression of CCND1 and PCNA in SMSCs transfected with a miR-148a-3p mimic, inhibitor, or negative control. Data are expressed as mean ± SEM (N = 3).

FIGURE 4

Effect of miR-148a-3p on differentiation of SMSCs. (A,B) Relative expression of mRNA of the genes Myf5, MyoD, MyHC, and MyoG, and abundance of proteins MyoG and Myosin as measured at 48 h of cell differentiation in SMSCs transfected with a miR-148a-3p mimic, inhibitor, or negative control. (C) Representative images of immunofluorescent staining of differentiated SMSCs (200×). Myosin: red, a molecular marker of myogenesis; DAPI: blue, cell nuclei; Merge: the fusion of SMSCs into primary myotubes. (D) Western blot detects protein levels of myogenic marker genes after inhibition and overexpression of miR-148a-3p. Data are expressed as mean ± SEM (N = 3). *P < 0.05; **P < 0.01 vs. NC.

FIGURE 5

Effect of miR-148a-3p on rate of apoptosis in SMSCs. Scattergram and rate of apoptosis in SMSCs transfected with miR-148a-3p inhibitor (A) or miR-148a-3p mimics (B) as analyzed using flow cytometry following staining with annexin V and PI. (C,D) Abundance of mRNA and proteins of caspase 3 and caspase 9 in SMSCs transfected with miR-148a-3p inhibitor and miR-148a-3p mimics as determined by use of Western blot analysis. Data are expressed as mean ± SEM (N = 3). **P < 0.01 vs. NC.

FIGURE 6

Meox2 as a target gene of miR-148a-3p. (A) Seed sequence of miR-148a-3p. (B) Prediction of target genes using TargetScan, miRDB, and Diana. (C) Dual-luciferase reporter gene (pEZX-FR02) with wild type (pEZX-Meox2-WT) or mutant (pEZX-Meox2-MT). (D) Luciferase assays were performed by co-transfection of wild-type or mutant Meox2 3′ UTR with a miR-148a-3p mimic or mimic-NC in SMSCs. (E) Expression of Meox2 following transfection with miR-148-30 by use of qRT-PCR. Data are expressed as mean ± SEM (N = 3). **P < 0.01 vs. NC.

FIGURE 7

Meox2 inhibits SMSCs differentiation. (A–C) Expression of Meox2, MyoD, MyoG, MyHC, and Myf5 mRNA and the protein abundance of MyoG and Myosin in SMSCs transfected with si-Meox2, pCD3.1-Meox2, or negative control. (D) Representative images of immunofluorescent staining of differentiated SMSCs (200×). Myosin: red, a molecular marker of myogenesis; DAPI: blue, cell nuclei; Merge: the fusion of SMSCs into the primary myotubes. Data are expressed as mean ± SEM (N = 3). *P < 0.05; **P < 0.01 vs. NC.

FIGURE 8

Effect of Meox2 on rate of apoptosis in SMSCs. Scattergram and rate of apoptosis in SMSCs transfected with pCD3.1-Meox2 (A) or siRNA-Meox2 (B) as analyzed using flow cytometry following staining with annexin V and PI. (C,D) Abundance of mRNA and proteins of caspase 3 and caspase 9 in SMSCs transfected with pCD3.1-Meox2 as determined by use of Western blot analysis. Data are expressed as mean ± SEM (N = 3). *P < 0.05; **P < 0.01 vs. NC.

FIGURE 9

Upregulation of miR-148a-3p suppresses the PI3K/AKT signaling pathways in SMSCs. (A) The miR-148a-3p mimic was transfected into SMSCs with pcDNA3.1 or pcDNA3.1-Meox2. At 48 h after transfection, western blot analysis was completed to assess expression of AKT, p-AKT and GAPDH. (B,C) Effect of miR-148a-3p mimics on differentiation and apoptosis of SMSCs in the absence or presence of LY294002. Data are expressed as mean ± SEM (N = 3).

FIGURE 10

miR-148a-3p-mediated skeletal muscle satellite cell regulatory pathway model. miR-148a-3p activates the PI3K/AKT pathway by directly inhibiting Meox2 expression, and then miR-148a-3p promotes differentiation of SMSCs and inhibits apoptosis of SMSCs by inhibiting Meox2.