miR-208b modulating skeletal muscle development and energy homoeostasis through targeting distinct targets

Abstract

Embryonic and neonatal skeletal muscles grow via the proliferation and fusion of myogenic cells, whereas adult skeletal muscle adapts largely by remodelling pre-existing myofibers and optimizing metabolic balance. It has been reported that miRNAs played key roles during skeletal muscle development through targeting different genes at post-transcriptional level. In this study, we show that a single miRNA (miR-208b) can modulate both the myogenesis and homoeostasis of skeletal muscle by distinct targets. As results, miR-208b accelerates the proliferation and inhibits the differentiation of myogenic cells by targeting the E-protein family member transcription factor 12 (TCF12). Also, miR-208b can stimulate fast-to-slow fibre conversion and oxidative metabolism programme through targeting folliculin interacting protein 1 (FNIP1) but not TCF12 gene. Further, miR-208b could active the AMPK/PGC-1a signalling and mitochondrial biogenesis through targeting FNIP1. Thus, miR-208b could mediate skeletal muscle development and homoeostasis through specifically targeting of TCF12 and FNIP1.

Keywords: FNIP1; TCF12; energy homeostasis; miR-208b; skeletal muscle development.

Figure 1.

miR-208b inhibits skeletal muscle growth at postnatal. (A) Body weight (g) and (B) GA (gastrocnemius) muscle weight (g) in WT and Myl1-208b mice at 1 month of age; (C) Immunohistochemistry analysis of GA muscle. Top panel: H&E staining; middle and bottom panel: laminin and DAPI staining. (D) Frequency of distribution for cross-section area (CSA, µm²) of myofibers from GA muscle. The results are presented as mean ± SEM (n = 3). *, P < 0.05.

Figure 2.

miR-208b promotes C2C12 myoblast proliferation and prevents differentiation. (A) EdU staining of the proliferating myoblasts after miR-208b overexpression. C2C12 myoblasts were transfected with miR-208b mimic, mut-miR-208b, or control mimics and incubated with EdU for 24 h. Scale bar: 100μm. (B) The percentage of dual positive cells of EdU and DAPI staining. (C) C2C12 cells transfected with miR-208b mimic or NC were collected for cell cycle analysis 24 h after transfection. Cells were stained by propidium iodide and subjected to fluorescence-activated cell sorting analysis by flow cytometry. (D) Representative images showing differentiated myotubes treated with different miRNA mimics or inhibitors. C2C12 myoblast cells were transfected 4 times at 24 h intervals with miRNA-208b, miRNA-499, NC, miRNA-208b inhibitor, miRNA-499 inhibitor, or NC inhibitor. MyHc staining was performed at 5 days of differentiation. Scale bar: 100μm. (E and F) The Q-PCR results of miR-208b in C2C12 cells with miR-208b mimic or inhibitor miR-208b transfected, respectively. (G) The creatine kinase activity at 72 h of C2C12 differentiation when miR-208b or miR-499 overexpressed. (H) Q-PCR analysis of myogenic genes in myotubes treated with different miRNA mimics. (I) Western blotting analysis of MyoD protein level in differentiated myotubes treated with different miRNA mimics or inhibitors. (J) Scan of master gel used for spot comparison and selection. Whole protein preparations were differentially labelled with Cydyes and separated by isoelectric focusing and apparent molecular weight. MYH6, Desmin, Myh9, LaminA, Fscn1 and Dlat1 were down-regulated protein expression in the miR-208b overexpressing myoblasts by mass spectrum analysis. The results are presented as mean ± SEM (n = 3). The mRNA of tubulin was used as internal control for the expression of functional genes. *, P < 0.05; **, P < 0.01.

Figure 3.

miR-208b represses TCF12 during skeletal muscle development. (A and B) Wild and mutant TCF12 3′ UTR were inserted into the dual-luciferase reporter vector psi-CHECK2 at the 3′ end of the Renilla gene (hRluc). Then, the constructs were co-transfected with one of the constructs of pEGFP-miR-208b, pEGFP-mut-miR-208b, pEGFP-miR-499, pEGFP-mut-miR-499, or pEGFP-C1 (C1) into BHK-21 cells. The luciferase activity was analysed after 24 h transfection. (C) The miR-208b-biotin pull-down assays were performed and the TCF12 mRNA level was detected by Q-PCR. (D and E) Q-PCR results of miR-208b and TCF12 during C2C12 differentiation. (F) Q-PCR results of TCF12 in C2C12 cells with or without miR-208b mimics transfection. (G and H) C2C12 myoblast cells transfected with different miRNA mimics or inhibitors miRNA were differentiated for 72 h. TCF12 protein expression was detected by western blotting. (I) The leg muscle protein level of TCF12 was detected in WT and Myl1-208b mice at 1 month of age. (J) A total of 654 genes were identified in the differentially expressed genes from both miR-208b and si-TCF12 transcriptome and was represented by a Venn diagram. (K and L) GO analysis of the 654 common differentially expressed genes indicates significant enrichment of genes involved in negative regulation of cell cycle and myogenic differentiation. The results are presented as mean ± SEM (n = 3). U6 mRNA was used as internal control for miR-208b expression. *, P < 0.05; **P < 0.01.

Figure 4.

TCF12 is a potent pro-differentiation factor in skeletal muscle. (A-C) ChIP-seq profile shows the co-localization of MyoD, MyoG, and TCF12 on MyoD, MyoG, and TCF12 loci in C2C12 myoblasts or myotubes. (D) C2C12 cells were transfected with pcDNA-Flag-TCF12 plasmid and pcDNA-HA-myoD, respectively. 72 h after differentiation, cell lysates were subjected to IP. Co-immunoprecipitated proteins of MyoD, MyoG, and TCF12 were detected by western blotting. (E) Combined analysis of the si-TCF12 transcriptome and TCF12 ChIP-seq showed that common differentially expressed genes were mainly involved in cell cycle and myogenesis regulation. (F) KEEG pathway analysis showed that cell cycle, actin cytoskeleton, and hypertrophic cardiomyopathy were enriched. (G and H) Immunofluorescence detection of MyHc (green) in C2C12 cells when TCF12 overexpression (pcDNA-TCF12) or TCF12 knockdown (si-TCF12). NC and pcDNA3.1 used as controls. Scale bar: 100 μm.

Figure 5.

miR-208b stimulates fast to slow muscle fibre conversion and mitochondrial energy metabolism. (A) Analysis of GA muscle from both miR-208b overexpression and control mice. MYH7 staining showed slow type fibre in cross-sections (top). Scale bar: 200μm. Electron microscope (EM) showing mitochondria distribution (white arrows) in the miR-208b overexpression and control muscles (bottom) Scale bar: 2000μm. (B) The mRNA expression of the MyHc-I, MyHc-IIa, MyHcIIb, and MyHcIIx genes in the leg muscle from WT and Myl1-208b mice at 2 months of age. (C) Genes involved in mitochondria biogenesis were upregulated in Myl1-208b mice. (D and E) C2C12 myoblasts were differentiated into myotubes for 96 h, and myotubes were transfected with miR-208b mimics or miR-208b inhibitor. Immunostaining of C2C12 myotubes was performed using myosin-fast and myosin-slow antibody, respectively. Scale bar: 100μm. (F) C2C12 myotubes were transfected with miR-208b mimic, miR-499 mimic, or NC. The mRNA expression of MyHc-I, MyHc-IIa, MyHcIIb, and MyHcIIx genes was detected by Q-PCR. (G) The MAplot revealed the genes that changed significantly between the miR-208b overexpression and NC. all_down: all the down-regulated genes, all_up: all the up-regulated genes, mt_up: up-regulated mitochondrial genes, mt_up: down-regulated mitochondrial genes, no: no difference gene, op: mitochondrial oxidative phosphorylation genes. (H-J) C2C12 myoblasts were transfected with miR-208b mimic or NC. (H) ATP production; (I) The mRNA expression of mitochondrial energy metabolism genes (PGC1a, NDUFB7, Sdhb, Cytob, Cox5b, and Atp5i) was detected by Q-PCR. (J) The copy number of mitochondria genes (ND1 and Cytob) was detected by Q-PCR. The results are presented as mean ± SEM (n = 3). Tubulin mRNA was used as internal control for the expression of functional genes. *, P < 0.05; **, P < 0.01.

Figure 6.

miR-208b regulates energy metabolism and fibre type conversion through targeting FNIP1 gene. (A and B) The wild and mutant FNIP1 3′ UTR were inserted into the dual-luciferase reporter vector psi-CHECK2 at the 3′ end of the Renilla luciferase gene (hRluc). The constructs were co-transfected with pEGFP-miR-208b, pEGFP-mut-miR-208b, pEGFP-miR-499, pEGFP-mut-miR-499, or pEGFP-C1 (C1) into BHK-21 cells, and normalized Renilla luciferase activity was measured. (C) The miR-208b-biotin pull-down assays were performed and the PNIP1 mRNA level was detected by Q-PCR. (D) C2C12 myotubes were transfected with miR-208b or NC after transfection, the mRNA expression of FNIP1 was detected by Q-PCR. (E and F) C2C12 myotubes transfected with miR-208b mimic, NC, miR-208b inhibitor, or NC were differentiated for 48 h. FNIP1 protein expression was detected by Western blotting. (G) The leg muscle protein level of FNIP1, phosphor-AMPKα (Thr172), PGC1a, and Cytob was examined in WT and Myl1-208b mice at 2 months of age by Western blotting. (H-J) C2C12 myoblasts were induced into myotubes for 96 h after induction, and myotubes were transfected with si-FNIP1 or NC sequentially differentiated for 48 h. (H) The mRNA expression of MyHc-I, MyHc-IIa, MyHc-IIb, and MyHc-IIx genes was detected by Q-PCR. (I) Immunostaining of C2C12 myotubes was performed using myosin-fast and myosin-slow antibody. (J) The mRNA expression of mitochondrial energy metabolism genes (PGC1a, NUDFB7 Sdhb, Cytob, Mtg1, Mrp155, Timm44, TIm8a1, Drp1, and Mfn1) was detected by Q-PCR. The results are presented as mean ± SEM (n = 3). Tubulin mRNA was used as internal control for the expression of functional genes. *, P < 0.05; **, P < 0.01.

Figure 7.

Model of miR-208b-mediated regulation of skeletal muscle development and homoeostasis.