Long non-coding RNA Irm enhances myogenic differentiation by interacting with MEF2D

Abstract

Recent studies suggest important roles for long non-coding RNAs as essential regulators of myogenic differentiation. Here, we report that lncRNA Irm is upregulated during myogenesis. Functional analyses show that the overexpression of Irm enhances myogenic differentiation, whereas the inhibition of Irm has completely opposite effects in vitro. Notably, the inhibition of Irm blocks damage-induced muscle regeneration in vivo. Mechanistically, Irm regulates the expression of myogenic genes by directly binding to MEF2D, which in turn promotes the assembly of MyoD/MEF2D on the regulatory elements of target genes. Collectively, we have identified a novel lncRNA that interacts with MEF2D to regulate myogenesis.

Fig. 1. Irm is a myogenesis relevant lncRNA.

a Left: the representative pictures of C2C12 cells at 0, 1, 3, and 5 days in differentiation medium; right: the protein levels of MyoD and myogenin were detected by western blotting in C2C12 cells at 0, 1, 3, and 5 days in DM. Scale bars, 50 mm. b, c The expression levels of Rian (b) and Irm (c) were detected by qRT-PCR in C2C12 cells at 0, 1, 3, and 5 days in DM. d The certification of the primary myoblasts freshly isolated from mouse limb muscles. Scale bars, 50 mm. e Left: the representative pictures of the primary myoblasts at 0, 1, 3, and 5 days in DM; right: the protein levels of MyoD and myogenin were detected by western blotting in primary myoblasts at 0, 1, 3, and 5 days in DM. Scale bars, 50 mm. f, g The expression levels of Rian (f) and Irm (g) were detected by qRT-PCR in primary myoblasts at 0, 1, 3, and 5 days in DM. h, i The mRNA levels of MyoD (h) and Irm (i) were detected by qRT-PCR during CTX-induced regeneration. j The expression levels of Irm were detected by qRT-PCR in muscles of postnatal mice. Data shown represent the mean ± SEM of three independent experiments. n.s., not significant; ^*P < 0.05, ^**P < 0.05, and ^***P < 0.05 by Student’s t test. CTX, cardiotoxin; DM, differentiation medium; qRT-PCR, quantitative real-time polymerase chain reaction

Fig. 2. Overexpression of Irm enhances myogenic differentiation.

a Relative expression levels of Irm in C2C12 cells expressing pcDNA3.1 (pc-Ctrl) or pcDNA3.1-Irm (pc-Irm), as detected by qRT-PCR. b The differentiation of Irm-overexpressed C2C12 cells was detected by staining for MHC at 48 h in DM. Fusion index was calculated. Scale bars, 50 mm. c, d The mRNA levels of myogenin (c) and MHC (d) were detected by qRT-PCR in Irm-overexpressed C2C12 cells at 0, 1, 3, and 5 days in DM. e, f The protein levels of myogenin (e) and MHC (f) were detected by western blotting in Irm-overexpressed C2C12 cells at 0, 1, 3, and 5 days in DM. Myogenin protein levels were normalized to the GAPDH protein levels. Data shown represent the mean ± SEM of three independent experiments. ^*P < 0.05, ^**P < 0.05, and ^***P < 0.05 by Student’s t test. DM, differentiation medium; MHC, myosin heavy chain; qRT-PCR, quantitative real-time polymerase chain reaction

Fig. 3. Knockdown of Irm inhibits myogenic differentiation.

a Relative expression levels of Irm in C2C12 cells expressing si-scramble or si-Irm, as detected by qRT-PCR. b The differentiation of Irm-knockdown C2C12 cells was detected by staining for MHC at 96 h in DM. Fusion index was calculated. Scale bars, 50 mm. c, d The mRNA levels of myogenin (c) and MHC (d) were detected by qRT-PCR in Irm-knockdown C2C12 cells at 0, 1, 3, and 5 days in DM. e, f The protein levels of myogenin (e) and MHC (f) were detected by western blotting in Irm-knockdown C2C12 cells at 0, 1, 3, and 5 days in DM. Myogenin protein levels were normalized to GAPDH protein levels. Data shown represent the mean ± SEM of three independent experiments. ^*P < 0.05, ^**P < 0.05, and ^***P < 0.05 by Student’s t test. DM, differentiation medium; MHC, myosin heavy chain; qRT-PCR, quantitative real-time polymerase chain reaction

Fig. 4. Knockdown of Irm inhibits injury-induced muscle regeneration in vivo.

a Injection scheme for si-scramble or si-Irm into CTX-injured muscles. n = 8 mice for each group. b Relative expression levels of Irm at multiple time points after CTX injection, as detected by qRT-PCR. c RNA-FISH for detecting Irm 6 days post-CTX injury in muscle tissues. Green: Irm. Scale bar, 50 μm. d, e Knockdown of Irm reduced the mRNA (d) and protein (e) levels of myogenin at multiple time points after CTX injection. Myogenin protein levels were normalized to the GAPDH protein levels. f Representative hematoxylin and eosin (H&E)-stained sections of TA muscle 3 or 6 days post-CTX injury (3 or 6 dpi) induced by a CTX injection. Scale bars, 100 μm. g The cross-sectional areas of regenerating fibers on day 6 post-CTX injury using ImageJ software. Only myofibers containing centralized nuclei were measured. h The average area of the cross-sections of regenerating fibers on day 6 post-CTX injury; approximately eight random fields were captured, and > 1500 fibers were measured per sample. Data shown represent the mean ± SEM. ^*P < 0.05, ^**P < 0.05, and ^***P < 0.05 by Student’s t test. CTX, cardiotoxin; qRT-PCR, quantitative real-time polymerase chain reaction; RNA-FISH, RNA fluorescence in situ hybridization; TA, tibialis anterior

Fig. 5. Irm directly binds to MEF2D.

a RNA-FISH for detecting Irm and GAPDH in undifferentiated and differentiated C2C12 cells. Red: Irm or GAPDH. Blue: DAPI staining. Scale bar, 50 μm. b Relative abundance of Irm in total and nuclear RNAs of differentiating C2C12 cells, as detected by qRT-PCR. U6, Xist, and GAPDH were used as endogenous controls. c A schematic representation of RNA pull-down assay. d Western blotting assay for the specific interaction of Irm with MEF2D. e A schematic representation of RNA immunoprecipitation (RIP) assay. f RIP assay showed the association of MEF2D with Irm in vivo, as detected by RT-PCR and qRT-PCR. g Deletion mapping of the MEF2D-binding region(s) in Irm. Top: western blotting for MEF2D in protein samples pulled down by the different truncated Irm constructs. Bottom: diagrams of the full-length and truncated Irms. nt: nucleotide. Data shown represent the mean ± SEM of three independent experiments. n.s., not significant; ^***P < 0.05 by Student’s t test. DAPI, 4′,6-diamidino-2-phenylindole; qRT-PCR, quantitative real-time polymerase chain reaction; RNA-FISH, RNA fluorescence in situ hybridization

Fig. 6. Irm enhances the transcriptional activity of MyoD/MEF2D.

a The effect of Irm knockdown on the expression of myogenin and miR-206 depended on MEF2D, which was detected by qRT-PCR. b The effect of Irm knockdown on the differentiation of C2C12 cells depended on MEF2D. Fusion index was calculated. Scale bars, 50 mm. c C2C12 cells were transfected with si-Irm or si-scramble, and the luciferase reporter plasmids were generated by inserting the promoter region of myogenin or miR-206. The luciferase activities were measured 48 h after differentiation. d C2C12 cells were transfected with pc-Irm or pc-Ctrl, and the luciferase reporter plasmids were generated by inserting the promoter region of myogenin or miR-206. The luciferase activities were measured 48 h after differentiation. e Knockdown of Irm impaired the binding ability of MEF2D to myogenin and miR-206 promoters, which was determined by ChIP and qRT-PCR assays. f Knockdown of Irm impaired the binding ability of MyoD to myogenin and miR-206 promoters, which was determined by ChIP and qRT-PCR assays. g Chromatin isolation by RNA purification (ChIRP) assay was performed using even and odd antisense oligos tiling Irm and, a significant amount of genomic DNAs corresponding to myogenin and miR-206 promoters but not in glyceraldehyde 3-phosphate dehydrogenase (GAPDH) locus was retrieved. LacZ ChIRP retrieved no signal. h Model for Irm-regulating myogenesis. Data shown represent the mean ± SEM of three independent experiments. n.s., not significant; ^*P < 0.05, ^**P < 0.05, and ^***P < 0.05 by Student’s t test. ChIP, chromatin immunoprecipitation; qRT-PCR, quantitative real-time polymerase chain reaction