A newly identified lncRNA MAR1 acts as a miR-487b sponge to promote skeletal muscle differentiation and regeneration

Abstract

Background: Skeletal muscle atrophy induced by either aging (sarcopenia) or mechanical unloading is associated with serious health consequences. Long non-coding RNAs (lncRNAs) are implicated as important regulators in numerous physiological and pathological processes.

Methods: Microarray analysis was performed to identify the differentially expressed lncRNAs in skeletal muscle between adult and aged mice. The most decreased lncRNA in aged skeletal muscle was identified. The C2C12 mouse myoblast cells were used to assess the biological function of the lncRNA in vitro. The target microRNA of lncRNA and the target protein of microRNA were predicted by bioinformatics analysis and validated in vitro. Furthermore, the biology function of the lncRNA in vivo was investigated by local overexpression or knockdown the lncRNA in skeletal muscle. The therapeutic effect of the lncRNA overexpression in age-related or mechanical unloading-induced muscle atrophy was also evaluated.

Results: We identified a novel lncRNA (muscle anabolic regulator 1, MAR1) which was highly expressed in mice skeletal muscle and positively correlated with muscle differentiation and growth in vitro and in vivo. We predicted and validated that microRNA-487b (miR-487b) was a direct target of MAR1. We also predicted and validated that Wnt5a, an important regulator during myogenesis, was a target of miR-487b in C2C12 cells. Our findings further demonstrated that enforced MAR1 expression in myoblasts led to derepression of Wnt5a. Moreover, MAR1 promoted skeletal muscle mass/strength and Wnt5a protein level in mice. Enforced MAR1 expression in mice attenuated muscle atrophy induced by either aging or unloading.

Conclusions: The newly identified lncRNA MAR1 acts as a miR-487b sponge to regulate Wnt5a protein, resulting in promoting muscle differentiation and regeneration. MAR1 could be a novel therapeutic target for treating muscle atrophy induced by either aging or mechanical unloading.

Keywords: Long non-coding RNA; Muscle differentiation; Muscle regeneration; Wnt5a; miR-487b.

Figure 1

Expression level of muscle anabolic regulator 1 (MAR1) in gastrocnemius muscle associated with age‐related or mechanical loading‐related changes in skeletal muscle mass of mouse (A) microarray (left) and RT‐PCR analysis (right) of MAR1 levels between adult and aged skeletal muscle. *P < 0.05 vs. adult. (B) Real‐time PCR analysis of MAR1 levels in different tissue/organs of mouse. U6 is used as internal control. *P < 0.05 vs. skeletal muscle. (C) Real‐time PCR analysis of MAR1 levels in different skeletal muscles of mouse. U6 is used as internal control. *P < 0.05 vs. gastrocnemius muscle. (D–F) Expression level of MAR1 (D), muscle weight of gastrocnemius/body weight ratio (M/B ratio) (E) and correlation analysis between MAR1 level and M/B ratio, (F) of gastrocnemius muscle from young (2‐month‐old), adult (6‐month‐old), and aged (24‐month‐old) mice. *P < 0.05 vs. young. ^# P < 0.05 vs. adult. (G–I) Expression level of MAR1 (G), M/B ratio (H) and correlation analysis between MAR1 level and M/B ratio. (I) of gastrocnemius muscle in adult mice with 28‐day hindlimb suspension (HS) and 28‐day reloading. *P < 0.05 vs. baseline. ^# P < 0.05 vs. unloading. n = 3 for microarray and n = 10 for animal study. Each sample was assessed in triplicate. Data are presented as mean ± SEM.

Figure 2

Muscle anabolic regulator 1 (MAR1) promoted myogenic differentiation of C2C12 myoblasts in vitro. (A) Real‐time PCR analysis of expression levels of MAR1 and myogenic markers (MyoD and MyoG) during differentiation of C2C12 myoblasts. C2C12 myoblast cells were maintained in growth medium (GM) and differentiated into myotubes in differentiation medium (DM) for 1–7 days. *P < 0.05 vs. GM. (B) Schematic of lentiviral vector used to produce lentivirus for overexpression or knockdown of MAR1. (C) Real‐time PCR analysis of MAR1 level in MAR1 infected C2C12 cells. (D) Real‐time PCR analysis of myogenic markers (MyoD, MyoG, Mef2c, and Myf5) mRNA levels in MAR1 infected C2C12 cells. (E) Western blot analysis of myogenic markers (MyoD, MyoG, Mef2c, and Myf5) protein levels in MAR1 infected C2C12 cells. (F) Microscopy observation of myotube formation in MAR1 infected C2C12 cells on day 7 in DM. Scale bar = 50 μm. Black arrow: Myotube. (G) Real‐time PCR analysis of MAR1 level in MAR1 shRNA infected C2C12 cells. (H) Real‐time PCR analysis of myogenic markers (MyoD, MyoG, Mef2c, and Myf5) mRNA levels in MAR1 shRNA infected C2C12 cells. (I) Western blot analysis of myogenic markers (MyoD, MyoG, Mef2c, and Myf5) protein levels in MAR1 shRNA infected C2C12 cells. (J) Microscopy observation of myotube formation in MAR1 shRNA infected C2C12 cells on day 7 in DM. Scale bar = 50 μm. Black arrow: Myotube. n = 5 for each group. Each sample was assessed in triplicate. U6 small nuclear RNA is used as the internal control of lncRNA and miRNA. GAPDH is used as the control for mRNA. β‐Actin is used as the internal control in western blot. Data are presented as mean ± SEM. *P < 0.05 vs. control.

Figure 3

Prediction and validation of miR‐487b as one of target binding miRNAs of MAR1 in C2C12 cells. (A) Bioinformatic prediction of miR‐487b as a target miRNA of MAR1 by RNAhybrid 2.12. MFE: Minimum free energy. (B) Pull‐down assay and real‐time PCR analysis of miR‐487b level in C2C12 cells transfected with biotin labelled MAR1 at different dosage. *P < 0.05 vs. 0.5 mM, ^# P < 0.05 vs. 5 mM. (C) Bioinformatic prediction of binding site of miR‐487b to MAR1. (D) Luciferase reporter assay of miR‐487b‐WT and miR‐487b‐Mut in C2C12 cells transfected with either MAR1‐WT or MAR1‐Mut. * P < 0.05 vs. MAR1‐NC. (E) Formation of myotube in C2C12 cells with enhanced MAR1 expression co‐transfected with either miR‐487b‐WT or miR487b–Mut. Scale bar = 50 μm. (F–G) Real‐time PCR and western blot analysis of mRNA and protein expression of myogenic markers (MyoD, MyoG, Mef2c, and Myf5) in C2C12 cells with enhanced MAR1 expression co‐transfected with either miR‐487b‐WT or miR487b–Mut. *P < 0.05 vs. control, ^# P < 0.05 vs. MAR1. n = 5 for each group. Each sample was assessed in triplicate. U6 small nuclear RNA is used as the internal control of lncRNA and miRNA. GAPDH is used as the control for mRNA. β‐Actin is used as the internal control in western blot. Data are presented as mean ± SEM.

Figure 4

Prediction and validation of Wnt5a as a target of miR‐487b in C2C12 cells. (A) Western blot analysis of expression level of Wnt5a in C2C12 cells treated with either agomiR‐487b or antagomiR‐487b. *P < 0.05 vs. control, ^# P < 0.05 vs. agomiR‐487b. (B) Bioinformatic prediction of binding site in the 3′ UTR region of Wnt5a to miR‐487b. (C) Luciferase reporter assay of Wnt5a 3'UTR and Wnt5a 3' UTR‐Mut in C2C12 cells transfected with either miR‐487b‐WT or agomiR‐487b‐Mut. * P < 0.05 vs. agomiR‐487b‐NC. (D) Luciferase reporter assay of Wnt5a 3′ UTR in C2C12 cells treated with antagomiR‐487b. (E–F) Real‐time PCR analysis of expression of myogenic markers (MyoD, MyoG, Mef2c, and Myf5) (E) and formation of myotube (F) in C2C12 cells transfected with either Wnt5a 3′ UTR‐WT or Wnt5a 3′ UTR‐Mut. (G) Western blot analysis of expression levels of Wnt5a in C2C12 cells transfected with Wnt5a 3′ UTR. (H–I) Real‐time PCR analysis of expression of myogenic markers (MyoD, MyoG, Mef2c, and Myf5) (H) and formation of myotube (I) in C2C12 cells treated Wnt5a siRNA, antagomiR‐487b, and combination of above two. n = 5 for each group. Each sample was assessed in triplicate. U6 small nuclear RNA is used as the internal control of lncRNA and miRNA. GAPDH is used as the control for mRNA. β‐Actin is used as the internal control in western blot. Scale bar = 50 μm. Data are presented as mean ± SEM. *P < 0.05 vs. corresponding control. ^# P < 0.05 vs. AntagomiR‐487b.

Figure 5

MAR1 promoted expression level of Wnt5a protein in C2C12 cells. (A) Western blot analysis of expression level of Wnt5a in C2C12 cells during differentiation. *P < 0.05 vs. GM. (B) Correlation analysis between expression level of Wnt5a protein and MAR1 in C2C12 cells during differentiation. (C) Western blot analysis of expression level of Wnt5a in C2C12 cells with enhanced MAR1 expression treated with agomiR‐487b. (D) Western blot analysis of expression level of Wnt5a in C2C12 cells with inhibited MAR1 expression treated with antagomiR‐487b. n = 5 for each group. Each sample was assessed in triplicate. U6 small nuclear RNA is used as the internal control of lncRNA and miRNA. GAPDH is used as the control for mRNA. β‐actin is used as the internal control in western blot. Data are presented as mean ± SEM. * P < 0.05 vs. control.

Figure 6

Muscle anabolic regulator 1 (MAR1) promoted skeletal muscle mass/strength and Wnt5a protein level in mice (A) real‐time PCR analysis of MAR1 level in gastrocnemius muscle of MAR1 overexpressed mice. (B–D) Gastrocnemius muscle‐to‐body weight ratio (B), cross‐sections from mid‐belly gastrocnemius muscle immunostained by anti‐dystrophin antibody (C), and mean muscle fibre cross‐sectional area (CSA) (D) in MAR1 overexpressed mice. Scale bar = 50 μm. (E) In situ muscle function testing of specific force in gastrocnemius muscle of MAR1 overexpressed mice. (F–H) Expression level of Wnt5a protein (F), mRNA (G) and protein levels (H) of myogenic markers (MyoD, MyoG, Mef2c, and Myf5) in gastrocnemius muscle of MAR1 overexpressed mice. (I) Real‐time PCR analysis of MAR1 level in gastrocnemius muscle of MAR1 shRNA infected mice. (J–L) Gastrocnemius muscle‐to‐body weight ratio (J), cross‐sections from mid‐belly gastrocnemius muscle immunostained by anti‐dystrophin antibody (K) and mean muscle fibre CSA (L) in MAR1 shRNA infected mice. Scale bar = 50 μm. (M) In situ muscle function testing of specific force in gastrocnemius muscle of MAR1 shRNA infected mice. (N–P) Expression level of Wnt5a protein (N), mRNA (O), and protein levels (P) of myogenic markers (MyoD, MyoG, Mef2c, and Myf5) in gastrocnemius muscle of MAR1 shRNA infected mice. For (D) and (L), the fibre CSA of each muscle was measured from an average of 300 muscle fibres. n = 10 for each group. Each sample was assessed in triplicate. U6 small nuclear RNA is used as the internal control of lncRNA and miRNA. GAPDH is used as the control for mRNA. β‐Actin is used as the internal control in western blot. Data are presented as mean ± SEM. *P < 0.05 vs. control.

Figure 7

Enforced muscle anabolic regulator 1 (MAR1) expression attenuated muscle atrophy in aged mice. (A) Real‐time PCR analysis of MAR1 level in gastrocnemius muscle of aged mice with enforced MAR1 expression. (B–D) Gastrocnemius muscle‐to‐body weight ratio (B), cross‐sections from mid‐belly gastrocnemius muscle immunostained by anti‐dystrophin antibody (C), and mean muscle fibre CSA (D) in aged mice with enforced MAR1 expression. The fibre CSA of each muscle was measured from an average of 300 muscle fibres. Scale bar = 50 μm. (E) In situ muscle function testing of twitch force and specific force in gastrocnemius muscle of aged mice with enforced MAR1 expression. (F–H) Expression level of Wnt5a protein (F), mRNA (G), and protein levels (H) of myogenic markers (MyoD, MyoG, Mef2c, and Myf5) in gastrocnemius muscle of aged mice with enforced MAR1 expression. n = 10 for each group. Each sample was assessed in triplicate. U6 small nuclear RNA is used as the internal control of lncRNA and miRNA. GAPDH is used as the control for mRNA. β‐Actin is used as the internal control in western blot. Data are presented as mean ± SEM. *P < 0.05 vs. baseline, ^# P < 0.05 vs. aged.

Figure 8

Enforced muscle anabolic regulator 1 (MAR1) expression attenuated muscle atrophy in hindlimb suspension (HS) mice. (A) Real‐time PCR analysis of MAR1 level in gastrocnemius muscle of HS mice with enforced MAR1 expression. (B–D) Gastrocnemius muscle‐to‐body weight ratio (B), cross‐sections from mid‐belly gastrocnemius muscle immunostained by anti‐dystrophin antibody (C), and mean muscle fibre CSA (D) in HS mice with enforced MAR1 expression. The fibre CSA of each muscle was measured from an average of 300 muscle fibres. Scale bar = 50 μm. (E) In situ muscle function testing of twitch force and specific force in gastrocnemius muscle of HS mice with enforced MAR1 expression. (F–H) Expression level of Wnt5a protein (F), mRNA (G), and protein levels (H) of myogenic markers (MyoD, MyoG, Mef2c, and Myf5) in gastrocnemius muscle of HS mice with enforced MAR1 expression. n = 10 for each group. Each sample was assessed in triplicate. U6 small nuclear RNA is used as the internal control of lncRNA and miRNA. GAPDH is used as the control for mRNA. β‐Actin is used as the internal control in western blot. Data are presented as mean ± SEM. *P < 0.05 vs. baseline, ^# P < 0.05 vs. HS.