miR-491 inhibits skeletal muscle differentiation through targeting myomaker

Archives of Biochemistry and Biophysics 625-626 (2017) 30e38

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miR-491 inhibits skeletal muscle differentiation through targeting myomaker Jian He a, Fei Wang b, *, Peng Zhang a, Wenjiong Li a, Jing Wang a, Jinglong Li a, Hongju Liu a, Xiaoping Chen a, b, ** a

State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center, No. 26 Beiqing Road, Beijing 100094, China National Key Laboratory of Human Factors Engineering, China Astronaut Research and Training Center, No. 26 Beiqing Road, Beijing 100094, China

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 March 2017 Received in revised form 10 May 2017 Accepted 31 May 2017 Available online 1 June 2017

The myogenesis of skeletal muscle has several stages, including satellite cell proliferation, differentiation, fusion and specific muscle formation. Recent studies have shown that myomaker, a muscle-specific transmembrane protein, was critical for myoblasts fusion. However, the regulatory mechanism of myomaker and its effects on myogenesis remain elusive. In this study, miR-491 was identified as a posttranscriptional regulator of myomaker, which binds specifically to its 30 untranslated region leading to its down-regulation. At the end of myotube differentiation, the expression levels of miR-491 increased drastically, while myomaker was significantly down-regulated, which indicated that miR-491 shut down the expression of myomaker. Functional studies showed that miR-491 overexpression suppressed muscle cell differentiation and adult muscle regeneration, while the inhibition of miR-491 promoted myotube differentiation. Taken together, our findings identified miR-491 as a novel negative regulator of myogenic differentiation through targeting myomaker. © 2017 Published by Elsevier Inc.

1. Introduction The development of skeletal muscle (myogenesis) is a complex and multi-stage process with many regulators cooperatively involved in these stages [1]. The myogenic progenitor cells give rise to proliferating cells that are committed to the myogenic lineage termed myoblasts. Proliferating myoblasts subsequently differentiate and then fuse to form multi-nucleated myofibers [2]. In adult muscle regeneration, the quiescent satellite cells are activated by trauma mechanically or biologically, then undergo proliferation, differentiation, and finally generate into new muscle fibers [3]. It is well known that the myogenic regulatory factors (MRFs) determine the process of myogenesis [4]. Among all the development stages, differentiation is crucial to muscle cell fates and final muscle formation. The regulatory network of muscle cell differentiation has

* Corresponding author. ** Corresponding author. State Key Laboratory of Space Medicine Fundamentals and Application, China Astronaut Research and Training Center, No. 26 Beiqing Road, Beijing 100094, China. E-mail addresses: [email protected] (F. Wang), [email protected] (X. Chen). http://dx.doi.org/10.1016/j.abb.2017.05.020 0003-9861/© 2017 Published by Elsevier Inc.

been studied sufficiently but still need to be perfected. And, the effect of miRNAs in myogenesis has not been fully understood. The determination and differentiation of muscle cells are governed by four key transcription factors: Myf5, MyoD, Myogenin and Myf4. Myf5 and MyoD are identified as determinants of myogenesis, whereas Myogenin and Myf4 are highly expressed during the terminal differentiation and trigger myoblasts to fuse to form myotubes [5]. In these studies, the C2C12 mouse myoblast model is widely applied. Although many genes have been shown to regulate muscle differentiation, the controlling of muscle cell fusion remains indistinct. TMEM8c was reported to be a myogenesis regulator with expression profiles similar to those of MyoD and Myogenin, also known as Myomaker, which contains seven membrane-spanning regions embedded in the plasma membrane and a required intracellular C-terminal tail [6]. It is the first muscle-specific protein identified as direct regulator of myoblasts fusion in mammals [7]. Myomaker is highly expressed in the developing skeletal muscle and subsequently down-regulated upon completion of muscle formation [8]. Genetic knockout of myomaker in mice results in a deficiency of fusion competency in myoblasts, and myomaker is transiently expressed in satellite cells in response to muscle

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injuries, established its essential role in adult muscle regeneration [9]. It has also been shown that mono-nucleated muscle cells failed to fuse to form multi-nucleated myotubes in the absence of myomaker in zebrafish [10]. During the avian myoblast differentiation, MyoD and Myogenin could bind directly to myomaker promotor and induce myomaker transcription [11]. The expression of MRFs must be precisely controlled both in space and time, by the complex regulatory networks of the transcription of their corresponding genes and the stabilization of their mRNAs. MicroRNAs (miRNAs) are about 22nt single-stranded non-coding RNAs that silence gene expression at post-transcriptional level by binding to the 30 -UTR of specific target mRNAs or promoting mRNA degradation [12,13]. A variety of studies have demonstrated that miRNAs play a critical role in skeletal muscle differentiation and disease, mostly act as negative regulators via repressing the myogenic factors [14,15]. The miRNAs involved in muscle differentiation comprise muscle-specific and non-muscle specific miRNAs. Muscle-specific miRNAs (myomiRs) are highly and specifically expressed during cardiac and skeletal muscle differentiation [16,17], including miR-1/206 and miR-133 families. The expression of these miRNAs is directly regulated by MRFs [18,19]. A recent study has reported that miR-140-3p inhibited myomaker expression and chicken myoblast fusion by binding to myomaker 30 -UTR in vitro [11], indicating that myomaker could be a key mediator of miRNAs to regulate skeletal muscle differentiation. In the present study, we identified the mammal miRNAs which target and regulate myomaker. Our data demonstrated that miR491 could suppress skeletal muscle differentiation via downregulation of myomaker. 2. Materials and methods 2.1. Animals All the C57BL/6 mice were purchased from Vital River Laboratories (Beijing, China) and the mouse experimental procedures were performed according to the guidelines from Committees of Animal Ethics and Experimental Safety of China Astronaut Research and Training Center. The dystrophin-deficient mdx mice and wildtype mice were purchased from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China).

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formaldehyde for 10 min and then stained as described below. 2.3. Target prediction Mouse myomaker (TMEM8c) coding sequence (CDS) was acquired from PubMed (www.ncbi.nlm.nih.gov/pubmed). Bioinformatics databases TargetScan6.2 (www.targetscan.org) and RNA22 (https://cm.jefferson.edu/rna22/) were used to predict potential miRNAs targeting to myomaker. 2.4. Plasmid construction 366-bp genomic fragment of myomaker 30 -UTR was amplified by PCR using the primer pairs: forward 50 -CTAGCTAGCGGCCCTGATGCTTCGATTCT-30 , reverse 50 -GCTCTAGAGAACCAGTGGGTCCCTAAGC-30 . The PCR products, containing the restriction enzyme Xhol and Xbal cutting site, were cloned into pmirGLO (Promega, USA), a Dual-Luciferase miRNA Target Expression Vector with multiple cloning site. The mutant Myomaker 30 UTR were produced from myomaker 30 -UTR by changing the miR491 binding site from CCCCCC to GGGGTG, which was accomplished using Fast Mutagenesis System (Stratagene, USA). Both of the plasmids were verified by sequencing (AuGCT, China). 2.5. RNA oligonucleotide and transfection The miRNA mimics, miRNA inhibitors, and the corresponding negative controls were purchased from RiboBio, PR China. Transfections were conducted using Lipofectamine 3000 Reagent (Invitrogen, USA). For functional study, the C2C12 cells and primary muscle cells were plated into Corning 6-well plates or FluoroDish (Wpiinc, USA). At the beginning of differentiation, 100 nM of negative controls for mimics (NC), miR-491 mimics, and negative controls for inhibitors (anti-NC), miR-491 inhibitors were transfected into cells. 200 nM of myomaker siRNA (TMEM8c siRNA) and scrambled siRNA (Santa Cruz, USA) were transfected or cotransfected with miR-491 inhibitors into cells. Cells were harvested or fixed at day 7 of differentiation for protein and Immunofluorescence assay. All the experiments were repeated for 3 times at least with 3 duplicates for each group, and the transfection procedure was performed according to the manufacturer's direction.

2.2. Cell culture 2.6. Dual-luciferase reporter assay C2C12 myoblast cells were cultured in growth medium (GM), DMEM medium (Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco, USA), 1% glutamine and penicillinstreptomycin (HyClone, USA), at 37  C under 5% CO2. To induce differentiation, cells were grown to 80% confluency, then switched into differentiation medium (DM), DMEM supplemented with 2% horse serum (Gibco, USA), 1% glutamine and penicillinstreptomycin. Primary mouse muscle cells were extracted from gastrocnemius muscle of 8-week-old C57BL/6 mice. Removed muscles were washed in HBSS (Hank's Balanced Salt Solution) for 3 times, and digested in 400 U/ml Collagenase Type 1 for 1.5 h at 37  C.The digestion was stopped by GM and the digested mixture was centrifuged at 1000 r/min speed for 5 min. The supernatant containing muscle cells were filtered by 70 mm cell strainer (Biologix, USA) for twice. The filtrate was centrifuged at 1000 r/min speed for 10 min and cells were precipitated. Then the precipitates were resuspended carefully in GM. The suspended cells were then plated into culture flask (Corning, USA) maintained in GM at 37  C under 5% CO2, and subcultured to remove fibroblasts. C2C12 myoblast cells or primary muscle cells were incubated in DM for differentiating into myotubes. Myotubes were fixed by 4%

C2C12 and 293T cells were plated into 24-well plates (Corning, USA) and grown to 90% confluency. For each well, 25 nM of NC, miR-491 mimics, and 50 nM of anti-NC, miR-491 inhibitors were transfected with 0.5 mg wild type or mutated myomaker 30 -UTR luciferase plasmids into cells. Cells were harvested 24 h after the transfections and the assays were performed using the DualLuciferase Reporter Assay System (Promega, USA). Renilla luciferase (hRluc-neo) acted as a control reporter for normalization. Each transfection was repeated for 3 times at least with 3 duplicates for each group. The measured course was performed according to kit protocols. 2.7. CTX injury model and administration of agomiRs or antagomiRs Cardiotoxin (CTX, Sigma, Germany) was dissolved in sterile saline to a concentration of 50 mM. 8-week-old C57BL/6 mice were anesthetized by 1% pentobarbital sodium (100 ml per kg body weight), the legs were shaved and disinfected with alcohol, and 20 ml CTX was injected intramuscularly into tibialis anterior (TA)

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muscle using a 25-ml microsyringe. Isopyknic saline injection was used as negative control. The mice were sacrificed and the TA muscles were harvested at days 3, 7, and 14 after CTX injection. Harvested muscles were frozen, cut at 8 mm and stained with hematoxylin and eosin (H&E). AgomiRs and antagomiRs of miR-491, synthesized from Ribobio, were chemically engineered oligonucleotides to mimic or block miRNA expression, and injected into TA at a dose of 1 nmol per side in the position where CTX was injected. A scramble miRNA agomiR and antagomiR was used as the negative control. The injections were repeated every 72 h to ensure the efficacy.

stained by Vectashield with DAPI (Vector, USA). Images were visualized using Leica TCS SPS III confocal microscope (Leica, Germany). The diameters of myotubes were measured and the fusion indexes (the percentage of nuclei under myotubes with three or more nuclei) were determined using Image-Pro Plus 6.0. 2.11. Statistical analysis All data are shown as mean ± S.D, from at least three independent experiments. Unpaired t-test and one-way ANOVA were used to determine the significance, accepting p < 0.05 as statistically significant.

2.8. RNA isolation and real-time PCR 3. Results Total RNA was extracted with TRIzol according the manufacturer's protocol (Invitrogen, USA). The PrimeScript RT reagent Kit (Takara, Japan) was used to reverse transcribes total RNA (500 ng) into complementary DNA with random hexamer primers. For determination of mature miR-491 expression, first strand cDNA was synthesized using First Strand cDNA Synthesis Kit (Thermo Scientific, USA). Real-time PCR was carried out in a Step-One Plus (ABI, USA) using Power SYBR® Green (Applied Biosystems). The results were compared with a standard curve and normalized to expression of GAPDH. The control expression level was set to 1 as described. The sequences of the primers used are listed below: GAPDH: forward 50 -TTGTGATGGGTGTGAACCACGAGA-30 , reverse 50 -CATGAGCCCTTCCACAATGCCAAA-30 , Myomaker: forward 50 ATCGCTACCAAGAGGCGTT-30 , reverse 50 -CACAGCACAGACAAACCAGG-3’. U6 snRNA was used for normalization. Both miR491 and U6 primers were purchased from Ribobio. All the experiments were repeated for 3 times at least with 3 duplicates for each group and expression levels calculated using the comparative CT method (2DDCT). 2.9. Western blot assay Protein extracted from TA muscles or cells of each group were prepared with RIPA lysis buffer (50 mM TriseHCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS) on ice for 30 min. Protein fractions were collected by centrifugation at 10,000 g at 4  C for 10 min and qualified using Bradford protein assay reagent (Bio-Rad, USA). For each lane, 20 mg proteins were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. The membranes were probed with primary antibodies in TBST overnight at 4  C: GAPDH (MW: 35 kDa, 1:2000 dilution, Santa Cruz Biotechnology, USA), TMEM8c (MW: 25 kDa, 1:500 dilution, Santa Cruz Biotechnology, USA) in 5% skim milk (Becton, Dickinson, USA). Then the membranes were incubated with secondary antibodies (anti-rabbit IgG or anti-Goat IgG, 1:2000 dilution, ZSGB-BIO, China) conjugated to horseradish peroxidase. Protein bands were visualized using enhanced chemiluminescence assay (ECL, Thermo Scientific, USA). GAPDH was used as the loading control. 2.10. Immunofluorescence and histological analysis Myotubes placed on Dish with Cover Glass Bottom (Wpiinc) were fixed, blocked with 5% goat serum (ZSGB-BIO, China) and incubated with anti-Myosin (1:100 dilution, ZSGB-BIO, China) in 5% sheep serum in PBS. TA muscles were harvested, frozen, sliced into sections at 8 mm. After being permeabilized by 0.3% Triton X-100 in PBS and blocked with 5% goat serum, sections on slides were incubated with primary antibody against Laminin (1:200 dilution, Abcam, UK), Myosin (1:100 dilution, Abcam, UK) and Desmin (1:200 dilution, Abcam, UK) in 5% sheep serum in PBS. Nuclei were

3.1. miR-491 suppresses myomaker through binding to its 30 -UTR To find out myomaker regulating miRNAs, we screened potential miRNAs targeting myomaker using TargetScan6.2 and RNA22 software. According to the matching results, miR-491-5p, miR-466i, miR-98-5p and let-7i were selected as candidates for targeting myomaker. The targeting miRNAs and predicted target sites were showed in Supplementary Table 1. To explore whether these miRNAs bind to the 30 -UTR of myomaker, we inserted mouse myomaker 30 -UTR in pmiRGLO luciferase reporter and then co-transfected miRNA mimics with constructed reporter plasmid into C2C12 cells. As was shown in Fig. 1A, miR-491 mimics was found to repress the myomaker luciferase activity by 50% compare with NC. Intuitively, the map of the mouse myomaker 30 -UTR including seeding area for miR-491 was showed in Supplementary Fig. 1A. The predicted binding site then was tested by RNA22 software both in mouse and human. Furthermore, the binding site was conserved between species, which indicated possible evolutionary significance (Supplementary Fig. 1B). To verify the interaction between myomaker 30 -UTR and miR491, we co-transfected wild type myomaker 30 -UTR luciferase reporter with miR-491 mimics or inhibitors into C2C12 and 293T cells. The results showed that the relative luciferase activities were drastically repressed by miR-491 mimics, while induced by miR491 inhibitors in both C2C12 and 293T cells with no significant differences (Fig. 1B and C). However, when the binding site was mutated (Fig. 1D), miR-491 mimics and inhibitors had no effects on the luciferase activities in both C2C12 and 293T cells (Fig. 1E and F). These results indicated that miR-491 can suppress myomaker by directly binding to the seeding area in 30 -UTR. 3.2. The expression patterns of miR-491 and myomaker during myoblast differentiation To investigate the expression of miR-491 during myogenesis in vitro, the expression level of miR-491 was examined during the differentiation of C2C12 myoblasts (Fig. 2A). We found that the expression of miR-491 was kept at low level during the differentiation process (day 0e5), and was dramatically up-regulated at the end of differentiation (day 6e7). At the same time, the expression profile of myomaker mRNA was detected (Fig. 2B). The results showed that myomaker mRNA increased significantly during the differentiation process (day 0e5), and dropped quickly at the end of differentiation (day 6e7). Meanwhile, the expression pattern of myomaker protein was similar to its mRNA (Fig. 2C). The level of myomaker protein gradually increased until day 5 of differentiation, and then it was down-regulated at day 6e7 of differentiation. Our results showed the opposite expression patterns of miR-491 and myomaker during and after C2C12 myoblast differentiation. Since myomaker plays a key role in myoblasts fusion [8], the low

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Fig. 1. Identification of the binding site of miR-491 in the myomaker 3′-UTR. (A) miR-466i, 491, 98-5p, Let-7i mimics or NC (negative control) were co-transfected with myomaker 30 -UTR reporter (Myk 30 UTR) into C2C12 cells. Dual-luciferase activities were normalized to NC, which was set as 1. Experiments were performed in 6 duplicates, and data are presented as mean ± S.D. Statistical significant differences between two groups was indicated by **p < 0.01. (B, C) Myomaker 30 -UTR luciferase reporter was co-transfected with miR-491 mimics, inhibitors or their negative controls (NC, anti-NC) into C2C12 and 293T cells. (D) Sequence containing the binding site of miR-491 to the myomaker 30 -UTR and the mutation of the binding region were shown. (E, F) Mutated myomaker 30 -UTR luciferase reporter was co-transfected with miR-491 mimics, inhibitors or their negative controls (NC, anti-NC) into C2C12 and 293T cells. Positive control was set using myomaker 30 -UTR co-transfected with miR-491 mimics. Dual-luciferase activities were normalized to NC, which was set as 1. Experiments were performed in 6 duplicates, and data are presented as mean ± S.D. Statistical significances are analyzed using one-way ANOVA and indicated by *p < 0.05, **p < 0.01.

expression level of miR-491 may promote myoblast differentiation, whereas the high expression level of miR-491 may contribute to the termination of myoblast differentiation. 3.3. miR-491 inhibits muscle cell fusion and myomaker expression To identify the role of miR-491 in myoblast differentiation, miR491 mimics or inhibitors were transfected into C2C12 cells at the first day of differentiation. Multi-nucleated myotubes were formed at the 7th day of differentiation. The myotubes were immunostained using anti-myosin (Fig. 3A). We found out that C2C12 cells transfected with miR-491 mimics were incapable of fusing to form myotubes, as compared with cells transfected with negative control (NC). On the contrary, miR-491 inhibitors (amiR-491) promoted myoblasts to form more and bigger myotubes (Fig. 3A). However, cells transfected with myomaker siRNA or co-transfected with amiR-491 failed to form mature myotubes. And, the miR-491 mimics led to a 50% decrease in myotube diameter and a 68% decrease in fusion index, compared with NC. Inversely, amiR-491 caused a 75% increase in myotube diameter and a 22% increase in fusion index, compared with anti-NC. Myomaker siRNA caused incapacities of myotube formation even in cells transfected with amiR-491 (Fig. 3B and C). Furthermore, we analyzed the proteins extracted from myotubes and expressions of myomaker in these six groups. The myomaker protein level showed a significant decrease

in miR-491 transfected cells and an increase in amiR-491 transfected cells, compared with their negative controls respectively. SiRNA blocked the myomaker protein expression largely, as showed in Fig. 3D. The effects of miR-491 on myoblasts differentiation were then examined in primary muscle cells, which were isolated directly from mice gastrocnemius muscles. Similarly, the transfection of miR-491 mimics inhibited the differentiation of primary muscle cells (Supplementary Fig. 2A), which was also reflected by the changes of diameters and fusion index (Supplementary Fig. 2B and C). And, the expression of myomaker protein was repressed by miR491and promoted by amiR-491 (Supplementary Fig. 2D). These results suggested that miR-491 can suppress muscle cell differentiation by targeting myomaker. 3.4. The expression profiles of miR-491 and myomaker in CTX injected and mdx mice To investigate the potential effects of miR-491 on myogenesis in vivo, an acute muscle injury model was established [9]. The tibialis anterior (TA) muscles were injured with cardiotoxin (CTX) and harvested at 3, 7, 14 days after injection. Hematoxylin and eosin (H&E) staining showed the process of muscle injury and regeneration (Fig. 4A). We observed that inflammatory infiltration was most acute on day 3 after injection and regeneration occurred on

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Fig. 2. The expression patterns of miR-491 and myomaker during myoblast differentiation. (A) The relative expressions of miR-491 during C2C12 myoblast differentiation were detected by Real-Time PCR. (B) The relative mRNA expressions of myomaker during C2C12 myoblast differentiation were detected by Real-Time PCR. (C) The protein levels of myomaker were analyzed by western blot assay and the densitometric analysis of each band was performed using Image-Pro Plus 6.0. Protein expression levels were normalized to GAPDH. Data are presented as mean ± S.D. of three independent experiments. Statistically significances are analyzed using one-way ANOVA and indicated by *p < 0.05, **p < 0.01.

Fig. 3. MiR-491 inhibits C2C12 myoblast differentiation and myomaker expression. (A) C2C12 myoblasts were transfected with NC, miR-491 mimics, anti-NC,miR-491 inhibitors, myomaker siRNA and myomaker siRNA co-transfected with inhibitors, and then induced to differentiation for 7 days. Myotubes were fixed and stained with anti-myosin (green) and DAPI (blue). Bars, 100 mM. (B, C) The diameter of myotubes and fusion index were counted by Image-Pro Plus 6.0. (D) Myomaker protein expressions were analyzed by western blot assay and the densitometric analysis of each band was performed using Image-Pro Plus 6.0. Protein expression levels were normalized to GAPDH. Data are presented as mean ± S.D of three independent experiments. Statistical significances are analyzed using one-way ANOVA and indicated by **p < 0.01.

day 7, when activated satellite cells were differentiated. The muscle was nearly restored on the day 14. We also found out that the

endogenous miR-491 was dramatically down-regulated during the muscle regeneration induced by injection of CTX and increased

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Fig. 4. The expression profiles of miR-491 and myomaker in CTX injected and mdx mice. (A) The TA muscles were injured by CTX, and harvested at 3, 7, 14 days. Frozen muscles were sectioned and stained with hematoxylin and eosin (H&E); the locations of injection were indicated by red arrow. Bars, 50 mm. Saline-injected TA muscle was used as control. (B) MiR-491(C) and myomaker mRNA expression were detected by Real-Time PCR. (D) Myomaker protein levels were analyzed by western blot assay. (E) Gastrocnemius muscle of 8 weeks old mdx and wild type mice were harvested. MiR-491 and (F) Myomaker mRNA expression were detected by Real-Time PCR. (G) Myomaker protein levels were analyzed by western blot assay. The densitometric analysis of each band was performed using Image-Pro Plus 6.0. Protein expression levels were normalized to GAPDH. Data are presented as mean ± S.D of three independent experiments. Statistical significances are analyzed using one-way ANOVA and indicated by *p < 0.05, **p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

gradually from day 3 to day 14 (Fig. 4B). However, the myomaker mRNA was rapidly up-regulated on day 3 and then declined until day 14 (Fig. 4C). Meanwhile, myomaker protein levels were

drastically increased at the 3rd and 7th day after CTX injection, and reversed to control level at the 14th day after CTX injection (Fig. 4D). The expression of myomaker during muscle regeneration

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Fig. 5. MiR-491 inhibits mouse muscle regeneration and myomaker expression. (A) The time course of agomiR-NC, agomiR-491, antagomiR-NC, antagomiR-491 and CTX injections. TA muscles were analyzed 14 d post-injury. (B) The sections stained with H&E showed the differences between groups. Bars, 50 mm. (C) Myosin and Desmin immunohistochemistry revealed a significant loss of regenerated muscle fibers in agomiR-491 treated mice. (D) Myomaker protein levels were analyzed by western blot assay. The densitometric analysis of each band was performed using Image-Pro Plus 6.0. Protein expression levels were normalized to GAPDH. Data are presented as mean ± S.D of three independent experiments. Statistical significances are indicated by **p < 0.01.

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was similar to a previous study [9]. Furthermore, we also detected the expression profiles of miR-491 and myomaker in mdx mice, which was characterized by cycles of muscle degeneration and regeneration [20], we found that the expression of miR-491 was reduced (Fig. 4E) while the mRNA and protein expression of myomaker were induced in TA muscles of mdx mice (Fig. 4F and G), compared with wild type mice. These results indicated that the low expression level of miR-491 may contribute to the expression of myomaker during adult muscle regeneration in vivo. 3.5. miR-491 inhibits muscle regeneration through targeting myomaker Since myomaker is necessary for muscle regeneration [9] and miR-491 regulates myomaker expression. The effects of agomiR491 and antagomiR-491, which were chemically engineered oligonucleotides to mimic or block miRNA expression, on the regeneration of TA muscle induced by CTX injection were assessed (Fig. 5A). H&E staining showed agomiR-491 inhibited the regenerative ability of TA muscle, which generated small and less myofibers containing multiple centralized nuclei, compared with the agomiR negative control (agomiR-NC) treated muscle. However, atagomiR-491 showed no significant effect on muscle regeneration (Fig. 5B) Furthermore, we immunostained regenerating TA muscles with anti-Myosin and anti-Desmin. The results revealed a remarkable loss of regenerative myofibers in regenerating muscles when treated with agomiR-491, also there were no distinct differences between antagomiR-491 treated muscles and their negative controls (antagomiR-NC) (Fig. 5C). Finally, we found a significant decrease of myomaker protein level in agomiR-491 treated muscle, compared with agomiR-NC (Fig. 5D). These results confirmed that miR-491 suppressed muscle regeneration by targeting myomaker. 4. Discussion As is well known, the differentiation of skeletal muscle is strictly regulated by positive and negative factors. Insulin-like growth factors (IGFs) and MRFs have been identified to positively regulate the muscle differentiation, while serum response factors (SRFs) and transforming growth factor-b (TGF-b) negatively regulated the muscle differentiation [15]. Furthermore, miRNAs have been reported to be involved in the regulation network of skeletal muscle differentiation [21]. Recently, some studies have demonstrated that myomaker was required for myoblast fusion and muscle regeneration [6,8e10]. However, the regulating mechanism of myomaker still remains elusive. Our results identified miR-491 as a crucial suppressor of skeletal muscle differentiation through binding to myomaker. Since myomaker is the only verified muscle-specific protein essential for muscle cell fusion both in embryonic muscle development and adult muscle regeneration, its down-regulation by miR-491 represents a novel pathway in regulating skeletal muscle differentiation. It has been reported that myomaker was regulated by miR-1403p in avian myoblasts [11]. As one target gene was usually controlled by multiple miRNAs [22,23]. The regulation of myomaker by miRNAs is largely unknown. Among the four predicted candidates, miR-491 displayed a 50% decrease in relative luciferase activity compared with the NC, which indicated miR-491 may bind to the mouse myomaker 30 -UTR. Moreover, the conservation of myomaker 30 -UTR between species revealed its potential functions. (Fig. 1and Fig. S1). In this study, myomaker was increased dramatically during the differentiation process (day 0e5), and dropped quickly at the end of differentiation (day 6e7). Myomaker has been proved to be a potent myoblast fusion protein [8], therefore elevated expression of

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myomaker was essential for myoblast differentiation. Simultaneously, the expression of miR-491 was maintained at low level. At the end of differentiation, the expression of miR-491 was sharply increased so as to down-regulate myomaker expression and terminate the process of differentiation (Fig. 2). Then we assessed the function of miR-491 during the differentiation of C2C12 myoblasts and primary muscle cells by in vitro assays. MiR-491 mimics significantly inhibited myoblast and primary cell differentiations, with less and smaller myotubes were formed, while miR-491 inhibitors promoted the process of differentiation (Fig. 3 and Fig. S2). In addition, we examined the expression patterns of miR-491 and myomaker in acute muscle injury and chronic mdx mice, which underwent adult muscle regeneration in vivo. Also, myomaker was induced during muscle regeneration when miR-491 was down-regulated. The opposite expression profiles of miR-491 and myomaker suggested miR-491 exerted its differentiation suppressor function by targeting myomaker. It has been reported that miR-491 functions as an important tumor suppressor by coordinating with various target genes involved in tumorigenesis, such as EGFR [24,25], Bcl-xL [26] CDK6 [27] and IGFBP2 [28,29]. However, we uncovered the function of miR-491 in skeletal muscle differentiation for the first time. To verify the function of miR-491 in muscle regeneration, we injected the agomiR-491, antagomiR-491, as well as their negative controls to TA muscle injured by CTX. Increased exogenous miR-491 by agomiR-491 revealed a large number of mono-nucleated muscle cells, instead of myofibers with multiple centralized nuclei (Figs. 4 and 5), suggesting that miR-491 is a suppressor in muscle regeneration. It was also been reported that the deletion of myomaker in satellite cells caused a similar impaired regeneration [9]. Then we examined the myomaker protein levels in agomiR-491 or antagomiR-491 injected muscles. AgomiR-491 caused a significant decrease of myomaker protein level. However, no enhanced muscle regeneration was observed in the TA muscle when the endogenous miR-491 was inhibited by antagomiR-491. Considering the low expression of miR-491 during muscle regeneration, it was easy to understand that no significant difference was found between antagomiR-491 treated muscle and negative control treated muscle. Interestingly, previous study has reported that miR-491 can be induced by TGF-b1 in rat proximal tubular epithelial cells [30]. It is well known that TGF-b1 signaling is crucial to skeletal myogenesis [31], so it is reasonable for us to suppose that miR-491 is involved in the regulation of TGF-b1 to skeletal muscle development, which needs to be further studied. In conclusion, we discovered for the first time that myomaker is a target gene of miR-491. Myomaker is a novel regulator of skeletal muscle differentiation, but its transcriptional regulation is still elusive. The regulation of myomaker by miR-491 and possibly other miRNAs may contribute to the understanding of skeletal muscle cell differentiation and regeneration. Author contributions J.H. conducted the experimental studies, interpreted the data and drafted the manuscript. W.F. and X.P.C contributed to the study design. P.Z. and W.J.L performed the animal experiments. J.W. and H.J.L contributed to cell culture experiments. J.L.L participated in data analysis. W.F. and X.P.C. provided funding for this study. All authors approved the final manuscript. Competing financial interests The authors declare no competing financial interests.

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