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MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation
Biochemical and Biophysical Research Communications xxx (2015) 1e6
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MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation Zhiyuan Ma, Xiaorui Sun, Dequan Xu, Yuanzhu Xiong, Bo Zuo* Key Laboratory of Swine Genetics and Breeding of the Ministry of Agriculture, College of Animal Sciences, Huazhong Agricultural University, Wuhan 430070, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 October 2015 Accepted 17 October 2015 Available online xxx
Myogenesis is a complex process including myoblast proliferation, differentiation and myotube formation and is controlled by myogenic regulatory factors (MRFs), MyoD, MyoG, Myf5 and Myf6 (also known as MRF4). MicroRNA is a kind of ~22 nt-long non-coding small RNAs, and act as key transcriptional or post-transcriptional regulators of gene expression. Identification of miRNAs involved in the regulation of muscle genes could improve our understanding of myogenesis process. In this study, we investigated the regulation of Myf6 gene by miRNAs. We showed that miR-374b specifically bound to the 3'untranslated region (UTR) of Myf6 and down-regulated the expression of Myf6 gene at both mRNA and protein level. Furthermore, miR-374b is ubiquitously expressed in the tissues of adult C57BL6 mouse, and the mRNA abundance increases first and then decreases during C2C12 myoblasts differentiation. Over-expression of miR-374b impaired C2C12 cell differentiation, while inhibiting miR-374b expression by 20 -O-methyl antisense oligonucleotides promoted C2C12 cell differentiation. Taken together, our findings identified miR-374b directly targets Myf6 and negatively regulates myogenesis. © 2015 Published by Elsevier Inc.
Keywords: Myogenesis Myf6 miR-374b C2C12 Skeletal muscle differentiation
1. Introduction In vertebrates, myogenic precursors originate from somite, proliferate and differentiate into myoblasts, myoblasts undergo several rounds of division and terminally differentiate into multinucleated myotubes [1,2]. This process is precisely controlled though the coordination of Myogenin, MyoD, Myf5 and Myf6, which are well known as myogenic regulatory factors (MRFs) [3]. These transcription factors orchestrate the expression of many muscle specific genes though recognize and bind to the promoter regions, which contain a simple consensus sequence of CANNTG (Ebox) [2,4]. Although these MRFs have overlap expression patterns, each MRFs plays distinct roles during myogenic differentiation [5,6]. While MyoD and Myf5 determine different muscle cell lineages [7], Myogenin and Myf6 are responsible for terminal differentiation and myotube formation [8,9]. In Myf5 and Myod doublemutant mice, Myf6 also acts as a determination factor [10]. MiRNAs are a set of evolutionarily conversed non-coding RNAs that inhibit translation or induce mRNA degradation by binding to
* Corresponding author. E-mail address: [email protected] (B. Zuo).
the 30 UTR of their target genes [11,12]. Researchers have demonstrated miRNAs take part in myogenesis. Some miRNAs are specially expression in muscle and play critical role in muscle development, including miR-1, miR-206 and miR-133 [13,14]. It is also found that non-muscle specific miRNAs regulate myogenesis. For example, miR-181, miR-26a, miR-148a and miR-146b enhanced myogenic differentiation [15e18], while miR-155, miR-23a and miR-199a-3p impaired myogenesis [19e21]. Identification of miRNA-mediated mechanism of myogenesis contributes to better understanding the complexity of gene regulation orchestrating the development of skeletal muscle. While the induction of miRNAs expression requires distinct MRFs during skeletal myogenesis [14], MRFs are also regulated by differential miRNAs. MiR-669a/miR-669q prevent skeletal myogenesis in cardiac progenitors by targeting MyoD 30 UTR [22]. MiR31 targets Myf5 in quiescent satellite cells thus preventing Myf5 protein accumulation [23]. MiR-186 inhibits myogenic differentiation by targeting myogenin [24]. However, there is no report about the regulation of Myf6 directly by miRNAs until now. In this study, we focused on the identification of miRNAs which could directly bind to and regulate Myf6 expression. We found that miR-374b is a regulator of Myf6 and negatively regulate C2C12 myoblasts differentiation.
http://dx.doi.org/10.1016/j.bbrc.2015.10.086 0006-291X/© 2015 Published by Elsevier Inc.
Please cite this article in press as: Z. Ma, et al., MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.086
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2. Material and methods 2.1. Cell culture C2C12 cells were grown in growth medium (GM) consisting of 90% Dulbecco's Modified Eagle's Medium (DMEM, Hyclone) and 10% fetal bovine serum (FBS, Gibco) at 37 C, 5% CO2. To induce differentiation, the sub-confluent cells were transferred to differentiation medium (DM), DMEM supplemented with 2% horse serum (Gibco). HeLa cells were cultured at the same condition of C2C12 cells in GM.
(1ug) was reverse transcribed into cDNA by using the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas) with random primers or mir-374b specific stem-loop primer (RT-miR-374: 50 CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCACTTAGC-30 ). Real-time quantitative PCR was performed using a SYBR green kit (Takara) on the Bio-Rad CFX-96>™ real time system (Bio-Rad). GAPDH was used to normalize the expression of individual mRNAs whereas U6 was used to normalize the mir-374b expression. Primers for expression analysis were provided in Supplementary Table S3. 2.4. Western blot analysis
2.2. Dual luciferase reporter assay We amplified 470 bp mouse Myf6 30 UTR from C2C12 cell line genomic DNA, and then the fragment was cloned into the XhoⅠ and SalⅠ restriction sites of pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega). Point mutation in the putative miR374b targeting site located in Myf6 30 UTR was generated using overlap-extension PCR. Primers used for plasmid construction and point mutation were listed in Supplementary Table S1. Vectors were confirmed by double enzyme digestion and sequencing (Sangon Biotech). MiRNA mimics, 20 -O-methyl antisense oligonucleotidesmiRNA inhibitor or negative control were synthesized by GenePharama (Shanghai, China), the sequences can be found in Supplementary Table S2. 50 nM of each miRNA mimics, miRNA inhibitor or negative control were co-transfected into HeLa cells in 24 well plates (Costar) with wild type or mutated Myf6 30 UTR DualLuciferase plasmid (200 ng) using Lipofectamine 2000 reagent (Invitrogen). 24 h later, cells were harvested and Dual Luciferase Reporter Assay System (promega) was used for Renilla and firefly luciferase activities assay in a luminometer (PerkinElmer). The relative luciferase activity was measured by firefly luciferase activity/renilla luciferase activity.
Cells were lysed with ice-cold RIPA buffer (Beyotime) containing 1 mM PMSF (Beyotime). The lysates were centrifuged and then boiled in 5 protein loading buffer for 5 min. Proteins were separated by 12% SDS-polyacrylamide gels and transferred to PVDF membranes (Millipore), then the membranes were blocked with 5% BSA. After blocking, the membranes were incubated with Myf6 (Santa Cruz), MHC (Santa Cruz) and b-tubulin (Santa Cruz) primary antibodies respectively, followed by horseradish peroxidaseconjugated specific secondary antibodies (Santa Cruz). The results were detected by ECL blotting detection reagents (Bio-Rad). 2.5. Immunofluorescence analysis C2C12 cells were grown in 12-well plats (Costar) and cross linked with 2% paraformaldehyde for 10 min at 72 h after differentiation. Then the cells were permeabilized with 0.1% Triton-X100 for 10 min and blocked with 5% BSA for 1 h. After blocking, the cells were incubated with MHC (Santa Cruz) primary antibodies for 2 h at room temperature. The Alexa Fluor 555 anti-mouse IgG (Beyotime) was incubated for 2 h at room temperature. Nuclei were visualized using DAPI staining.
2.3. cDNA synthesis and real-time quantitative PCR
2.6. Statistical analysis
Total RNA was extracted from tissues or cell by TRIzol reagent (Ambion) following the manufacturer's instruction. RNA quality and concentration were assayed by 0.8% agarose gel electrophoresis and NarnoDrop 2000 (Thermo Scientific), respectively. Each sample
Each experiment was repeated three times, all data are represented as mean ± standard deviation (SD). Student's t test was used to perform significant test. The level of significance was set at p < 0.05.
Fig. 1. MiR-374b targets 30 UTR of Myf6. (A) The analysis of the relative luciferase activity of Myf6 wt-30 UTR in HeLa cells after transfection with candidate miRNA mimics. MiR-374b and miR-376c caused a significant decrease in relative luciferase activity compared to negative control. (B) Predicted target site of miR-374b and the 8 bp mutation site of Myf6 mut30 UTR. (C) The Myf6 mut-30 UTR dual luciferase report vector was co-transfected with miR-374b into HeLa cells could not cause a decrease of relative luciferase activity compared to negative control. UN: untreated group. *p < 0.05, and **p < 0.01.
Please cite this article in press as: Z. Ma, et al., MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.086
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Fig. 2. MiR-374b repressed the endogenous Myf6 in C2C12 cells. A and B, cells were induced differentiation at 12 h after transfected with miR-374b mimics or negative control. (A) Total RNA were extracted and analyzed for Myf6 mRNA expression using real-time quantitative PCR at 24 h of differentiation. (B) Whole cell extracts were harvested and analyzed for protein expression using immunoblots at 72 h of differentiation. C and D, cells were induced differentiation at 12 h after transfected with miR-374b inhibitor or inhibitor control. Similar determination of Myf6 mRNA level (C) and protein level (D) were performed at 24 h or at 72 h of differentiation respectively. *p < 0.05, and **p < 0.01.
3. Results 3.1. MiR-374b directly binding to Myf6 30 UTR To identify miRNAs regulating Myf6, online prediction software miRanda was used to predict miR-374b, miR-376c, miR-377, miR499 and miR-669f as candidates. To verify whether these candidate miRNAs targeted Myf6 30 UTR, we performed luciferase reporter
assay. HeLa cells were grown with a confluence of ~70% in 24 well plate and then co-transfected with wild type dual luciferase reporter plasmid and candidate miRNA mimics or negative control. Of the five candidates, miR-374b most efficiently repressed the luciferase activity (Fig. 1A). In order to determine the specificity of miR374b and Myf6 binding site, we generated a mutated dual luciferase reporter plasmid with 8 bp mutation at the putative miR-374b target site in Myf6 30 UTR (Fig. 1B). However, over-expression of
Fig. 3. Expression profile of miR-374b. (A) The mature sequence and name of mmu-miR-374b in selected species. Underlines indicate seed sequence of miR-374b. Mmu, Musmusculus; Hsa, Homo sapiens; Cfa, Canisfamiliaris; Bta, Bos Taurus; Eca, Equuscaballus; Ssc, Susscrofa; Oar, Ovisaries; Chi, Capra hircus. (B) Tissue expression profile of miR-374b in adult C57BL6 mouse. (C) mRNA level of miR-374b in soleus and Gastrocnemius muscle. (D) mRNA level of miR-374b during C2C12 myoblasts differentiation. *p < 0.05, and **p < 0.01.
Please cite this article in press as: Z. Ma, et al., MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.086
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miR-374b no longer repressed the mutant luciferase activity in HeLa cells (Fig. 1C). These results illustrated that miR-374b specially binds to the putative site of Myf6 30 UTR. 3.2. MiR-374b regulates Myf6 expression during myogenesis We further examined whether miR-374b regulates the endogenous Myf6 level in C2C12 cells. Cells were cultured in growth medium (GM) and transferred to differentiation medium (DM) to induce myogenic differentiation 12 h after miR-374b mimics (or inhibitor) transfection, then we detected the Myf6 mRNA level at 24 h of differentiation and protein level at 72 h of differentiation. We found that over-expression of miR-374b leads to the decrease at mRNA and protein level of Myf6 (Fig. 2, A and B). Conversely, when miR-374b was knocked-down by 20 -O-methyl antisense oligonucleotides, the mRNA and protein level of Myf6 were increased (Fig. 2, C and D). These results indicated miR-374b could regulate endogenous Myf6 expression at both mRNA and protein level.
3.3. The conservative and expression profile analysis of miR-374b MiR-374b is highly conserved during species. The mature sequence of selected species, including mouse, human, dog, bovine, horse, swine, sheep and goat are the same (Fig. 3A). Real-time quantitative PCR was performed to detect miR-374b mRNA level during nine tissues of adult C57BL6 mice. MiR-374b was universally expressed in all nine tissues (Fig. 3B), including skeletal muscle. Consider of Myf6 preferentially expressed in slow muscle fibers [25], we also measured miR-374b mRNA level in different fiber types. Results showed that miR-374b have a lower expression level in typically slow fiber type muscle the soleus than that of mix fiber type muscle gastrocnemius (Fig. 3C), suggested miR-374b may have functions in fiber type conversion. We detected the expression profile of miR-374b at 0, 24, 72 and 96 h of C2C12 cell differentiation, as shown in Fig. 3D, miR-374b abundance increases firstly and then decreases during myogenesis of C2C12 myoblasts. These results implied that miR-374b may play a role in myogenesis.
Fig. 4. MiR-374b negatively regulates C2C12 cells differentiation. AeD, C2C12 cells were induced differentiation at 12 h after transfected with miR-374b mimics or negative control. (A) Immunofluorescence of MHC in C2C12 myotubes at 72 h of differentiation. The nucleus was strained with DAPI. Scale bar, 200 mm. (B) Fusion index was measured by MHC positive cells/nuclei. (C) Immunoblotting for MHC and MyoG at 0 h, 24 h, 72 h and 96 h of differentiation. (D) RT-qPCR for relative mRNA level of MyoG, MyoD and Myf5 at 24 h of differentiation. EeG, C2C12 cells were induced differentiation at 12 h after transfected with miR-374b inhibitor or inhibitor control. (E) Immunofluorescence of MHC in C2C12 myotubes at 72 h of differentiation. Scale bar, 200 mm. (F) Fusion index was measured by MHC positive cells/nuclei. (G) Immunoblotting for MHC and MyoG at 0 h, 24 h, 72 h and 96 h of differentiation. *p < 0.05, and **p < 0.01.
Please cite this article in press as: Z. Ma, et al., MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.086
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3.4. MiR-374b suppress myogenesis in C2C12 cells To elucidate the function of miR-374b in myogenic differentiation, we transfected miR-374b mimics into C2C12 myoblasts, which were induced to differentiation after 12 h of transfection. Overexpression of miR-374b largely decreased fusion index (Fig. 4, A and B) and the protein level of MHC and MyoG (Fig. 4C) at 72 h of differentiation. However, at 24 h of differentiation, the mRNA level of Myogenin was increased, while MyoD and Myf5 have no remarkable changes (Fig. 4D). The results implied that miR-374b suppress myoblast differentiation. Further study was taken to investigate the role of miR-374b in myogenesis, we treated C2C12 cells with 20 -O-methyl antisense oligonucleotides against miR374b. On the contrary, inhibition of miR-374b promotes myogenic differentiation, as indicated by an increase of fusion index (Fig. 4, D and E) and protein level of MHC and MyoG (Fig. 4F). Taken together, it clearly indicated that miR-374b acts as a critical negative regulator of myogenesis. 4. Discussion Skeletal muscle development is a high coordinated process and is controlled by MRFs, including MyoD, MyoG, Myf5 and Myf6. Previous studies have discovered the importance of miRNAs in the regulation of skeletal muscle development though directly regulating MyoD [22], MyoG [24] and Myf5 [23]. In this study, miR-374b was identified as a novel miRNA that acts as a post-transcriptional regulator of Myf6 during myogenesis. The function and targets of miR-374b have been reported in several studies. Qian et al. found that miR-374b can target two AKT pathway-associated genes (AKT1 and Wnt16) and suppresses proliferation in T-cell lymphoblastic lymphoma [26]. Xie et al. showed miR-374b-5p promotes gastric cancer cell invasion and metastasis though suppressing RECK expression [27]. Pan et al. reported that miR-374b can regulate metabolism in Meishan pigs by targeting C/EBP-b [28]. But our findings identified Myf6 as a new target for miR-374b. MiR-374b showed efficiency for inhibiting the relative luciferase activity of Myf6 30 UTR wild type but not mutational type, indicating that miR-374b was a specific regulator of Myf6. Additionally, over-expression of miR-374b decreased Myf6 mRNA and protein expression level and knocked-down of miR374b by 20 -O-methyl antisense oligonucleotides increased expression level, suggesting that miR374b was a major regulator of endogenous Myf6 during C2C12 myoblasts differentiation. Skeletal myogenesis includes three major steps, specification of myoblasts, terminal differentiation and contractile muscle fibers assemble [29]. Myf6 not only act as a determination gene to direct embryonic cells into myoblasts, but also drives the terminal differentiation and assembly of the contractile muscle fibers [30,31]. In the process of cardiomyocyte progenitor cells differentiation, human miR-374 showed a four-fold increase in expression level. Considering the multiple roles of Myf6 during myogenesis and the expression pattern of miR-374 in cardiomyocyte progenitor cells, we strongly suggested miR-374b may also be critical for myogenesis. Non-muscle specific miRNAs also have myogenic functions, such as miR-24 [32], miR-181 [15] and miR-214 [33], and their expression levels change during myogenesis. Expression profile analysis showed that miR-374b is universally expressed in adult C57BL6 mouse tissues and up-regulated firstly then down-regulated during C2C12 myoblasts differentiation. It was found that over-expression of miR-374b during C2C12 myoblasts differentiation, inhibited myotube formation. On the contrary, suppressing the expression of miR-374b though 20 -O-methyl antisense oligonucleotides promote myotube formation. These results confirmed that miR-374b have a
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negative regulation role during C2C12 myoblasts differentiation. Our study illustrated that miR-374b significantly repressed myogenesis and reduced the expression of Myf6 during C2C12 cells differentiation. Myf6 is one of MRFs which control terminal differentiation, though regulating the expression of muscle-specific genes. Thus, the control of Myf6 expression mediated by miR374b may be a novel regulatory mechanism by which myogenesis was negatively regulated. Further studies should be performed to validate the effect of miR-374b on myogenesis in vivo by generating muscle injury model and miR-374b can be considered as a novel target for treating skeletal muscle disease. Conflicts of interest The authors declare no conflict of interest. Acknowledgments This work was supported by grants from the Key National High Technology Development Project of China (2013AA102502), the Fundamental Research Funds for the Central Universities (2014PY038) and the National Project for Breeding of Transgenic Pig (2013ZX08006-002). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.10.085. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.10.086. References [1] A.E. Brent, R. Schweitzer, C.J. Tabin, A somitic compartment of tendon progenitors, Cell 113 (2003) 235e248. [2] M.H. Parker, P. Seale, M.A. Rudnicki, Looking back to the embryo: defining transcriptional networks in adult myogenesis, Nat. Rev. Genet. 4 (2003) 497e507. [3] D.A. Sassoon, Myogenic regulatory factors: dissecting their role and regulation during vertebrate embryogenesis, Dev. Biol. 156 (1993) 11e23. [4] H. Weintraub, The MyoD family and myogenesis: redundancy, networks, and thresholds, Cell 75 (1993) 1241e1244. [5] B. Kablar, K. Krastel, C. Ying, A. Asakura, S.J. Tapscott, M.A. Rudnicki, MyoD and Myf-5 differentially regulate the development of limb versus trunk skeletal muscle, Development 124 (1997) 4729e4738. [6] X. Jin, J.G. Kim, M.J. Oh, H.Y. Oh, Y.W. Sohn, X. Pian, J.L. Yin, S. Beck, N. Lee, J. Son, H. Kim, C. Yan, J.H. Wang, Y.J. Choi, K.Y. Whang, Opposite roles of MRF4 and MyoD in cell proliferation and myogenic differentiation, Biochem. Biophys. Res. Commun. 364 (2007) 476e482. [7] B. Kablar, A. Asakura, K. Krastel, C. Ying, L.L. May, D.J. Goldhamer, M.A. Rudnicki, MyoD and Myf-5 define the specification of musculature of distinct embryonic origin, Biochem. Cell Biol. 76 (1998) 1079e1091. [8] M. Buckingham, S.D. Vincent, Distinct and dynamic myogenic populations in the vertebrate embryo, Curr. Opin. Genet. Dev. 19 (2009) 444e453. [9] C.F. Bentzinger, Y.X. Wang, M.A. Rudnicki, Building muscle: molecular regulation of myogenesis, Cold Spring Harb. Perspect. Biol. 4 (2012). [10] L. Kassar-Duchossoy, B. Gayraud-Morel, D. Gomes, D. Rocancourt, M. Buckingham, V. Shinin, S. Tajbakhsh, Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice, Nature 431 (2004) 466e471. [11] N. Bushati, S.M. Cohen, microRNA functions, Annu. Rev. Cell Dev. Biol. 23 (2007) 175e205. [12] D.P. Bartel, MicroRNAs: genomics, biogenesis, mechanism, and function, Cell 116 (2004) 281e297. [13] H.K. Kim, Y.S. Lee, U. Sivaprasad, A. Malhotra, A. Dutta, Muscle-specific microRNA miR-206 promotes muscle differentiation, J. Cell Biol. 174 (2006) 677e687. [14] J.F. Chen, E.M. Mandel, J.M. Thomson, Q. Wu, T.E. Callis, S.M. Hammond, F.L. Conlon, D.Z. Wang, The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation, Nat. Genet. 38 (2006) 228e233. [15] I. Naguibneva, M. Ameyar-Zazoua, A. Polesskaya, S. Ait-Si-Ali, R. Groisman, M. Souidi, S. Cuvellier, A. Harel-Bellan, The microRNA miR-181 targets the
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Please cite this article in press as: Z. Ma, et al., MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.086
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MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation Zhiyuan Ma, Xiaorui Sun, Dequan Xu, Yuanzhu Xiong, Bo Zuo* Key Laboratory of Swine Genetics and Breeding of the Ministry of Agriculture, College of Animal Sciences, Huazhong Agricultural University, Wuhan 430070, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 October 2015 Accepted 17 October 2015 Available online xxx
Myogenesis is a complex process including myoblast proliferation, differentiation and myotube formation and is controlled by myogenic regulatory factors (MRFs), MyoD, MyoG, Myf5 and Myf6 (also known as MRF4). MicroRNA is a kind of ~22 nt-long non-coding small RNAs, and act as key transcriptional or post-transcriptional regulators of gene expression. Identification of miRNAs involved in the regulation of muscle genes could improve our understanding of myogenesis process. In this study, we investigated the regulation of Myf6 gene by miRNAs. We showed that miR-374b specifically bound to the 3'untranslated region (UTR) of Myf6 and down-regulated the expression of Myf6 gene at both mRNA and protein level. Furthermore, miR-374b is ubiquitously expressed in the tissues of adult C57BL6 mouse, and the mRNA abundance increases first and then decreases during C2C12 myoblasts differentiation. Over-expression of miR-374b impaired C2C12 cell differentiation, while inhibiting miR-374b expression by 20 -O-methyl antisense oligonucleotides promoted C2C12 cell differentiation. Taken together, our findings identified miR-374b directly targets Myf6 and negatively regulates myogenesis. © 2015 Published by Elsevier Inc.
Keywords: Myogenesis Myf6 miR-374b C2C12 Skeletal muscle differentiation
1. Introduction In vertebrates, myogenic precursors originate from somite, proliferate and differentiate into myoblasts, myoblasts undergo several rounds of division and terminally differentiate into multinucleated myotubes [1,2]. This process is precisely controlled though the coordination of Myogenin, MyoD, Myf5 and Myf6, which are well known as myogenic regulatory factors (MRFs) [3]. These transcription factors orchestrate the expression of many muscle specific genes though recognize and bind to the promoter regions, which contain a simple consensus sequence of CANNTG (Ebox) [2,4]. Although these MRFs have overlap expression patterns, each MRFs plays distinct roles during myogenic differentiation [5,6]. While MyoD and Myf5 determine different muscle cell lineages [7], Myogenin and Myf6 are responsible for terminal differentiation and myotube formation [8,9]. In Myf5 and Myod doublemutant mice, Myf6 also acts as a determination factor [10]. MiRNAs are a set of evolutionarily conversed non-coding RNAs that inhibit translation or induce mRNA degradation by binding to
* Corresponding author. E-mail address: [email protected] (B. Zuo).
the 30 UTR of their target genes [11,12]. Researchers have demonstrated miRNAs take part in myogenesis. Some miRNAs are specially expression in muscle and play critical role in muscle development, including miR-1, miR-206 and miR-133 [13,14]. It is also found that non-muscle specific miRNAs regulate myogenesis. For example, miR-181, miR-26a, miR-148a and miR-146b enhanced myogenic differentiation [15e18], while miR-155, miR-23a and miR-199a-3p impaired myogenesis [19e21]. Identification of miRNA-mediated mechanism of myogenesis contributes to better understanding the complexity of gene regulation orchestrating the development of skeletal muscle. While the induction of miRNAs expression requires distinct MRFs during skeletal myogenesis [14], MRFs are also regulated by differential miRNAs. MiR-669a/miR-669q prevent skeletal myogenesis in cardiac progenitors by targeting MyoD 30 UTR [22]. MiR31 targets Myf5 in quiescent satellite cells thus preventing Myf5 protein accumulation [23]. MiR-186 inhibits myogenic differentiation by targeting myogenin [24]. However, there is no report about the regulation of Myf6 directly by miRNAs until now. In this study, we focused on the identification of miRNAs which could directly bind to and regulate Myf6 expression. We found that miR-374b is a regulator of Myf6 and negatively regulate C2C12 myoblasts differentiation.
http://dx.doi.org/10.1016/j.bbrc.2015.10.086 0006-291X/© 2015 Published by Elsevier Inc.
Please cite this article in press as: Z. Ma, et al., MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.086
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2. Material and methods 2.1. Cell culture C2C12 cells were grown in growth medium (GM) consisting of 90% Dulbecco's Modified Eagle's Medium (DMEM, Hyclone) and 10% fetal bovine serum (FBS, Gibco) at 37 C, 5% CO2. To induce differentiation, the sub-confluent cells were transferred to differentiation medium (DM), DMEM supplemented with 2% horse serum (Gibco). HeLa cells were cultured at the same condition of C2C12 cells in GM.
(1ug) was reverse transcribed into cDNA by using the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas) with random primers or mir-374b specific stem-loop primer (RT-miR-374: 50 CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCACTTAGC-30 ). Real-time quantitative PCR was performed using a SYBR green kit (Takara) on the Bio-Rad CFX-96>™ real time system (Bio-Rad). GAPDH was used to normalize the expression of individual mRNAs whereas U6 was used to normalize the mir-374b expression. Primers for expression analysis were provided in Supplementary Table S3. 2.4. Western blot analysis
2.2. Dual luciferase reporter assay We amplified 470 bp mouse Myf6 30 UTR from C2C12 cell line genomic DNA, and then the fragment was cloned into the XhoⅠ and SalⅠ restriction sites of pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega). Point mutation in the putative miR374b targeting site located in Myf6 30 UTR was generated using overlap-extension PCR. Primers used for plasmid construction and point mutation were listed in Supplementary Table S1. Vectors were confirmed by double enzyme digestion and sequencing (Sangon Biotech). MiRNA mimics, 20 -O-methyl antisense oligonucleotidesmiRNA inhibitor or negative control were synthesized by GenePharama (Shanghai, China), the sequences can be found in Supplementary Table S2. 50 nM of each miRNA mimics, miRNA inhibitor or negative control were co-transfected into HeLa cells in 24 well plates (Costar) with wild type or mutated Myf6 30 UTR DualLuciferase plasmid (200 ng) using Lipofectamine 2000 reagent (Invitrogen). 24 h later, cells were harvested and Dual Luciferase Reporter Assay System (promega) was used for Renilla and firefly luciferase activities assay in a luminometer (PerkinElmer). The relative luciferase activity was measured by firefly luciferase activity/renilla luciferase activity.
Cells were lysed with ice-cold RIPA buffer (Beyotime) containing 1 mM PMSF (Beyotime). The lysates were centrifuged and then boiled in 5 protein loading buffer for 5 min. Proteins were separated by 12% SDS-polyacrylamide gels and transferred to PVDF membranes (Millipore), then the membranes were blocked with 5% BSA. After blocking, the membranes were incubated with Myf6 (Santa Cruz), MHC (Santa Cruz) and b-tubulin (Santa Cruz) primary antibodies respectively, followed by horseradish peroxidaseconjugated specific secondary antibodies (Santa Cruz). The results were detected by ECL blotting detection reagents (Bio-Rad). 2.5. Immunofluorescence analysis C2C12 cells were grown in 12-well plats (Costar) and cross linked with 2% paraformaldehyde for 10 min at 72 h after differentiation. Then the cells were permeabilized with 0.1% Triton-X100 for 10 min and blocked with 5% BSA for 1 h. After blocking, the cells were incubated with MHC (Santa Cruz) primary antibodies for 2 h at room temperature. The Alexa Fluor 555 anti-mouse IgG (Beyotime) was incubated for 2 h at room temperature. Nuclei were visualized using DAPI staining.
2.3. cDNA synthesis and real-time quantitative PCR
2.6. Statistical analysis
Total RNA was extracted from tissues or cell by TRIzol reagent (Ambion) following the manufacturer's instruction. RNA quality and concentration were assayed by 0.8% agarose gel electrophoresis and NarnoDrop 2000 (Thermo Scientific), respectively. Each sample
Each experiment was repeated three times, all data are represented as mean ± standard deviation (SD). Student's t test was used to perform significant test. The level of significance was set at p < 0.05.
Fig. 1. MiR-374b targets 30 UTR of Myf6. (A) The analysis of the relative luciferase activity of Myf6 wt-30 UTR in HeLa cells after transfection with candidate miRNA mimics. MiR-374b and miR-376c caused a significant decrease in relative luciferase activity compared to negative control. (B) Predicted target site of miR-374b and the 8 bp mutation site of Myf6 mut30 UTR. (C) The Myf6 mut-30 UTR dual luciferase report vector was co-transfected with miR-374b into HeLa cells could not cause a decrease of relative luciferase activity compared to negative control. UN: untreated group. *p < 0.05, and **p < 0.01.
Please cite this article in press as: Z. Ma, et al., MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.086
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Fig. 2. MiR-374b repressed the endogenous Myf6 in C2C12 cells. A and B, cells were induced differentiation at 12 h after transfected with miR-374b mimics or negative control. (A) Total RNA were extracted and analyzed for Myf6 mRNA expression using real-time quantitative PCR at 24 h of differentiation. (B) Whole cell extracts were harvested and analyzed for protein expression using immunoblots at 72 h of differentiation. C and D, cells were induced differentiation at 12 h after transfected with miR-374b inhibitor or inhibitor control. Similar determination of Myf6 mRNA level (C) and protein level (D) were performed at 24 h or at 72 h of differentiation respectively. *p < 0.05, and **p < 0.01.
3. Results 3.1. MiR-374b directly binding to Myf6 30 UTR To identify miRNAs regulating Myf6, online prediction software miRanda was used to predict miR-374b, miR-376c, miR-377, miR499 and miR-669f as candidates. To verify whether these candidate miRNAs targeted Myf6 30 UTR, we performed luciferase reporter
assay. HeLa cells were grown with a confluence of ~70% in 24 well plate and then co-transfected with wild type dual luciferase reporter plasmid and candidate miRNA mimics or negative control. Of the five candidates, miR-374b most efficiently repressed the luciferase activity (Fig. 1A). In order to determine the specificity of miR374b and Myf6 binding site, we generated a mutated dual luciferase reporter plasmid with 8 bp mutation at the putative miR-374b target site in Myf6 30 UTR (Fig. 1B). However, over-expression of
Fig. 3. Expression profile of miR-374b. (A) The mature sequence and name of mmu-miR-374b in selected species. Underlines indicate seed sequence of miR-374b. Mmu, Musmusculus; Hsa, Homo sapiens; Cfa, Canisfamiliaris; Bta, Bos Taurus; Eca, Equuscaballus; Ssc, Susscrofa; Oar, Ovisaries; Chi, Capra hircus. (B) Tissue expression profile of miR-374b in adult C57BL6 mouse. (C) mRNA level of miR-374b in soleus and Gastrocnemius muscle. (D) mRNA level of miR-374b during C2C12 myoblasts differentiation. *p < 0.05, and **p < 0.01.
Please cite this article in press as: Z. Ma, et al., MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.086
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miR-374b no longer repressed the mutant luciferase activity in HeLa cells (Fig. 1C). These results illustrated that miR-374b specially binds to the putative site of Myf6 30 UTR. 3.2. MiR-374b regulates Myf6 expression during myogenesis We further examined whether miR-374b regulates the endogenous Myf6 level in C2C12 cells. Cells were cultured in growth medium (GM) and transferred to differentiation medium (DM) to induce myogenic differentiation 12 h after miR-374b mimics (or inhibitor) transfection, then we detected the Myf6 mRNA level at 24 h of differentiation and protein level at 72 h of differentiation. We found that over-expression of miR-374b leads to the decrease at mRNA and protein level of Myf6 (Fig. 2, A and B). Conversely, when miR-374b was knocked-down by 20 -O-methyl antisense oligonucleotides, the mRNA and protein level of Myf6 were increased (Fig. 2, C and D). These results indicated miR-374b could regulate endogenous Myf6 expression at both mRNA and protein level.
3.3. The conservative and expression profile analysis of miR-374b MiR-374b is highly conserved during species. The mature sequence of selected species, including mouse, human, dog, bovine, horse, swine, sheep and goat are the same (Fig. 3A). Real-time quantitative PCR was performed to detect miR-374b mRNA level during nine tissues of adult C57BL6 mice. MiR-374b was universally expressed in all nine tissues (Fig. 3B), including skeletal muscle. Consider of Myf6 preferentially expressed in slow muscle fibers [25], we also measured miR-374b mRNA level in different fiber types. Results showed that miR-374b have a lower expression level in typically slow fiber type muscle the soleus than that of mix fiber type muscle gastrocnemius (Fig. 3C), suggested miR-374b may have functions in fiber type conversion. We detected the expression profile of miR-374b at 0, 24, 72 and 96 h of C2C12 cell differentiation, as shown in Fig. 3D, miR-374b abundance increases firstly and then decreases during myogenesis of C2C12 myoblasts. These results implied that miR-374b may play a role in myogenesis.
Fig. 4. MiR-374b negatively regulates C2C12 cells differentiation. AeD, C2C12 cells were induced differentiation at 12 h after transfected with miR-374b mimics or negative control. (A) Immunofluorescence of MHC in C2C12 myotubes at 72 h of differentiation. The nucleus was strained with DAPI. Scale bar, 200 mm. (B) Fusion index was measured by MHC positive cells/nuclei. (C) Immunoblotting for MHC and MyoG at 0 h, 24 h, 72 h and 96 h of differentiation. (D) RT-qPCR for relative mRNA level of MyoG, MyoD and Myf5 at 24 h of differentiation. EeG, C2C12 cells were induced differentiation at 12 h after transfected with miR-374b inhibitor or inhibitor control. (E) Immunofluorescence of MHC in C2C12 myotubes at 72 h of differentiation. Scale bar, 200 mm. (F) Fusion index was measured by MHC positive cells/nuclei. (G) Immunoblotting for MHC and MyoG at 0 h, 24 h, 72 h and 96 h of differentiation. *p < 0.05, and **p < 0.01.
Please cite this article in press as: Z. Ma, et al., MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.086
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3.4. MiR-374b suppress myogenesis in C2C12 cells To elucidate the function of miR-374b in myogenic differentiation, we transfected miR-374b mimics into C2C12 myoblasts, which were induced to differentiation after 12 h of transfection. Overexpression of miR-374b largely decreased fusion index (Fig. 4, A and B) and the protein level of MHC and MyoG (Fig. 4C) at 72 h of differentiation. However, at 24 h of differentiation, the mRNA level of Myogenin was increased, while MyoD and Myf5 have no remarkable changes (Fig. 4D). The results implied that miR-374b suppress myoblast differentiation. Further study was taken to investigate the role of miR-374b in myogenesis, we treated C2C12 cells with 20 -O-methyl antisense oligonucleotides against miR374b. On the contrary, inhibition of miR-374b promotes myogenic differentiation, as indicated by an increase of fusion index (Fig. 4, D and E) and protein level of MHC and MyoG (Fig. 4F). Taken together, it clearly indicated that miR-374b acts as a critical negative regulator of myogenesis. 4. Discussion Skeletal muscle development is a high coordinated process and is controlled by MRFs, including MyoD, MyoG, Myf5 and Myf6. Previous studies have discovered the importance of miRNAs in the regulation of skeletal muscle development though directly regulating MyoD [22], MyoG [24] and Myf5 [23]. In this study, miR-374b was identified as a novel miRNA that acts as a post-transcriptional regulator of Myf6 during myogenesis. The function and targets of miR-374b have been reported in several studies. Qian et al. found that miR-374b can target two AKT pathway-associated genes (AKT1 and Wnt16) and suppresses proliferation in T-cell lymphoblastic lymphoma [26]. Xie et al. showed miR-374b-5p promotes gastric cancer cell invasion and metastasis though suppressing RECK expression [27]. Pan et al. reported that miR-374b can regulate metabolism in Meishan pigs by targeting C/EBP-b [28]. But our findings identified Myf6 as a new target for miR-374b. MiR-374b showed efficiency for inhibiting the relative luciferase activity of Myf6 30 UTR wild type but not mutational type, indicating that miR-374b was a specific regulator of Myf6. Additionally, over-expression of miR-374b decreased Myf6 mRNA and protein expression level and knocked-down of miR374b by 20 -O-methyl antisense oligonucleotides increased expression level, suggesting that miR374b was a major regulator of endogenous Myf6 during C2C12 myoblasts differentiation. Skeletal myogenesis includes three major steps, specification of myoblasts, terminal differentiation and contractile muscle fibers assemble [29]. Myf6 not only act as a determination gene to direct embryonic cells into myoblasts, but also drives the terminal differentiation and assembly of the contractile muscle fibers [30,31]. In the process of cardiomyocyte progenitor cells differentiation, human miR-374 showed a four-fold increase in expression level. Considering the multiple roles of Myf6 during myogenesis and the expression pattern of miR-374 in cardiomyocyte progenitor cells, we strongly suggested miR-374b may also be critical for myogenesis. Non-muscle specific miRNAs also have myogenic functions, such as miR-24 [32], miR-181 [15] and miR-214 [33], and their expression levels change during myogenesis. Expression profile analysis showed that miR-374b is universally expressed in adult C57BL6 mouse tissues and up-regulated firstly then down-regulated during C2C12 myoblasts differentiation. It was found that over-expression of miR-374b during C2C12 myoblasts differentiation, inhibited myotube formation. On the contrary, suppressing the expression of miR-374b though 20 -O-methyl antisense oligonucleotides promote myotube formation. These results confirmed that miR-374b have a
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negative regulation role during C2C12 myoblasts differentiation. Our study illustrated that miR-374b significantly repressed myogenesis and reduced the expression of Myf6 during C2C12 cells differentiation. Myf6 is one of MRFs which control terminal differentiation, though regulating the expression of muscle-specific genes. Thus, the control of Myf6 expression mediated by miR374b may be a novel regulatory mechanism by which myogenesis was negatively regulated. Further studies should be performed to validate the effect of miR-374b on myogenesis in vivo by generating muscle injury model and miR-374b can be considered as a novel target for treating skeletal muscle disease. Conflicts of interest The authors declare no conflict of interest. Acknowledgments This work was supported by grants from the Key National High Technology Development Project of China (2013AA102502), the Fundamental Research Funds for the Central Universities (2014PY038) and the National Project for Breeding of Transgenic Pig (2013ZX08006-002). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.10.085. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.10.086. References [1] A.E. Brent, R. Schweitzer, C.J. Tabin, A somitic compartment of tendon progenitors, Cell 113 (2003) 235e248. [2] M.H. Parker, P. Seale, M.A. Rudnicki, Looking back to the embryo: defining transcriptional networks in adult myogenesis, Nat. Rev. Genet. 4 (2003) 497e507. [3] D.A. Sassoon, Myogenic regulatory factors: dissecting their role and regulation during vertebrate embryogenesis, Dev. Biol. 156 (1993) 11e23. [4] H. Weintraub, The MyoD family and myogenesis: redundancy, networks, and thresholds, Cell 75 (1993) 1241e1244. [5] B. Kablar, K. Krastel, C. Ying, A. Asakura, S.J. Tapscott, M.A. Rudnicki, MyoD and Myf-5 differentially regulate the development of limb versus trunk skeletal muscle, Development 124 (1997) 4729e4738. [6] X. Jin, J.G. Kim, M.J. Oh, H.Y. Oh, Y.W. Sohn, X. Pian, J.L. Yin, S. Beck, N. Lee, J. Son, H. Kim, C. Yan, J.H. Wang, Y.J. Choi, K.Y. Whang, Opposite roles of MRF4 and MyoD in cell proliferation and myogenic differentiation, Biochem. Biophys. Res. Commun. 364 (2007) 476e482. [7] B. Kablar, A. Asakura, K. Krastel, C. Ying, L.L. May, D.J. Goldhamer, M.A. Rudnicki, MyoD and Myf-5 define the specification of musculature of distinct embryonic origin, Biochem. Cell Biol. 76 (1998) 1079e1091. [8] M. Buckingham, S.D. Vincent, Distinct and dynamic myogenic populations in the vertebrate embryo, Curr. Opin. Genet. Dev. 19 (2009) 444e453. [9] C.F. Bentzinger, Y.X. Wang, M.A. Rudnicki, Building muscle: molecular regulation of myogenesis, Cold Spring Harb. Perspect. Biol. 4 (2012). [10] L. Kassar-Duchossoy, B. Gayraud-Morel, D. Gomes, D. Rocancourt, M. Buckingham, V. Shinin, S. Tajbakhsh, Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice, Nature 431 (2004) 466e471. [11] N. Bushati, S.M. Cohen, microRNA functions, Annu. Rev. Cell Dev. Biol. 23 (2007) 175e205. [12] D.P. Bartel, MicroRNAs: genomics, biogenesis, mechanism, and function, Cell 116 (2004) 281e297. [13] H.K. Kim, Y.S. Lee, U. Sivaprasad, A. Malhotra, A. Dutta, Muscle-specific microRNA miR-206 promotes muscle differentiation, J. Cell Biol. 174 (2006) 677e687. [14] J.F. Chen, E.M. Mandel, J.M. Thomson, Q. Wu, T.E. Callis, S.M. Hammond, F.L. Conlon, D.Z. Wang, The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation, Nat. Genet. 38 (2006) 228e233. [15] I. Naguibneva, M. Ameyar-Zazoua, A. Polesskaya, S. Ait-Si-Ali, R. Groisman, M. Souidi, S. Cuvellier, A. Harel-Bellan, The microRNA miR-181 targets the
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Please cite this article in press as: Z. Ma, et al., MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.086