Cellular Signalling 27 (2015) 1895–1904

Contents lists available at ScienceDirect

Cellular Signalling

j ourna l homepage: www.e lsev ie r .com/ locate /ce l l s ig

MicroRNA-128 targets myostatin at coding domain sequence to regulatemyoblasts in skeletal muscle development

Lei Shi a,c,1, Bo Zhou a,c,⁎,1, Pinghua Li a,c,1, Allan P. Schinckel b, Tingting Liang a, Han Wang a, Huizhi Li a,Lingling Fu a, Qingpo Chu a, Ruihua Huang a,c,⁎a Institute of Swine Science, Nanjing Agricultural University, Nanjing 210095, Chinab Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-2054, USAc Huaian Academy of Nanjing Agricultural University, Huaian 223001, China

Abbreviations: CDS, coding domain sequence; DM, dethynyl-2′-deoxyuridine; GM, Growth medium; miRNAmTOR, mammalian target of rapamycin; Myf5, myogenicchain;MyoG,myogenin; Pax, paired box; UTR, untranslate⁎ Corresponding authors at: Institute of Swine Science,

Nanjing 210095, China. Tel.: +86 25 84395362; fax: +86E-mail addresses: zhoubo@njau.edu.cn (B. Zhou), rhhu

1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.cellsig.2015.05.0010898-6568/© 2015 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 24 January 2015Received in revised form 1 April 2015Accepted 1 May 2015Available online 6 May 2015

Keywords:C2C12 myoblastsMicroRNA-128MyostatinProliferation and differentiation

MicroRNAs (miRNAs ormiRs) play a critical role in skeletalmuscle development. In a previous studyweobservedthat miR-128 was highly expressed in skeletal muscle. However, its function in regulating skeletal muscle devel-opment is not clear. Our hypothesis was thatmiR-128 is involved in the regulation of the proliferation and differ-entiation of skeletal myoblasts. In this study, through bioinformatics analyses, we demonstrate that miR-128specifically targetedmRNA ofmyostatin (MSTN), a critical inhibitor of skeletal myogenesis, at coding domain se-quence (CDS) region, resulting in down-regulating of myostatin post-transcription. Overexpression of miR-128inhibited proliferation of mouse C2C12 myoblast cells but promoted myotube formation; whereas knockdownof miR-128 had completely opposite effects. In addition, ectopic miR-128 regulated the expression of myogenicfactor 5 (Myf5),myogenin (MyoG), paired box (Pax) 3 and 7. Furthermore, an inverse relationshipwas found be-tween the expression of miR-128 andMSTN protein expression in vivo and in vitro. Taken together, these resultsreveal that there is a novel pathway in skeletal muscle development in which miR-128 regulates myostatin atCDS region to inhibit proliferation but promote differentiation of myoblast cells.

© 2015 Elsevier Inc. All rights reserved.

1. Introduction

MicroRNAs (miRNAs or miRs) are endogenous non-coding RNAs, 21to 23nucleotides in length, imperfectly bind andpartially block targetedmessenger RNAs (mRNAs) via inhibiting translation or increasing deg-radation [1]. Some muscle-specific miRNAs, such as miR-1, miR-133[2] and miR-206 [3,4], have important roles in regulating myogenesisand differentiation of skeletal muscle. Myostatin (MSTN) is a memberof the transforming growth factor-β (TGF-β) super family and playskey roles in activation, proliferation, and self-renewal of skeletal musclecells [5]. As a negative regulator in muscle growth, several animal stud-ies have shown that amutation ofMSTN causes proliferation and hyper-trophy of skeletal muscle cells and increases muscle mass [6,7]. Aprevious study demonstrated that MSTN allele of Texel sheep is charac-terized by a G to A transition in the 3′ UTR that creates a target site for

ifferentiation medium; EdU, 5-, microRNA; MSTN, myostatin;factor 5; MyHC, myosin heavyd region.Nanjing Agricultural University,2584395314.ang@njau.edu.cn (R. Huang).

miR-1 and miR-206, resulting in highly expressed miRNAs in skeletalmuscle. This causes translational inhibition of MSTN gene and hencecontributes to the muscular hypertrophy of Texel sheep [8]. In C2C12cells,miR-27a targets the 3′ untranslated region (3′-UTR) ofMSTN tran-script and down-regulates MSTN expression, which promotes myoblastproliferation [9]. Also, previous studies have shown that somemiRNAs,such asmiR-125 [10], miR-26 [11], miR-29 [12], miR-181 [13], andmiR-214 [14], are highly expressed in skeletal muscle [15] and play a notablyimportant role in skeletal muscle development. Therefore, it is impor-tant to detect the molecular mechanisms regulated by miRNAs duringthe proliferation and differentiation of skeletal muscle cells, whichcould provide a theoretical basis for the treatment of skeletal-relateddiseases and molecular breeding of livestock.

In a previous study, we observed thatmiR-128 is highly expressed inthe skeletal muscle bymiRNAmicroarray [16]. In addition, our bioinfor-matic analysis showed that miR-128 potentially targeted mRNA ofMSTN at coding domain sequence (CDS) region. Previous studies ongene expression regulation by miRNAs have mainly focused on sites lo-cated in 3′-UTRs [17]. Although previous studies have shown thatmiRNAs bind to CDS and 3′-UTR with comparable frequency [18], fewstudies on skeletal muscle have evaluated miRNAs binding to CDS.Thus, this study was designed to test the hypothesis that miR-128 is in-volved in the regulation of the proliferation and differentiation of skele-tal myoblasts via targeting the MSTN CDS region.

1896 L. Shi et al. / Cellular Signalling 27 (2015) 1895–1904

2. Materials and methods

2.1. Ethic statement

All experimental procedures were reviewed and approved by theAnimal Care and Use Committee of Nanjing Agricultural University.

2.2. Animals and cells

Male Balb/c mice were purchased from Qinglongshan LaboratoryAnimal Company (Nanjing, China). Five groups of mice (four miceeach) at different ages (2 days, 2, 4, 6, and 8 weeks) were used. Threeskeletal muscle samples from the hind legs of each mouse were collect-ed. Twomuscle sampleswere snap-frozen in liquid nitrogen, and storedat−80 °C for quantitative real-time RT-PCR and western blotting anal-ysis. The third sample was fixed in 4% paraformaldehyde for immuno-fluorescence assay.

Mouse C2C12 myoblasts were cultured in growth medium (GM)consisting of Dulbecco's modified Eagle's medium (DMEM, Gibco,Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Hyclon,Logan, UT, USA) and 1% penicillin–streptomycin (Gibco, Carlsbad, CA,USA) at 37 °C and 5% CO2. Near-confluent cells (80% to 90%) were in-duced to differentiate in differentiation medium (DM), consistingof DMEM plus 2% horse serum (Gibco, Carlsbad, CA, USA) and 1%Penicillin–Streptomycin solution for 1, 3 or 5 days.

2.3. Plasmid constructs and transfection

Fragments of mouse MSTN CDS region mRNA containing putativemiR-128 binding sites were commercially synthesized by Invitrogenand cloned into the pmirGLO dual-luciferase miRNA target expressionvector (Promega, Madison,WI, USA) between SacI and XhaI sites down-stream of the Renilla luciferase gene. Pointmutations, a 2-base substitu-tion, were introduced into the seed-matched sequences of putativemiR-128 binding sites of MSTN CDS (MSTN CDS Mut), which was syn-thesized by Invitrogen. The ligated vectors were transformed intoDH5α competent cells and then plated on ampicillin-resistant platesovernight. Plasmid DNA was extracted by the alkaline lysis methodand purified by using Endo Free Plasmid Maxi Kit (Tian Gen Biotech,Beijing, China). All constructs were verified by DNA sequencing.

RNA oligonucleotides, including miR-128 mimics (mmu-miR-128-3p, GenePharma, Shanghai, China), 2′-O-methylatedmiR-128 inhibitors(GenePharma, Shanghai, China), NC-nonspecific miRNA (GenePharma,Shanghai, China) or miR-128 precursor expression vectors (Cat.MmiR3449-MR04,Mus musculus miR-128-1 stem-loop, GeneCopoeia,Rockville, MD, USA) were transfected with Lipofectamine 2000(Invitrogen, Carlsbad, CA, USA) following the manufacturer's instruc-tions. RNA oligonucleotides were listed in Supplementary Table 1.

2.4. Cell proliferation analysis

EdU (5-ethynyl-2′-deoxyuridine) proliferation assay: 6 h after trans-fection with miR-128 inhibitor, NC (non-specific miRNA), miR-128mimics or pre-miR-128 were cultured with fresh growth mediumcontaining EdU (final concentration, 10 μM) for 24 h before fixation,permeabilization, and EdU staining (Click-iT EdU Imaging Kit, Lifetechnologies). EdU is a nucleoside analog of thymidine that is incorpo-rated into DNA during active DNA synthesis only by proliferating cells.Cell nuclei were stained with DAPI (Invitrogen) at a concentration of5 μg/ml for 30 min.

Cell cycle flow cytometry: 36 h after transfection with miR-128 in-hibitor, NC, miR-128 mimics or pre-miR-128, trypsinized cells werefixed in 70% (v/v) ethanol at−20 °C overnight. Cellswere then incubat-ed in 50mg/ml propidium iodide solution (100mg/mlRNase A and 0.2%(v/v) Triton X-100) for 30 min at 4 °C. Samples were run on a BDFACSCalibur flow cytometry (Becton Dickinson, Franklin Lakes, NJ,

USA) and data were analyzed with ModFit software (Verity SoftwareHouse, Topsham, ME, USA). The proliferation index (PI) stands for theproportion of mitotic cells from a total of 20,000 cells examined.

2.5. Immunofluorescence analysis

After transfection and differentiation, cell cultures were analyzed byimmunofluorescence as previously described [19]. Sampleswere subse-quently examined using a confocal microscopy (Zeiss LSM 700 META),and imaged on a CCD camera. Cell scoring was carried out using thepublic domain software ImageJ.

2.6. Quantitative real-time RT-PCR

In order to determine the expression of miR-128 and myogenicmarker genes myosin heavy chain (MyHC) and myogenin (MyoG) dur-ing the C2C12 myoblast differentiation, cells were transferred to DM.RNA and protein were sampled at 0, 1, 3 and 5 days of myoblast differ-entiation, respectively. Total RNA was extracted from mouse tissues orC2C12 cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). TotalRNA concentration and integrity were assessed by the NanoDrop 2000(Thermo, Waltham, MA, USA) and denatured gel electrophoresis.Reverse transcription was performed using PrimeScript reverse tran-scription reagent kit (Takara Biotech. Co., Ltd., Dalian, China). For quan-titative real-time RT-PCR of miRNA, total RNA was reverse transcribedusing a stem-loop primer as described elsewhere [40]. Randomprimers,oligo (dT)12–18 or miRNA specific primers were added to initiate cDNAsynthesis. The PCR reaction was carried out in the ABI Stepone Plusreal-time PCR system (Applied Biosystems,USA), and the reaction mix-ture used SYBR Premix Ex TaqTM II (Takara Biotech. Co., Ltd., Dalian,China).

The reaction conditions were optimized respectively as follows:95 °C for 30 s for initial denaturation, then 40 cycles of 95 °C for 5 s fordenaturation, 60 °C for 30 s for annealing, and 72 °C for 30 s for exten-sion, followed by 72 °C for 5 min for final extension. Relative gene ex-pression calculated as 2−ΔΔCt after normalization to U6 for miR-128 orGAPDH for other genes. Primer sequences are shown in SupplementaryTable 2.

2.7. Western blot analysis

Total protein extracts were prepared and used as previously de-scribed [20]. Cells were harvested in PBS, and whole-cell lysates wereprepared by re-suspending cell pellets in ice-cold RIPA buffer containing1% PMSF (protease inhibitor), and incubating the suspension on ice for30min. Supernatant lysateswere collected followinghigh-speed centri-fugation (12,700 ×g) for 5 min at 4 °C. Supernatant lysates protein con-centration was determined by BCA protein assay kit (Solarbio, BeiJing,China), and after adding 6× SDS sample buffer boiled at 100 °C for10 min. Proteins were separated on 10% SDS-PAGE gels and transferredto polyvinylidene fluoride membranes (Milipore, Billerica, MA, USA).The membranes were blocked with 2% BSA, incubated overnight at4 °C with primary antibodies for MyHC, MSTN (Abcam, USA; 1:1000 di-lution) andα-tubulin (Abcam, USA; 1:1000 dilution), and then incubat-ed with HRP-labeled anti-rabbit/mouse IgG secondary antibody(Beyotime, Jiangsu, China; 1:1000 dilution) for 2 h. Images acquisitionwas performed on an enhanced chemiluminesence detection system(Amersham, Piscataway, NJ, USA) and analyzed using ImageJ software.

Skeletal muscle was dissected from hind legs of Balb/c mice andground to powder in liquid nitrogen. Proteins from mouse muscle tis-sues were extracted in RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mMsodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS,2 mM EDTA and 1mMPMSF). The concentration of proteins was deter-mined using the BCA protein assay kit (Pierce Biotechnology Inc.,Rockford, IL, USA). Proteins were separated on 10% SDS-PAGE gels andtransferred to nitrocellulose membranes.

1897L. Shi et al. / Cellular Signalling 27 (2015) 1895–1904

2.8. Dual-luciferase reporter assays

Cells of C2C12 andHEK 293 T lineswere cultured (8× 104 cells/cm2)in 12-well plates. When cells reached 70% to 80% confluence, 1 μg ofplasmid pmirGLO-MSTN luciferase vector and miR-128 mimics or con-trol 50 nM were added to each well. Transfections were performedwith Lipofectamine 2000 (Invitrogen). The cells were harvested, anddual-luciferase assays were performed 24 h after transfection usingthe Dual-Luciferase Reporter Assay System (Promega, Madison, WI,USA). Renilla luciferase activity was normalized to Firefly luciferase ex-pression for each sample to account for differences in transfectionefficiency.

The next day, 1000 ng/ml pmirGLO luciferase vector, including thewild-type or mutant miR-128 binding site and 50 nmol/ml miR-128mimic or scrambled oligonucleotide, was transfected using Lipofecta-mine 2000. Luciferase assays were performed using the dual-luciferasereporter assay system 24 h after transfection.

2.9. Bioinformatic and statistical analysis

Prediction ofmiRNA target siteswas performedby RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/) [21] and TargetScan 6.2Mouse (http://www.targetscan.org) [22]. One-way ANOVA or two-sample t-test was performed using the statistical software IBM SPSS 21(IBM SPSS Statistics, Chicago, IL, USA). Data are presented as themean ± S.E.M (n = 3) and subjected to Student's t test; a value ofP b 0.05 was considered significant.

3. Results

3.1. MSTN CDS region is a direct target of miR-128

The MSTN transcript was predicted to contain a miR-128 target siteon exon 3 using RNAhybrid, suggesting that MSTNmay be a regulatorytarget of miR-128. Thematched region of themiRNA-mRNA interactionis conserved in different species, such as rat, cattle, sheep, dog, pig, andhorse (Fig. 1A). A minimum free energy−24.2 kcal/mol was found in abinding site of miR-128 in MSTN by the software RNAhybrid (Fig. 1B),whichmeans that it has a stable structure andmay be a potential target

Fig. 1. Identification of a functional miR-128 binding site in the myostatin (MSTN) CDS. (A)aligned sequences are indicated by stars. (B) MiRNA mRNA hybridization structures and fold(housing a 2-base mutation), or NC was co-transfected with the pMSTN vector into C2C12 andas mean ± S.E.M. (n = 3). Statistical significance was analyzed by the Student's t-tests. *P b 0.

site of miR-128 in MSTNmRNA. MiR-128 is highly conserved in variousspecies. MSTN gene and miR-128 are also located on the same chromo-some in different species, such asmouse, pig, cattle, sheep, chicken, andhuman.

The fragments of the MSTN CDS containing the potential target sitewere inserted downstream of the firefly luciferase reporter gene(luc2) in the pmirGLO vector (pMSTN). Activity of the luciferase re-porters with the antisense sequence of miR-128 (miR-128-luc) was ab-rogated by miR-128 in a positive control. A significant reduction ofluciferase activity was caused after co-transfection of the reporter con-struct containing wild-type target site along with the miR-128 expres-sion vector. In contrast, the repression by miR-128 was abolished afterintroduction of mutations in the seed-matched region. The dual lucifer-ase reporter plasmid pMSTN and miR-128 mimics were co-transfectedinto C2C12 myoblasts and HEK293T cells. Compared with the controland mutant groups, the luciferase activity was lower after being co-transfected with dual luciferase reporter plasmid pMSTN and miR-128mimics both in the C2C12 cells and in HEK293T cells (Fig. 1C). This sug-gests that miR-128 could regulate MSTN by binding the CDS region inskeletal muscle cells.

3.2. Inverse expression relationship between miR-128 and MSTN proteinin vivo and in vitro

MSTNmRNA andmiR-128 expression inmurinemuscle increased asanimal age increased from 2 days to 8 weeks (Fig. 2A). However, MSTNprotein expression was significantly down-regulated from 2 days to8 week old animals (Fig. 2B), which was further verified by the immu-nofluorescence detection of MSTN in murine muscle sections (Fig. 2C).

In addition, miR-128 expression, but not MSTN mRNA, showed anincreasing trend during C2C12 cell differentiation from 0 day of GM to5 days of DM (Fig. 2D). MSTN protein expression was significantlydown-regulated during C2C12 cell differentiation (Fig. 2E). The post-transcriptional regulation of MSTN by miR-128 showed an inverse ex-pression relationship: the increase of miR-128 in skeletal muscle withage coincided with the corresponding decrease in MSTN proteinexpression.

MyoG mRNA expression showed an increasing trend during C2C12cell differentiation from 0 day of GM to 5 days of DM; and MyHC

Seed-matched sequences in the MSTN CDS are in red and conserved regions betweening energy was predicted by RNAhybrid. (C) Pooled miR-128 mimics, miR-128 mutantHEK293T cells, and normalized luciferase activity was assayed. The results are presented05; **P b 0.01.

Fig. 2.Reciprocal expression ofMyostatin (MSTN) andmiR-128 duringmuscle development and C2C12 cell differentiation. (A) Expression ofmiR-128 andMSTN in the hind-limbmusclesof postnatal Balb/c mice was determined by RT-PCR. The ages of themice were 2 days, 2, 4, 6, and 8 weeks. Fold changewas in relation to expression in 2-day-old mice. (B) MSTN proteinexpression was normalized to α-tubulin expression using western blotting assays. (C) Immunofluorescence detection with MSTN antibody was used to identify MSTN protein (red).The nuclei were stained blue with DAPI. The scale bar stands for 50 μm. The fluorescence intensity of is presented as mean ± S.E.M. (D) Expression of miR-128, MyoG, MyHC andMSTN mRNA in C2C12 cells was determined by RT-PCR at 0 day of GM, 1 day, 3 days and 5 days of DM. Fold change was in relation to expression in 0-day of GM. (E) Myostatin andMyHC protein expression of C2C12 cells at 0 day of GM, 1 day, 3 days and 5 days of DMwas normalized toα-tubulin expression using western blotting assays. The results are presentedas mean ± S.E.M. (n = 3). Statistical significance was analyzed by the Student's t-tests. *P b 0.05; **P b 0.01.

1898 L. Shi et al. / Cellular Signalling 27 (2015) 1895–1904

mRNA expression reached the highest level on the 3 days of DM(Fig. 2D), whereasMyHC protein expression gradually increased duringmyoblast differentiation (Fig. 2E). These results suggest that miR-128expression was up-regulated with C2C12 cell differentiation.

3.3. miR-128 inhibited proliferation of C2C12 cells

To study the effects of miR-128 overexpression on C2C12 cell prolif-eration, themiR-128 precursor expression vector was transfected to the

1899L. Shi et al. / Cellular Signalling 27 (2015) 1895–1904

C2C12 cells. Since there is a fluorescent protein eGFP in the miR-128precursor expression vector, transfection efficiency can be detected bya fluorescence microscope (Fig. 3A). The RT-PCR results showed thatmiR-128 expression was increased at 48 and 72 h after transfection(Fig. 3B), which suggests that the transfection of miR-128 precursor ex-pression vector was effective.

Expression of myogenic factor 5 (Myf5), myogenin gene (MyoG),paired box (Pax) 3, and 7 was significantly increased in the miR-128transected group than in theNCgroup at both 48 and 72 h post transfec-tion (Fig. 3B). Western blotting assays (Fig. 3C) and immunofluores-cence detection (Fig. 3D) showed that MSTN protein expression wasdecreased in both the miR-128 precursor and mimics groups than inthe NC group, which suggests that miR-128 plays a role in the C2C12cell proliferation by inhibiting MSTN translation.

To study the effects of inhibition of miR-128 on the proliferation ofC2C12 cells, the 2′-O-methylated miR-128 inhibitor was transfected toC2C12 cells. The RT-PCR results showed that expression of miR-128was notably decreased at 48 and 72 h after transfection (Fig. 3B).Compared with NC group at 48 h, the mRNA expression of Myf5 andPax3 decreased in themiR-128 inhibitor group (Fig. 3B). ThemRNA ex-pression of Myf5, MyoG and Pax3 was less for the miR-128 inhibitorgroup than the NC group at 72 h (Fig. 3B). MSTN protein expression

Fig. 3. Effects of miR-128 on the proliferation of C2C12 cells. (A) Cells were transfectedwith mifection efficiency of miR128 precursor vectors were detected using a fluorescencemicroscope. (miR-128 overexpression group (transfected with mmu-miR-128 precursor expression vector)malized to tubulin expression using western blotting assays at 48 and 72 h of transfection. (D)group, miR-128 overexpression group (mmu-miR-128 precursor expression vector) and miR-50 μm. The results are presented as mean ± S.E.M. (n = 3). Statistical significance was analyze

was increased at 48 and 72 h after transfection in themiR-128 inhibitorgroup than in the NC group (Fig. 3C and D).

The proliferation of C2C12 cells was measured by the 5-ethynyl-2′-deoxyuridine (EdU) proliferation assay and propidium iodine flow cy-tometry. The results showed that both miR-128 mimics and pre-miR-128 repressed proliferation of C2C12 cells; contrarily, miR-128 inhibitoraccelerated proliferation of cells (Fig. 4A). Quantitative analysis alsodemonstrated that these changes were statistically significant. The re-sults of cell cycle analysis showed that both miR-128 mimics and pre-miR-128 increased the percentage of C2C12 cells in the G1 and G2phases, and decreased the percentage of cells in the S phase (Fig. 4B, Cand D). Whereas after miR-128 inhibition the percentage of cells wasdecreased in the G1 phase but increased in the S phase (Fig. 4C andD). These results demonstrated the proliferation of C2C12 cells can bereduced by miR-128 overexpression or increased via inhibition ofmiR-128, which suggests that miR-128 inhibits the proliferation ofC2C12 cells.

3.4. miR-128 promoted differentiation of C2C12 cells

To study the effects of miR-128 overexpression on the differentia-tion of C2C12 cells, the mmu-miR-128 precursor expression vector

R-128 precursor expression vectors (GeneCopoeia, Rockville, MD, USA) for 24 h, the trans-B) Expression levels ofmiR-128, MyoG, Myf5, Pax7 and Pax3 determined by RT-PCR in theand inhibitor group at 48 and 72 h of transfection. (C) MSTN protein expression was nor-Immunofluorescence detection of MSTN (red) of negative control group, miR-128 mimics128 inhibitor group in C2C12 cells at 48 and 72 h of transfection. The scale bar stands ford by the Student's t-tests. *P b 0.05; **P b 0.01.

Fig. 4.miR-128 inhibits proliferation of C2C12 cells. (A) Proliferating C2C12 cells were labeled with EdU (red) after transfection with miR-128 inhibitor, NC, miR-128 mimics orpre-miR-128 according to the manufacturer's instructions. Cell nuclei were stained with DAPI (blue). The percentage of EdU-positive C2C12 cells were quantified. The scale barstands for 100 μm. (B) C2C12 cells were transiently transfectedwith pooledmiR-128 inhibitor, NC, miR-128mimics or pre-miR-128, and the cell cycle phase (C) and proliferationindex (D) were analyzed by propidium iodine flow cytometry. The results are presented as mean ± S.E.M. (n = 3). Statistical significance was analyzed by the Student's t-tests.*P b 0.05; **P b 0.01.

1900 L. Shi et al. / Cellular Signalling 27 (2015) 1895–1904

1901L. Shi et al. / Cellular Signalling 27 (2015) 1895–1904

and miR-128 mimics were transfected to C2C12 cells. At 12 h aftertransfection, cells were induced to differentiate in DM. The RT-PCR re-sults showed that miR-128 expression increased at 3 days of DM(Fig. 5A). The mRNA expression of MyHC and MyoG was greater forthe miR-128 precursor and mimics groups than the NC group at3 days of DM (Fig. 5A). MSTN protein expression was less (Fig. 5B andD) but MyHC protein expression was greater (Fig. 5C and D) for themiR-128 precursor and mimics groups than in the NC group.

Fig. 5.miR-128 promotes the differentiation of C2C12 cells. (A) Expression levels of miR-128, Mwithmmu-miR-128 precursor expression vector) and inhibitor group (transfectedwithmiR-12ofMSTN (red) of negative control group,miR-128mimics group, miR-128 overexpression groupat 3 days of DM. The scale bar stands for 50 μm. The fluorescence intensity is presented as meamiR-128mimics group, miR-128 overexpression group (mmu-miR-128 precursor expression v50 μm. (D) Protein levels of MSTN and MyHC were detected post transfection with pre-miR-12western blotting assays. The fluorescence intensity of is presented as mean ± S.E.M. (n = 3). S

To study the effects of miR-128 inhibition on the differentiation ofC2C12 cells, the miR-128 inhibitor was transfected to C2C12 cells. At12 h after transfection, cells were induced to differentiate in DM. TheRT-PCR results showed that miR-128 expression increased at 3 days ofDM (Fig. 5A). Compared with NC group at 3 days, the mRNA expressionof MyHC and MyoG decreased notably (Fig. 5A). MSTN protein expres-sion was greater (Fig. 5B and D) but MyHC protein expression wasless (Fig. 5C and D) at 3 days of DM for the miR-128 inhibitor group

yoG andMyHC determined by RT-PCR in themiR-128 overexpression group (transfected8 inhibitor) at 3 days of differentiationmedium (DM). (B) Immunofluorescence detection(mmu-miR-128 precursor expression vector) andmiR-128 inhibitor group in C2C12 cells

n ± S.E.M. (C) Immunofluorescence detection of MyHC (green) of negative control group,ector) andmiR-128 inhibitor group in C2C12 cells at 3 days of DM. The scale bar stands for8, miR-128 inhibitor, or NC. Comparable α-tubulin levels served as loading controls in alltatistical significance was analyzed by the Student's t-tests. *P b 0.05; **P b 0.01.

1902 L. Shi et al. / Cellular Signalling 27 (2015) 1895–1904

than theNC group. These results suggest thatmiR-128 promoted C2C12myoblast differentiation.

4. Discussion

One of the main objectives of this experiment was to test the hy-pothesis that miR-128 is involved in the regulation of the proliferationand differentiation of skeletal myoblasts. An inverse expression rela-tionship was found between miR-128 and MSTN protein via bothin vivo and in vitro experiments. Both bioinformatics analysis anddual-luciferase reporter gene assay showed that MSTN CDS region is adirect target of miR-128. In addition, the current results collected viamiR-128 overexpression or inhibition suggest that miR-128 inhibitsproliferation but promotes differentiation in C2C12 cells, which sup-ports our hypothesis.

Previous studies reported that miRNA binding to the CDS mainlyleads to translation inhibition [17,18]. In this study, the MSTN proteinexpression was reduced after miR-128 overexpression, but MSTNmRNA abundance did not change, which suggests that inhibitingmRNA translation rather than degradation is the main method of miR-128 to regulate MSTN gene expression.

In our study, the abundance of myostatin protein was very low attwo days of age, increased to the highest level at two weeks of age,and then decreased from 2 weeks to 8 weeks of age in mice. Our dataare in agreement with previous study results from Marco Patrunoet al. [23] who found that in porcine SM (Semimembranosus) musclethe myostatin protein abundance was very low at birth, increased atone month of age, and reduced in adult. Skeletal muscle mass is the re-sult of a dynamic balance coordinately regulated by twomajor branchesof AKT signaling pathways: the AKT (also known as protein kinase B)/mammalian target of rapamycin (mTOR) pathway that controls proteinsynthesis and the AKT/forkhead box O (FOXO) pathway that controlsprotein degradation [24]. Except for myostatin/Smad2,3/AKT signalingpathways [25], the IGF1/PI3K/AKT signaling pathways also can affectmuscle mass [26]. The expression level of IGF1, a positive regulator inmuscle hypertrophy, was lower during the end of growth phase thanduring the rapid growth phase [27]. Myostatin, a regulator that nega-tively regulatesmuscle hypertrophy and positively regulates muscle at-rophy in muscle [28], also should be down-regulated to reach anappropriate abundance during the end of growth phase. Therefore, itis understandable that a low abundance of myostatin protein was de-tected at 8 weeks of age (when growth has leveled off) in our study.

MSTN is regulated bymiRNAs in the development of skeletalmuscle.Previous studies have shown thatmiR-27b [29] negatively regulated themRNA expression of MSTN in skeletal muscle. Further, a “G” to “A” con-version in the 3′-UTR region of MSTN may form miR-1 and miR-206target sites, which represses MSTN gene expression at the post-transcription level, resulting in muscle hypertrophy in sheep [8]. Inthe current study, we first demonstrated that miR-128 directly targetsMSTN CDS region to affect its translation.

Previous studies showed that some miRNAs affect the proliferationand differentiation of skeletal muscle. Wei, He [19] demonstrated thatoverexpression of miR-29 repressed the proliferation of skeletal musclecells. Zhang, Ying [30] reported that miR-148a affected the cell cyclingduring the proliferation of skeletal muscle cells. Also, miR-1 promotedmyogenesis and miR-133 stimulated the proliferation of skeletal myo-blast [2]. Feng, Niu [31] found a feedback circuit between miR-133 andthe ERK1/2 pathway involving an exquisite mechanism for regulatingmyoblast proliferation and differentiation. Our findings that miRNA-128 targets MSTN at coding domain sequence to inhibit proliferationbut stimulate differentiation of myoblasts provide more clues tomiRNA regulation of skeletal muscle development.

Previous studies on gene expression regulation by miRNAs in mam-mals have mainly evaluated target sites located in 3′-UTRs becausemiRNAs are believed to predominantly act on 3′-UTRs [32]. However,accumulating experimental [33,34] and computational evidence [35,

36] supports the view thatmiRNAs bind to CDS and 3′-UTRwith compa-rable frequency [18]. Translation inhibition is themain regulatory effectofmiRNAbinding sites located in the CDS [18,37] comparingwith in the3′-UTR. The hypothesis agrees with our results, which indicates thatMSTN protein levels are negatively regulated by miR-128 targeting theCDS region of MSTN, without affecting MSTN mRNA levels.

In this study, the expression level of miR-128 was increased and theMSTN protein level was decreased with C2C12 myoblasts differentia-tion or as mice age increased, suggesting that miR-128 plays an impor-tant role in the development of skeletal muscle. And this inverseexpression relationship betweenmiRNA and protein was also previous-ly reported in miR-148a [30] and miR-29 [19] during the developmentof skeletal muscle. Our previous studies showed that miR-128 washighly expressed in skeletal muscle [16]. Similarly, in the currentstudy miR-128 expression also gradually increased during C2C12 myo-blast differentiation or with mice growth. Thus, it is possible that miR-128 is involved in skeletal muscle development.

As towhetherMSTN promotes or inhibitsmyoblast proliferation, re-sults of previous studies are contradictive. Earlier studies suggested thatMSTN inhibits C2C12 proliferation using very high doses of recombinantMSTN generated in bacteria [38–40]. But a latest study reported thatMSTN stimulates, not inhibits, C2C12 myoblast proliferation using 2 in-dependent sources of recombinant MSTN generated in eukaryotic cells[41]. In our study, the EdU proliferation assay, and propidium iodineflow cytometry were used to test the C2C12 cell proliferation aftermiR-128 overexpression or inhibition. The two experimental resultsshowed thatmiR-128 repressedmyoblast proliferation,which is consis-tentwith the previous study [41,42]. In the future, it is valuable to inves-tigate if miR-128 knockout mice have similar phenotypes as MSTNoverexpression mice.

Previous studies on miRNA in skeletal muscle were carried outusing miRNA mimics [19,30,31]. In this study, both miR-128 precursor(per-miR-128) and miR-128 mimics were used. Compared withmiRNA mimics, miRNA precursor, with a high specific sequence struc-ture, has lower interferon effect [43], could avoid excessively competingagainst endogenousmiRNAs for the cell internal resources [44] andmoreefficiently form mature miRNA [45]. Our results also showed that per-miR-128 had a better experimental effect than miR-128 mimics.

In this study, the expression levels of skeletal muscle proliferationand differentiation related genes (Pax3/7, Myf5, MyoG, and MyHC)were detected when miR-128 was over-expressed or inhibited. Thesemyogenic-related genes play important roles in the regulation of skele-talmuscle cell development, for example, deletion of Pax3/7 led tomus-cle atrophy [46]. Cycling skeletal myoblasts express the transcriptionfactors Pax7 and Pax3 [47] as well as the myogenic regulatory factorsMyf5 and MyoD [46]. Once committed to differentiation, myoblastsstop cycling and loose expression of Pax7, Pax3 and Myf5 [25]. In ourstudy, mRNA expression levels of Pax3/7, MyoG, Myf5 and MyHCwere up-regulated after miR-128 overexpression and down-regulatedwhen miR-128 was repressed respectively, which suggests that miR-128 affected the development of skeletal muscle.

McFarlane, Hennebry [48] demonstrated that MSTN signals throughPax7 to regulate satellite cell self-renewal and the expression of Pax7was increased when MSTN was inhibited. Myf5 regulates the skeletalmuscle growth, as well as Pax3/7 [47]. Steelman, Recknor [49] reportedthat the expression of Myf5 was increased inMSTN-knockoutmice. Theinhibition of MyoG represses myoblast differentiation and muscle fiberformation because MyoG promotes the integration of fiber cells [50].MSTN inhibits myoblast differentiation by down-regulating MyoD ex-pression [40]. MSTN also inhibits MyoG through Smad2/3 signal path-way, affecting myoblast differentiation [51]. MSTN-knockout inducesthe phosphorylation of the Smad family of transcription factors, whichregulates gene expression via the canonical TGF-β signaling pathway[51,52]. Thus, we conclude that miR-128 inhibits the MSTN expression,which promotes Myf5, MyoG, and Pax3/7 expression through Smad2/3signal pathway (Fig. 6).

Fig. 6.Model of themyostatin/miR-128negative regulatory circuit in skeletalmuscle.miR-128 represses myostatin at the posttranscriptional level and negatively regulates theSmad2/3 signaling pathway, which is involved in skeletal muscle proliferation, differenti-ation and hypertrophy.

1903L. Shi et al. / Cellular Signalling 27 (2015) 1895–1904

In this study, we observed thatmiR-128 stimulated differentiation ofC2C12 cells, similar to the functions of miR-148a [30] and miR-29 [19]reported in previous studies. Skeletal muscle differentiation is often ac-companied by the expression of myogenic marker genes, such as MyHCand MyoG [53]. Simultaneously, we observed that overexpression ofmiR-128 increased the expression of MyHC during the differentiationof C2C12 cells, which increased the generation of myotubes and cell dif-ferentiation and subsequently induced the formation of longer, largermyotubes; conversely, expression levels of MyoG and MyHC were de-creased when miR-128 was inhibited. Thus, our results demonstratedthat miR-128 promoted the differentiation of C2C12 myoblasts bytargeting MSTN.

In summary, our experimental results showed that miR-128inhibited proliferation but promoted differentiation in C2C12 cells.Bioinformatics analyses and dual luciferase reporter gene assay sug-gested that MSTN CDS region is a direct target of miR-128. Inversed ex-pression relationships between miR-128 and MSTN protein in vivo andin vitro also imply that miR-128 potentially inhibits MSTN. Taken to-gether, our experimental results demonstrated that miR-128 involvesin the regulation of the proliferation and differentiation of skeletalmyo-blasts by targeting MSTN CDS region.

Disclosure statement

No potential conflicts of interest were disclosed.

Acknowledgments

We are grateful to Dr. Hengwei Cheng (Purdue University) and Dr.Shihuan Kuang (Purdue University) for reviewing our article. Thiswork was supported by grants from the Natural Science Foundation ofJiangsu Province (BK2011650), the Fundamental Research Funds forthe Central Universities (KYZ201532), the foundations from JiangsuProvince (BE2014396 and BK20140688) and Youth Science and

Technology Innovation Fund (KJ2013020) of Nanjing Agricultural Uni-versity in China.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.cellsig.2015.05.001.

References

[1] P.D. Zamore, B. Haley, Science 309 (2005) 1519–1524.[2] J.F. Chen, E.M. Mandel, J.M. Thomson, Q. Wu, T.E. Callis, S.M. Hammond, F.L. Conlon,

D.Z. Wang, Nat. Genet. 38 (2006) 228–233.[3] E. Bober, T. Franz, H.-H. Arnold, P. Gruss, P. Tremblay, Development 120 (1994)

603–612.[4] H.K. Kim, Y.S. Lee, U. Sivaprasad, A. Malhotra, A. Dutta, J. Cell Biol. 174 (2006)

677–687.[5] S. Rachagani, Y. Cheng, J.M. Reecy, BMC Res. Notes 3 (2010) 297.[6] S.W. Cho, S. Kim, J.M. Kim, J.S. Kim, Nat. Biotechnol. 31 (2013) 230–232.[7] Q. Wang, A.C. McPherron, J. Physiol. 590 (2012) 2151–2165.[8] A. Clop, F. Marcq, H. Takeda, D. Pirottin, X. Tordoir, B. Bibe, J. Bouix, F. Caiment, J.M.

Elsen, F. Eychenne, C. Larzul, E. Laville, F. Meish, D. Milenkovic, J. Tobin, C. Charlier,M. Georges, Nat. Genet. 38 (2006) 813–818.

[9] Z. Huang, X. Chen, B. Yu, J. He, D. Chen, Biochem. Biophys. Res. Commun. 423 (2012)265–269.

[10] Y. Ge, Y. Sun, J. Chen, J. Cell Biol. 192 (2011) 69–81.[11] C.F. Wong, R.L. Tellam, J. Biol. Chem. 283 (2008) 9836–9843.[12] T.E. Callis, K. Pandya, H.Y. Seok, R.-H. Tang, M. Tatsuguchi, Z.-P. Huang, J.-F. Chen, Z.

Deng, B. Gunn, J. Shumate, J. Clin. Invest. 119 (2009) 2772–2786.[13] I. Naguibneva, M. Ameyar-Zazoua, A. Polesskaya, S. Ait-Si-Ali, R. Groisman, M.

Souidi, S. Cuvellier, A. Harel-Bellan, Nat. Cell Biol. 8 (2006) 278–284.[14] J. Liu, X.J. Luo, A.W. Xiong, Z.D. Zhang, S. Yue, M.S. Zhu, S.Y. Cheng, J. Biol. Chem. 285

(2010) 26599–26607.[15] S.S. Xie, T.H. Huang, Y. Shen, X.Y. Li, X.X. Zhang, M.J. Zhu, H.Y. Qin, S.H. Zhao, Anim.

Genet. 41 (2010) 179–190.[16] B. Zhou, H.L. Liu, F.X. Shi, J.Y. Wang, Anim. Genet. 41 (2010) 499–508.[17] A. Brummer, J. Hausser, Bioessays 36 (2014) 617–626.[18] J. Hausser, A.P. Syed, B. Bilen, M. Zavolan, Genome Res. 23 (2013) 604–615.[19] W.Wei, H.B. He, W.Y. Zhang, H.X. Zhang, J.B. Bai, H.Z. Liu, J.H. Cao, K.C. Chang, X.Y. Li,

S.H. Zhao, Cell Death Dis. 4 (2013) e668.[20] X.L. Chen, Z.Q. Huang, D.W. Chen, T. Yang, G.M. Liu, Int. J. Mol. Sci. 14 (2013)

14076–14084.[21] J. Kruger, M. Rehmsmeier, Nucleic Acids Res. 34 (2006) W451–W454.[22] R.C. Friedman, K.K. Farh, C.B. Burge, D.P. Bartel, Genome Res. 19 (2009) 92–105.[23] M. Patruno, F. Caliaro, L. Maccatrozzo, R. Sacchetto, T. Martinello, L. Toniolo, C.

Reggiani, F. Mascarello, Differentiation 76 (2008) 168–181.[24] M.A. Egerman, D.J. Glass, Crit. Rev. Biochem. Mol. Biol. 49 (2014) 59–68.[25] A.U. Trendelenburg, A. Meyer, D. Rohner, J. Boyle, S. Hatakeyama, D.J. Glass, Am. J.

Physiol. Cell Physiol. 296 (2009) C1258–C1270.[26] S.C. Bodine, T.N. Stitt, M. Gonzalez, W.O. Kline, G.L. Stover, R. Bauerlein, E.

Zlotchenko, A. Scrimgeour, J.C. Lawrence, D.J. Glass, G.D. Yancopoulos, Nat. CellBiol. 3 (2001) 1014–1019.

[27] N.O. Kanbur, O. Derman, E. Kinik, J. Bone Miner. Metab. 23 (2005) 76–83.[28] J. Rodriguez, B. Vernus, I. Chelh, I. Cassar-Malek, J.C. Gabillard, A. Hadj Sassi, I. Seiliez,

B. Picard, A. Bonnieu, Cell. Mol. Life Sci. 71 (2014) 4361–4371.[29] D.L. Allen, A.S. Loh, Am. J. Physiol. Cell Physiol. 300 (2011) C124–C137.[30] J. Zhang, Z.Z. Ying, Z.L. Tang, L.Q. Long, K. Li, J. Biol. Chem. 287 (2012) 21093–21101.[31] Y. Feng, L.L. Niu, W. Wei, W.Y. Zhang, X.Y. Li, J.H. Cao, S.H. Zhao, Cell Death Dis. 4

(2013) e934.[32] D.P. Bartel, Cell 136 (2009) 215–233.[33] S.W. Chi, J.B. Zang, A. Mele, R.B. Darnell, Nature 460 (2009) 479–486.[34] M. Hafner, M. Landthaler, L. Burger, M. Khorshid, J. Hausser, P. Berninger, A.

Rothballer, M. Ascano Jr., A.C. Jungkamp, M. Munschauer, A. Ulrich, G.S. Wardle, S.Dewell, M. Zavolan, T. Tuschl, Cell 141 (2010) 129–141.

[35] B.P. Lewis, I.H. Shih, M.W. Jones-Rhoades, D.P. Bartel, C.B. Burge, Cell 115 (2003)787–798.

[36] M. Schnall-Levin, Y. Zhao, N. Perrimon, B. Berger, Proc. Natl. Acad. Sci. U. S. A. 107(2010) 15751–15756.

[37] H. Guo, N.T. Ingolia, J.S. Weissman, D.P. Bartel, Nature 466 (2010) 835–840.[38] M. Thomas, B. Langley, C. Berry, M. Sharma, S. Kirk, J. Bass, R. Kambadur, J. Biol.

Chem. 275 (2000) 40235–40243.[39] W.E. Taylor, S. Bhasin, J. Artaza, F. Byhower, M. Azam, D.H. Willard Jr., F.C. Kull Jr., N.

Gonzalez-Cadavid, Am. J. Physiol. Endocrinol. Metab. 280 (2001) E221–E228.[40] B. Langley, M. Thomas, A. Bishop, M. Sharma, S. Gilmour, R. Kambadur, J. Biol. Chem.

277 (2002) 49831–49840.[41] B.D. Rodgers, B.D. Wiedeback, K.E. Hoversten, M.F. Jackson, R.G. Walker, T.B.

Thompson, Endocrinology 155 (2014) 670–675.[42] N. Motohashi, M.S. Alexander, Y. Shimizu-Motohashi, J.A. Myers, G. Kawahara, L.M.

Kunkel, J. Cell Sci. 126 (2013) 2678–2691.[43] C.A. Sledz, M. Holko, M.J. de Veer, R.H. Silverman, B.R. Williams, Nat. Cell Biol. 5

(2003) 834–839.[44] D. Castanotto, K. Sakurai, R. Lingeman, H.T. Li, L. Shively, L. Aagaard, H. Soifer, A.

Gatignol, A. Riggs, J.J. Rossi, Nucleic Acids Res. 35 (2007) 5154–5164.

1904 L. Shi et al. / Cellular Signalling 27 (2015) 1895–1904

[45] R.I. Gregory, T.P. Chendrimada, N. Cooch, R. Shiekhattar, Cell 123 (2005)631–640.

[46] F. Relaix, D. Rocancourt, A. Mansouri, M. Buckingham, Nature 435 (2005)948–953.

[47] F. Relaix, D. Montarras, S. Zaffran, B. Gayraud-Morel, D. Rocancourt, S. Tajbakhsh, A.Mansouri, A. Cumano, M. Buckingham, J. Cell Biol. 172 (2006) 91–102.

[48] C. McFarlane, A. Hennebry, M. Thomas, E. Plummer, N. Ling, M. Sharma, R.Kambadur, Exp. Cell Res. 314 (2008) 317–329.

[49] C.A. Steelman, J.C. Recknor, D. Nettleton, J.M. Reecy, FASEB J. 20 (2006) 580–582.[50] C.F. Bentzinger, Y.X. Wang, M.A. Rudnicki, Cold Spring Harb. Perspect. Biol. 4 (2012).[51] X. Zhu, S. Topouzis, L.-f. Liang, R.L. Stotish, Cytokine 26 (2004) 262–272.[52] D.L. Allen, T.G. Unterman, Am. J. Physiol. Cell Physiol. 292 (2007) C188–C199.[53] L.A. Sabourin, M.A. Rudnicki, Clin. Genet. 57 (2000) 16–25.