MEF2A Regulates the MEG3-DIO3 miRNA Mega Cluster-Targeted PP2A Signaling in Bovine Skeletal Myoblast Differentiation

Understanding the molecular mechanisms of skeletal myoblast differentiation is essential for studying muscle developmental biology. In our previous study, we reported that knockdown of myocyte enhancer factor 2A (MEF2A) inhibited myoblast differentiation. Here in this study, we further identified that MEF2A controlled this process through regulating the maternally expressed 3 (MEG3)—iodothyronine deiodinase 3 (DIO3) miRNA mega cluster and protein phosphatase 2A (PP2A) signaling. MEF2A was sufficient to induce MEG3 expression in bovine skeletal myoblasts. A subset of miRNAs in the MEG3-DIO3 miRNA cluster was predicted to target PP2A subunit genes. Consistent with these observations, MEF2A regulated PP2A signaling through its subunit gene protein phosphatase 2 regulatory subunit B, gamma (PPP2R2C) during bovine myoblast differentiation. MiR-758 and miR-543 in the MEG3-DIO3 miRNA cluster were down-regulated in MEF2A-depleted myocytes. Expression of miR-758 and miR-543 promoted myoblast differentiation and repressed PPP2R2C expression. Luciferase activity assay showed that PPP2R2C was post-transcriptionally targeted by miR-758 and miR-543. Taken together, these results reveal that the MEG3-DIO3 miRNAs function at downstream of MEF2A to modulate PP2A signaling in bovine myoblast differentiation.


Introduction
Skeletal muscle is an important constituent in maintaining metabolic homeostasis [1]. Vertebrate skeletal muscle development is a complex process that is controlled sequentially by somite commitment into progenitors, myoblasts proliferation, migration, fusion, and final adaptation into fast-twitch and slow-twitch muscle fibers [2]. During myogenesis, the cascade control of multiple transcription factors provides considerable effects [3,4].
Myocyte enhancer factor 2A (MEF2A), a basic helix-loop-helix (bHLH) transcription factor, which is highly expressed in the brain, heart, and skeletal muscle, is required for embryonic development, cardiomyocyte proliferation, skeletal muscle differentiation, and regeneration [5][6][7]. In mice, MEF2A-knockedout myoblasts are unable to differentiate and cause severe disruption of skeletal muscle regeneration, suggesting the key roles of MEF2A in skeletal muscle [8]. Similarly, our previous study shows that MEF2A is indispensable for bovine skeletal myoblast differentiation [9]. However, despite the findings that MEF2A participates in calcineurin, mitogen-activated protein

Myocyte Enhancer Factor 2A (MEF2A) Is Sufficient to Induce Maternally Expressed 3 (MEG3) Expression
In our previous study, we reported that transcript expression of MEF2A was obviously induced during skeletal muscle development and myoblast differentiation [9]. Here in this study, we found that the total MEF2A protein expression level was also elevated during myoblast differentiation ( Figure 1A). These results suggest that MEF2A is an essential inducer for myogenesis.
Previous studies reported that the mouse Gtl2-Dio3 miRNA cluster had a close correlation with myoblast differentiation. MEF2A activated the transcription of these miRNAs through binding to the MEF2 motif on the Gtl2 promoter [8]. To test this role in bovine skeletal myoblasts, we firstly detected the expression correlation between MEF2A and MEG3. We found that the expression of MEF2A obviously up-regulated MEG3 expression, and inversely, the interference of MEF2A reduced MEG3 expression ( Figure 1B,C). To explore whether MEF2A regulated MEG3 expression through directly activating the MEG3 promoter, first of all, transcription factor binding motifs were analyzed by using online MatInspector software. Surprisingly, unlike the mouse Gtl2 promoter, there were no potential MEF2A binding sites identified on the bovine MEG3 promoter ( Figure 1D). Given the complex regulation of MEF2A in skeletal muscle, it is supposed that MEF2A might regulate bovine MEG3 expression through interacting with other proteins [26].
In this study, according to the miRNA-mRNA prediction, one gene of protein phosphatase 2 scaffold subunit A, beta (PPP2R1B) that encoded subunit A protein and six genes including protein phosphatase 2 regulatory subunit B, alpha/beta/gamma (PPP2R2A, PPP2R2B, PPP2R2C) protein phosphatase 2 regulatory subunit B alpha/epsilon (PPP2R5A, PPP2R5E), and protein phosphatase 2 regulatory subunit B" alpha (PPP2R3A) that encoded subunit B proteins, were predicted by ten miRNAs in the MEG3-DIO3 miRNA cluster ( Figure 2C). These results imply that MEF2A might regulate myoblast differentiation through modulating MEG3-DIO3 miRNAs-targeted PP2A signaling.

MEF2A Regulates Protein Phosphatase 2A (PP2A) Signaling During Myoblast Differentiation
To explore the roles of PP2A signaling in myoblast differentiation, PP2A inhibitor LB-100 rescue assay was performed. As shown in Figure 3A, the results revealed that PP2A signaling absolutely participated in MEF2A regulated myoblast differentiation. Interference of MEF2A severely inhibited myogenesis as observed by the immunostaining of actinin alpha (ACTN), the myogenic differentiation marker. Surprising, when LB-100 was added, the impaired myoblast differentiation was nearly completely rescued. Based on the prediction frequency ( Figure 2C), PPP2R2A, PPP2R2C, PPP2R3A, and PPP2R5A were subjected to further analyses. Real time PCR (RT-PCR) analysis showed that the expression of PPP2R2C and PPP2R3A, except for PPP2R2A and PPP2R5A, were up-regulated in MEF2A-depleted myoblasts ( Figure 3B). In particular, the mRNA expression of PPP2R2C was sharply elevated nearly four times as much as the control group ( Figure 3B). Moreover, in this rescue assay, LB-100 efficiently inhibited expression of PPP2R2C and PPP2R3A ( Figure 3C), indicating that PPP2R2C and PPP2R3A might have pivotal roles in the  To explore the roles of PP2A signaling in myoblast differentiation, PP2A inhibitor LB-100 rescue assay was performed. As shown in Figure 3A, the results revealed that PP2A signaling absolutely participated in MEF2A regulated myoblast differentiation. Interference of MEF2A severely inhibited myogenesis as observed by the immunostaining of actinin alpha (ACTN), the myogenic differentiation marker. Surprising, when LB-100 was added, the impaired myoblast differentiation was nearly completely rescued. Based on the prediction frequency ( Figure 2C), PPP2R2A, PPP2R2C, PPP2R3A, and PPP2R5A were subjected to further analyses. Real time PCR (RT-PCR) analysis showed that the expression of PPP2R2C and PPP2R3A, except for PPP2R2A and PPP2R5A, were up-regulated in MEF2A-depleted myoblasts ( Figure 3B). In particular, the mRNA expression of PPP2R2C was sharply elevated nearly four times as much as the control group ( Figure 3B). Moreover, in this rescue assay, LB-100 efficiently inhibited expression of PPP2R2C and PPP2R3A ( Figure 3C), indicating that PPP2R2C and PPP2R3A might have pivotal roles in the MEF2A-regulated PP2A signaling pathway during myoblast differentiation. Due to the strong effects of MEF2A and LB-100 on PPP2R2C expression, PPP2R2C was subjected to further analysis.
(MYF6) act together to modulate myoblast differentiation [2]. MYF5 induces the myogenic commitment of progenitor cells and highly expresses in the proliferating myoblasts. MYOD1 activates the myogenic determination and early differentiation. MYF6 and MYOG regulate the late differentiation of myogenesis [4]. To investigate the role of PPP2R2C in myoblast differentiation, PPP2R2C silencing assay was performed by using specific short interference RNA (siRNA). As shown in Figure 3D, inhibition of PPP2R2C promoted myotube formation to some extent. At the transcriptional level, MYOD1, MYF5, and MYF6 except for MYOG were all up-regulated in PPP2R2C-silienced myocytes ( Figure 3E). These results indicate that PPP2R2C is an inhibitor of myogenic differentiation and mainly regulates the myogenic commitment and early differentiation stage.

MEF2A Regulates MEG3-DIO3 miRNAs During Myoblast Differentiation
Because miR-758, miR-543, and miR-495 were the miRNAs that might target PPP2R2C ( Figure  2C), in this study we investigated whether and how these three miRNAs affected myoblast differentiation. RT-PCR analysis showed that the expression of miR-758, miR-543, and miR-495 were down-regulated in MEF2A-depleted myoblasts ( Figure 4A). The transfection of miR-758 mimics (758m) and miR-543 mimics (543m) obviously promoted myoblast differentiation compared with the mimic negative control (mNC) group ( Figure 4B). Inversely, in comparison with the inhibitor negative control (INC) group, miR-758 inhibitor (758I) and miR-543 inhibitor (543I) repressed myoblast differentiation ( Figure 4B). However, miR-495 had no obvious effects on myoblast differentiation. Consistent with these observations, RT-PCR analysis showed that transfection of miR-758 mimics and miR-543 mimics significantly up-regulated MYOD1 and MYOG expression ( Figure 4C,D). miR-758 mimics also inhibited expression of MYF5 which is usually In skeletal muscle, four myogenic regulatory factors (MRFs) including myogenic differentiation 1 (MYOD1), myogenin (MYOG), myogenic factor 5 (MYF5), and myogenic factor 6 (MYF6) act together to modulate myoblast differentiation [2]. MYF5 induces the myogenic commitment of progenitor cells and highly expresses in the proliferating myoblasts. MYOD1 activates the myogenic determination and early differentiation. MYF6 and MYOG regulate the late differentiation of myogenesis [4]. To investigate the role of PPP2R2C in myoblast differentiation, PPP2R2C silencing assay was performed by using specific short interference RNA (siRNA). As shown in Figure 3D, inhibition of PPP2R2C promoted myotube formation to some extent. At the transcriptional level, MYOD1, MYF5, and MYF6 except for MYOG were all up-regulated in PPP2R2C-silienced myocytes ( Figure 3E). These results indicate that PPP2R2C is an inhibitor of myogenic differentiation and mainly regulates the myogenic commitment and early differentiation stage.

Protein Phosphatase 2 Regulatory Subunit B, Gamma (PPP2R2C) Expression Is Inhibited by miR-758 and miR-543
On the basis of the results obtained in this study, next the expression correlations between PPP2R2C and the three miRNAs were investigated. RT-PCR analysis showed that PPP2R2C expression was down-regulated in the presence of miR-758 mimics and miR-543 mimics ( Figure  5A,B), while miR-495 had no effects on PPP2R2C expression ( Figure 5C). To explore whether these miRNAs modulated PPP2R2C expression through post-transcriptional regulation, the 3′ untranslated region (3′UTR) of PPP2R2C mRNA was subjected to detailed analysis. As shown in Figure 5D, two miR-543 binding sites, two miR-758 binding sites, and one miR-495 binding site were predicted in the PPP2R2C 3′UTR. Subsequently, sequence alignments of the five miRNA binding sites were analyzed to determine the sequence conservation among mammals. The results showed that the miRNA binding sites of miR-543-1624bp, miR-758-2417bp, and miR-543-2486bp were completely conserved among cows, humans, goats, and mice ( Figure 5E). The other two miRNA binding sites including miR-495-1628bp and miR-758-2197bp were less conserved when compared the transcriptome database between cows and mice ( Figure 5E). Notably, among the five miRNA binding sites, miR-543-1624bp and miR-495-1628bp had an overlap of three nucleotides (GUU, red letters in Figure 5E), suggesting that in this region, there might be only one miRNA that could sufficiently bind to PPP2R2C mRNA. Taken together, these findings indicate that PPP2R2C might be post-transcriptionally regulated by miR-758, miR-543, and miR-495.

Protein Phosphatase 2 Regulatory Subunit B, Gamma (PPP2R2C) Expression Is Inhibited by miR-758 and miR-543
On the basis of the results obtained in this study, next the expression correlations between PPP2R2C and the three miRNAs were investigated. RT-PCR analysis showed that PPP2R2C expression was down-regulated in the presence of miR-758 mimics and miR-543 mimics ( Figure 5A,B), while miR-495 had no effects on PPP2R2C expression ( Figure 5C). To explore whether these miRNAs modulated PPP2R2C expression through post-transcriptional regulation, the 3 untranslated region (3 UTR) of PPP2R2C mRNA was subjected to detailed analysis. As shown in Figure 5D, two miR-543 binding sites, two miR-758 binding sites, and one miR-495 binding site were predicted in the PPP2R2C 3 UTR. Subsequently, sequence alignments of the five miRNA binding sites were analyzed to determine the sequence conservation among mammals. The results showed that the miRNA binding sites of miR-543-1624bp, miR-758-2417bp, and miR-543-2486bp were completely conserved among cows, humans, goats, and mice ( Figure 5E). The other two miRNA binding sites including miR-495-1628bp and miR-758-2197bp were less conserved when compared the transcriptome database between cows and mice ( Figure 5E). Notably, among the five miRNA binding sites, miR-543-1624bp and miR-495-1628bp had an overlap of three nucleotides (GUU, red letters in Figure 5E), suggesting that in this region, there might be only one miRNA that could sufficiently bind to PPP2R2C mRNA. Taken together, these findings indicate that PPP2R2C might be post-transcriptionally regulated by miR-758, miR-543, and miR-495.

PPP2R2C Is a Direct Target of miR-758 and miR-543
To investigate whether PPP2R2C was a downstream target of miR-758, miR-543, and miR-495, luciferase activity assays were introduced by using the psiCHECK-2 reporter vector. Ten reporter vectors containing the wide type (WT) or mutant (MUT) miRNA binding sites were generated and transfected together with the corresponding miRNA mimics in 293T cells ( Figure 6A). The mutant miR-543-1624 and miR-495-1628 reporters were generated by mutating GAAT to TAGC and TGTT to TCAG, respectively, without altering the overlapping nucleotides GTT. Luciferase activity assays showed that three out of the five potential miRNA binding sites interacted with the miRNAs (Figure 6B-F). Activities of the WT 3′UTR-1624bp vector and the WT 3′UTR-2197bp vector were down-regulated in the presence of miR-543 mimics ( Figure 6B) and miR-758 mimics ( Figure 6D), respectively, while activity of the WT 3′UTR-2417bp vector was elevated in the presence of miR-758 mimics ( Figure 6E). It is possible that this up-regulating effect might be achieved by unconventional miRNA functions [35]. For the other two remaining vectors containing the predicted miRNA binding sites 3′UTR-1628bp and 3′UTR-2486bp, miR-495 and miR-543 were not capable of regulating their activities ( Figure 6C,F). These results suggest that PPP2R2C is a direct target of the MEG3-DIO3 miRNA cluster that can be regulated by miR-758 and miR-543.
Taken together, our study firstly demonstrates a connection between MEF2A and PP2A signaling in myoblast differentiation. This study reveals that the MEG3-DIO3 miRNA cluster functions downstream of MEF2A to modulate PP2A signaling in myoblast differentiation ( Figure  7).

PPP2R2C Is a Direct Target of miR-758 and miR-543
To investigate whether PPP2R2C was a downstream target of miR-758, miR-543, and miR-495, luciferase activity assays were introduced by using the psiCHECK-2 reporter vector. Ten reporter vectors containing the wide type (WT) or mutant (MUT) miRNA binding sites were generated and transfected together with the corresponding miRNA mimics in 293T cells ( Figure 6A). The mutant miR-543-1624 and miR-495-1628 reporters were generated by mutating GAAT to TAGC and TGTT to TCAG, respectively, without altering the overlapping nucleotides GTT. Luciferase activity assays showed that three out of the five potential miRNA binding sites interacted with the miRNAs (Figure 6B-F). Activities of the WT 3 UTR-1624bp vector and the WT 3 UTR-2197bp vector were down-regulated in the presence of miR-543 mimics ( Figure 6B) and miR-758 mimics ( Figure 6D), respectively, while activity of the WT 3 UTR-2417bp vector was elevated in the presence of miR-758 mimics ( Figure 6E). It is possible that this up-regulating effect might be achieved by unconventional miRNA functions [35]. For the other two remaining vectors containing the predicted miRNA binding sites 3 UTR-1628bp and 3 UTR-2486bp, miR-495 and miR-543 were not capable of regulating their activities ( Figure 6C,F). These results suggest that PPP2R2C is a direct target of the MEG3-DIO3 miRNA cluster that can be regulated by miR-758 and miR-543.
Taken together, our study firstly demonstrates a connection between MEF2A and PP2A signaling in myoblast differentiation. This study reveals that the MEG3-DIO3 miRNA cluster functions downstream of MEF2A to modulate PP2A signaling in myoblast differentiation (Figure 7).

Discussion
In our previous study, we reported that MEF2A was required for skeletal myoblast differentiation. MEF2A depletion in differentiating myocytes severely inhibited myotube formation [9]. Here in this study, our results established a novel molecular role for MEF2A to regulate the MEG3-DIO3 miRNA cluster and PP2A signaling in skeletal myoblast differentiation. To our knowledge, this is the first evidence that PP2A signaling is involved in MEF2A-regulated skeletal muscle development.
Skeletal muscle development is a complex process which is coordinated with dramatic alteration of protein phosphorylation [25]. During myogenesis, a sustained elevation of intracellular free calcium activates calcineurin, a calcium/calmodulin-dependent serine/threonine phosphatase [36]. As a transcription activator in myocytes, MEF2A activity is stimulated by the dephosphorylation of the calcium-activated, calcineurin-dependent mechanism [36]. In this study, we found that total MEF2A protein was elevated during myoblast differentiation. Meanwhile, two protein bands with different molecular weights of MEF2A were detected on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. According to what Wu et al. reported, the upper band was the phosphorylated form and the lower band was the dephosphorylated form of MEF2A [36]. During myogenesis, the phosphorylation of MEF2A was attenuated and the dephosphorylation of MEF2A was enhanced, indicating that MEF2A activity was elevated. It is postulated that the trans-activation of MEF2A protein by dephosphorylation is the key in regulating myoblast differentiation. This is also approved by our previous study reporting that the overexpression of MEF2A results in the enrichment of numerous dephosphorylated proteins [9].
The MEF2A protein contains a highly conserved MCM1, Agamous, Deficiens, Serum Response factor (MADS) domain at its N-termini that mediates dimerization and binding to the DNA sequences. Adjacent to the MADS domain is a MEF2 domain that influences DNA-binding affinity and cofactor interactions [10,37]. The mouse Gtl2-Dio3 miRNA cluster was firstly reported to be regulated by MEF2A in muscle regeneration by Snyder et al. [8]. They demonstrated that MEF2A directly regulated the mouse Gtl2 promoter upstream of all miRNAs in the cluster by binding to the −39 bp MEF2 binding site. However, this hypothesis was challenged by another study suggesting that MEF2A did not depend on an intact DNA binding domain to stimulate expression of the Gtl2-Dio3 miRNA cluster, because a MADS domain-truncated MEF2A isoform still efficiently induces expression of the Gtl2-Dio3 miRNA cluster [16]. In our study, we found a positive regulation of MEF2A on the expression of MEG3 and miRNAs within the MEG3-DIO3 gene cluster. However, through transcription factor binding motif prediction by Genomatix software, no potential MEF2A binding sites were identified on the bovine MEG3 promoter. In such a situation, it is hard to verify the interactions between MEF2A and MEG3 through electrophoretic mobility shift assays or chromatin immunoprecipitation-PCR. Since MEF2A activates its target genes relying on the recruitment of, and cooperation with, other transcription factors [37,38], it is possible that the regulation of MEF2A on MEG3 expression is likely due to interacting with other transcription factors.
In this study, TargetScan release 6.2 (2012 to 2015) and 7.2 (2016 to 2018) were used to predict the target genes of the bovine MEG3-DIO3 miRNA cluster. There were two reasons why we selected such approaches: (1) Among the frequently-used prediction algorithms, including miRanda, TargetScan, and miRDB, only TargetScan contains a database for cows; (2) in this study, the prediction analysis was initially performed in 2015 by using release 6.2. When the latest version was updated in 2018, the MEG3-DIO3 miRNA cluster was subjected to prediction again by using release 7.2. The release 7.2 developed an improved quantitative model of canonical targeting to minimize confounding biases and predict the most effectively targeted mRNAs [39]. In the present study, each miRNA was predicted twice by using the two databases and the commonly predicted genes were regarded as potential targets. What should be noted is that, for some miRNAs, only few mRNAs are commonly predicted by the two databases (Table S3). Thus, it is necessary to combine different prediction algorithms when performing miRNA-mRNA prediction analysis.
It is intriguing that PP2A subunit genes emerge as the top predicted targets by the MEG3-DIO3 miRNA cluster and PPP2R2C is directly targeted by miR-758 and miR-543. This is the first evidence that PP2A participates in MEF2A-regulated myoblast differentiation. PP2A holoenzyme contains three subunits (A, B, and C), and each subunit is encoded by various genes [22]. The diversity of PP2A derives from the fact that over 200 PP2A complexes can be assembled due to different interactions and combinations of the three subunits with other proteins [40]. The regulatory subunit is the key to mediate PP2A activity. In different mammalian tissues, cells express different regulatory subunit proteins at different developmental stages [41]. However, it is unknown how PP2A activities occur in skeletal muscle and whether there are specific subunit proteins regulating skeletal muscle development. In our present study, we find that PP2A signaling is under the control of MEF2A, and absolutely participates in myoblast differentiation. Meanwhile, PPP2R2C plays essential roles in MEF2A-regulated myoblast differentiation. It is confirmed that PP2A signaling is a negative regulator in modulating myogenesis, which is consistent with its dephosphorylation function to its substrates.
Given the identification of the MEF2A-MEG3/DIO3-PP2A signaling cascade regulation in myoblast differentiation, it is interesting to speculate that this regulatory pathway also plays an important role in skeletal muscle regeneration. MEF2A is an important activator in muscle regeneration [5,8]. Previous researches have illustrated the role of the MEF2A-Gtl2/Dio3-WNT pathway in regulating mouse muscle regeneration [8]. In this study, we introduced the PP2A signaling to MEF2A-MEG3/DIO3 axis-regulated myoblast differentiation, which will provide new insights into the molecular mechanisms of skeletal muscle myogenesis.

Cell Culture
Bovine primary skeletal myoblasts were isolated as described previously [9]. The experiments were performed in accordance with the guiding principles for the experimental animals adopted by the Ministry of Science and Technology. Animal care was approved by the Institutional Animal Care and Use Committee of Northwest A&F University (approved code: No.142, 13 June, 2014). Myoblasts were cultured in DMEM/F-12 (Gibco, Shanghai, China) supplemented with 20% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Hyclone, ThermoFisher Scientific, Shanghai, China). For myogenic differentiation, myoblasts were cultured in DMEM/F-12 supplemented with 2% horse serum (Gibco) and 1% penicillin/streptomycin. 293T cells were cultured in DMEM/High Glucose (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin. The cell culture medium was changed every two days.

Adenovirus, Vectors, and RNA Oligonucleotides
Adenoviruses carrying the full-length bovine MEF2A gene coding sequence (termed as OE-2A) and specific short hairpin RNA (shRNA) targeting MEF2A mRNA (termed as sh-2A) as well as the negative control adenoviruses (termed as OE-NC and sh-NC) were generated as described previously [9]. The ten reporter vectors were generated by cloning the 3 UTR containing the WT or MUT miRNA binding sites into the psiCHECK-2 vector (Promega, Beijing, China). miRNA mimics were used to increase the expression of miR-758, miR-543, and miR-495. miRNA inhibitors were used to reduce the expression of miR-758, miR-543, and miR-495. The RNA oligonucleotides of the three miRNAs and NC were chemically synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China).

Transfection and Luciferase Activity Assay
For the transfection of miRNA mimics in myoblasts, cells at 70% confluence were transfected with the miRNA oligonucleotides by using lipofectamine™ 3000 transfection reagent (Invitrogen, Shanghai, China) at a final concentration of 30 nM. For co-transfection of miRNA oligonucleotides and psiCHECK-2 reporter vectors, 293T cells were seeded in 24-well cell culture plates (Corning, Shanghai, China) in triplicate wells. For each treatment, 100 µg plasmids and 5 µL of 20 µM miRNA oligonucleotides were mixed and transfected into cells. Transfection was performed according to the manufacturer's instructions.
The luciferase activity was measured 40 h after transfection by using the Dual-Luciferase Reporter Assay System (Promega) on Infinite1 200 PRO NanoQuant spectrophotometer (TECAN, Shanghai, China). The firefly luciferase activity was measured at 560 nm and the Renilla luciferase activity was measured at 480 nm. The relative activity of the reporter vector was determined as the ratio of renilla luciferase activity to firefly luciferase activity.

Quantitative Real Time-PCR
Total RNAs from the myocytes (n = 3) were isolated by using the RNAiso Plus kit (TaKaRa). RNA quantification was detected on Infinite1 200 PRO NanoQuant spectrophotometer. The cDNAs for mRNAs were reversely transcribed by using the Prime-Script TM RT reagent Kit with gDNA Eraser (TaKaRa) and for miRNAs were synthesized by using the miRcute Plus miRNA First-Strand cDNA Synthesis Kit (TIANGEN, Beijing, China). Quantitative RT-PCR was performed in triplicate wells with the 7500 Real Time PCR System (Applied Biosystems, Shanghai, China). TB Green TM Premix Ex Taq TM II (Tli RNaseH Plus, TaKaRa) was used for protein-coding gene analysis and the miRcute Plus miRNA qPCR Detection Kit (SYBR Green, TIANGEN) was used for miRNA analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 served as internal control for protein-cording genes and miRNAs respectively. Sequence information for the primers used in this study is provided in Table S3.

Cell Culture Immunofluorescence
Cells grown in 6-well cell culture plates were fixed with 4% paraformaldehyde for 15 min, rinsed with phosphate buffer solution (PBS) three times, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, Shanghai, China) for 15 min and then incubated in 10% normal donkey serum (Solarbio, Beijing, China)/1% BSA (Sigma-Aldrich)/0.3 M glycine (Sigma-Aldrich) for 1 h to block non-specific protein-protein interactions. The primary antibody was diluted in the blocking buffer to incubate cells overnight at 4 • C. After being washed with PBS three times, cells were incubated with secondary antibody protected from light at 37 • C for 1 h. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) in the dark at room temperature for 10 min. The antibodies were used as follows: anti α-actinin (H-300) (1:200, Santa Cruz Biotechnology, Shanghai, China), and donkey anti-rabbit IgG H&L (Alexa Fluor ® 555, 1:1000, abcam). DAPI was used at the final concentration of 1 µg/mL. Phase-contrast and fluorescent microscopy were performed by using an inverted fluorescence microscope (Olympus IX71, Olympus Corporation, Beijing, China).

Computational Analysis
According to the National Center for Biotechnology Information database, bovine MEG3-DIO3 locus is located on chromosome 21 and contains 56 miRNAs. The potential target mRNAs of all the 56 miRNAs were predicted twice by using the TargetScan cow database release 6.2 and release 7.2.
For each miRNA, the two subsets of the predicted mRNAs generated from the two databases were subjected to overlap diagram analysis. The commonly predicted mRNAs were regarded as potential targets of the corresponding miRNA. For each target gene, the prediction frequency was also analyzed. Transcription factor binding motif enrichment analysis for the mouse and bovine MEG3 promoter was performed with MatInspector from the Genomatix software [7].

PP2A Inhibitor LB-100 Rescue Assay
Myoblasts were cultured in 6-well cell culture plates and infected with sh-2A and sh-NC at multiplicity of infection (MOI) 50. The PP2A inhibitor LB-100 (Selleck, Shanghai, China) was added 24 h post infection of adenoviruses at a final concentration of 1 µM. Myoblasts were induced to myogenic differentiation for 6 days before microscopy and RT-PCR analysis.

Statistical Analysis
All data were presented as mean ± SEM. Statistically significant differences between two groups were analyzed using independent-samples t-test (IBM SPSS Statistics version 18.0, IBM, Xi'an, China). p values of ≤ 0.05 were considered to be statistically significant.