LncR-133a Suppresses Myoblast Differentiation by Sponging miR-133a-3p to Activate the FGFR1/ERK1/2 Signaling Pathway in Goats

Long noncoding RNAs (lncRNAs) are involved in a variety of biological processes and illnesses. While a considerable number of lncRNAs have been discovered in skeletal muscle to far, their role and underlying processes during myogenesis remain mostly unclear. In this study, we described a new functional lncRNA named lncR-133a. Gene overexpression and interference studies in goat skeletal muscle satellite cells (MuSCs) were used to establish its function. The molecular mechanism by which lncR-133a governs muscle differentiation was elucidated primarily using quantitative real-time PCR (qRT-PCR), Western blotting, dual-luciferase activity assays, RNA immunoprecipitation, biotin-labeled probe, and RNA fluorescence in situ hybridization analyses. LncR-133a was found to be substantially expressed in longissimus thoracis et lumborum muscle, and its expression levels changed during MuSC differentiation in goats. We validated that lncR-133a suppresses MuSC differentiation in vitro. Dual-luciferase reporter screening, Argonaute 2 (AGO2) RNA immunoprecipitation assays, biotin-labeled lncR-133a capture, and fluorescence in situ hybridization showed that lncR-133a interacted with miR-133a-3p. Additionally, miR-133a-3p facilitated MuSC differentiation, but lncR-133a reversed this effect. The luciferase reporter assay and functional analyses established that miR-133a-3p directly targets fibroblast growth factor receptor 1 (FGFR1). Moreover, lncR-133a directly reduced miR-133a-3p’s capacity to suppress FGFR1 expression, and positively regulated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). In summary, our results suggested that lncR-133a suppresses goat muscle differentiation by targeting miR-133a-3p and activating FGFR1/ERK1/2 signaling pathway.


Introduction
Skeletal muscle is the primary source of animal protein for human consumption, and its growth and development have a direct effect on meat quantity and quality [1]. Skeletal myogenesis is a well-regulated process that is regulated by a number of muscle-specific transcription factors, including the paired box family (Pax3 and Pax7), myogenic regulatory factor family (Myf5, Mrf4, MyoD, and MyoG), and myocyte enhancer factor 2 (MEF2) family [2][3][4][5][6]. However, muscle development processes, as well as the activities of novel regulatory elements and molecular mechanisms, must be investigated further. In recent years, a rising number of non-coding RNAs with the ability to regulate myogenesis have been found, including microRNAs, long non-coding RNAs, and circular RNAs. [7][8][9][10].
RNA (~1 µg) was reverse-transcribed into cDNA using the PrimeScript TM RT Reagent Kit with gDNA Eraser (Takara, Dalian, China) (for mRNA and lncRNA detection) and the Mir-X TM miRNA First-Strand Synthesis Kit (Takara, Dalian, China) (for miRNA assays). Quantitative real-time PCR (qRT-PCR) was performed using the SYBR Premix Ex Taq TM II or Mir-X TM miRNA qRT-PCR SYBR ® Kit (Takara, Dalian, China). PCR was performed in a 10 µL volume containing 5 µL of SYBR, 0.4 µL of each primer (10 µM), 0.8 µL of normalized template cDNA (~2 µg/µL), and 3.4 µL of sterile water. Amplification conditions were 95 • C for 2 min followed by 40 cycles at 95 • C for 10 s and annealing temperature for 30 s, as shown in Supplementary Table S1. Melting curve analysis was performed from 65 • C to 95 • C with an incremental increase of 0.5 • C per second. Each experiment was performed with three biological replicates and repeated independently three times. Relative expression levels were calculated using the 2 −∆∆Ct method [25]. The genes ACTB and U6 (for miRNA) served as internal controls. Supplementary Table S1 contains a list of the primers used in this work. Primers were designed using Primer-BLAST (http://www.ncbi. nlm.nih.gov/tools/primer-blast/, accessed on 8 May 2021), and were synthesized by the Sangon Biotechnology Company (Shanghai, China).

Vector Construction, RNA Oligonucleotides, and Transfection
The construction of overexpression plasmids involved the synthesis of the full-length lncR-133a by TsingKe (Beijing, China) followed by cloning into the pEGFP-N1 vector (Promega, Madison, WI, USA) using the restriction enzymes XhoI and BamHI to obtain the expression plasmid pEGFP-lncR-133a. The primers used for plasmid construction are listed in Supplementary Table S1. Three siRNAs independently targeting lncR-133a (si-lncR-133a: CCATCTACTAGCTGAATAA, CCTGATGTGTTTCAGTATT, CCGTCTG-GTCAGTCTCTAA) were designed and synthesized by RiboBio (Guangzhou, China). Nonspecific siRNA sequences were used as negative controls, and a mixture of three siRNAs was used for lncR-133a interference experiments. MiR-133a-3p mimics, negative control mimics, a miR-133a-3p inhibitor, and a negative control inhibitor were designed and synthesized by RiboBio (Guangzhou, China). Goat skeletal muscle satellite cells (MuSCs) were transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's specifications. The final concentration of plasmids and RNA oligos used in transfections or co-transfections was 50 nM.
To assess gain or loss of function, GM was replaced with DMEM containing 15% FBS when cells reached 80-90% confluence, followed by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) transfection. After 6 h of transfection, GM was replaced with DM. After 48 h, the transfected cells were collected for qRT-PCR and Western blot assays.

Immunofluorescence Analyses
MuSCs (seeded at~2 × 10 4 cells per dish) were transfected in 3.5-cm Petri dishes and cultured in DM. After 96 h of transfection, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature and washed three times with 1 mL of PBS. The cells were permeabilized with 1 mL of 0.5% Triton X-100 at 4 • C for and washed with PBS three times. Samples were blocked with 1 mL of 2% bovine serum albumin for 30 min at 37 • C and incubated overnight with a monoclonal antibody to myosin heavy chain (MyHC) (1:150; Abcam, Cat. No. ab51263, Cambridge Science Park, Cambridge, UK) at 4 • C with gentle shaking. The cells were washed three times with PBS (5 min each) and incubated with Cy3-conjugated IgG (H + L) (1:150, Cell Signaling Technology, Cat. No. 4410, Beverly, MA, USA) at 37 • C for 2 h. The cells were rinsedthree times with PBS and stained with 0.05 µg/mL DAPI (4 ,6 -diamidino-2-phenylindole; Invitrogen) at 37 • C for 10 min in a humidified dark chamber. The cells were washed three times with 1 mL of PBS and imaged on a fluorescent microscope (Leica, Wetzlar, Germany). The differentiation index was calculated as the percentage of nuclei in MyHC-positive cells, and the fusion index was calculated as the percentage of nuclei in fused myotubes with two or more nuclei. At least three samples were examined separately for each treatment, and five areas per sample were randomly selected.

Luciferase Activity Assay
Wild-type and mutant lncR-133a fragments and the 3 -untranslated region (UTR) of the FGFR1 gene were amplified and subcloned into the psiCHECK-2 vector (Promega, Madison, WI, USA) using XhoI and NotI restriction enzymes (Takara, Dalian, China). LncR-133a and FGFR1 mutants were generated by changing the binding site from GACCAA to GAATGC and from GGACCAA to GGTGAGC, respectively. The primers used for plasmid construction are listed in Supplementary Table S1. All constructs were confirmed by Sanger sequencing. For the luciferase reporter assays, MuSCs (~1 × 10 4 cells per well) were seeded in 24-well plates containing GM under experimental conditions similar to those for MuSCs, followed by transfection when the cells reached 80-90% confluence. GM was replaced with DM after 6 h of transfection. HEK293T cells were cultured in 48-well plates until they reached 70-80% confluence and were transfected with the plasmids. Transfected cells were harvested and lysed using cell lysis buffer (NEB, Ipswich, MA, USA), and 48 h after differentiation, luciferase activity was determined using a dual-luciferase reporter assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity.

Nuclear and Cytoplasmic RNA Fractionation
Nuclear/cytoplasmic fractionation of MuSCs was performed using a cytoplasmic/nuclear RNA purification kit (Norgen Biotek, Thorold, ON, Canada) according to the manufacturer's instructions. Briefly, MuSCs (seeded at~2 × 10 7 cells per dish) cultured on a 10-cm culture dish were washed twice using cold PBS after 3 days of differentiation and were lysed in a hypotonic buffer (10 mM Tris pH 8.0, 1 mM EDTA). To separate nuclei and debris, cells were centrifuged at 500× g for 5 min. Total RNA was extracted separately from the supernatant (cytosolic fraction) and the pellet (nuclei) using RNAiso Plus reagent (TaKaRa, Dalian, China) and reverse-transcribed into cDNA using the PrimeScript TM RT Reagent Kit and gDNA Eraser (Takara, Dalian, China). RNA was quantified by qRT-PCR. 18S ribosomal RNA (18S rRNA) and U6 were used as cytoplasmic and nuclear controls, respectively. All procedures were performed at 4 • C under RNase-free conditions.
2.9. RNA-Binding Protein Immunoprecipitation (RIP) Assay RIP assays were performed using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) following the manufacturer's protocol. MuSCs (~2 × 10 7 cells) grown for 72 h were harvested and lysed in RIP lysis buffer. The cells were divided into two equal parts and incubated overnight at 4 • C with magnetic beads conjugated with an antibody IgG (Millipore, Billerica, MA, USA) or AGO2 (Abcam, Cambridge Science Park, Cambridge, UK). LncR-133a and miR-133a-3p in immunoprecipitated RNA were quantified by qRT-PCR.

Biotin-Labeled lncR-133a Capture
MuSCs were transfected with a biotinylated lncR-133a probe and harvested at 48 h after transfection, as previously described [19]. The cells were lysed on ice for 30 min in 250 µL of cell lysis buffer (10 mM KCl, 1.5 mM MgCl 2 , 10 mM Tris-HCl pH 7.5, 5 mM dithiothreitol). The supernatant was centrifuged for 5 min at 12,000× g, and the beads were washed five times with solution A (0.1 M NaOH and 0.05 M NaCl) and thrice with 0.1 M NaCl. The beads were blocked with 1 mg/mL bovine serum albumin (Roche, Basel, Switzerland) and 1 mg/mL yeast transfer RNA (Thermo Fisher Scientific, Waltham, MA, USA) overnight. Subsequently, 500 µL NaCl (1 M) and 30 µL beads were added to the supernatant. The beads were washed five times in washing buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl) and mixed with the lysate for 4 h at 4 • C. The RNA complexes were bound to the beads were eluted and extracted for qRT-PCR. PCR assays were performed as described in the subheading RNA extraction and quantitative real-time PCR.

Statistics Analysis
Data are means ± standard error of the mean of at least three biological replicates. Statistical significance was analyzed using the Student's t-test for comparisons of two groups, and one-way analysis of variance with Tukey's post-hoc test for multiple groups using SAS software version 9.0 (SAS, Cary, NC, USA). The significance level was set at p < 0.05.

Coding Potential and Expression Profile of lncR-133a
Our previous study found that several lncRNAs, including the 1581-nucleotide lncR-133a (TCONS_00067594), were differentially expressed in fetal and neonatal muscle tissue of goats [24] and that lncR-133a was located on chromosome 16 (Supplementary Figure S1). The present study evaluated the coding potential of lncR-133a using CPC2 online software (http://cpc2.cbi.pku.edu.cn/, accessed on 12 March 2021) and revealed that the coding potential of lncR-133a was weaker than that of the controls Myod1 and linc-MD1 ( Figure 1a). Moreover, all goat tissues examined (heart, liver, spleen, lung, kidney, and LTL) expressed lncR-133a and expression was highest in the LTL muscle (p < 0.05) (Figure 1b). In LTL tissue, lncR-133a expression peaked on day 60 of gestation (p < 0.05) (Figure 1c). The expression level of four marker genes was also measured in said tissues (Supplementary Figure S2a,b), and the expression levels of myogenesis markers (Pax7, MyoD, MyoG, MyHC) during MuSC differentiation were quantified (Supplementary Figure S2c). The expression pattern of these markers during MuSC differentiation agreed with a previous study [27] and demonstrated that lncR-133a expression could be quantified accurately. The expression levels of lncR-133a during goat MuSC differentiation were also measured in vitro (growth medium [GM] and differentiation medium [DM] for 1, 3, 5, and 7 d). The results indicated that lncR-133a was upregulated in the early stage of differentiation and reached its peak in MuSCs differentiated for 3 d (p < 0.05) followed by a gradual decrease (Figure 1d).
Our previous study found that several lncRNAs, including the 1581-nucleotide lncR-133a (TCONS_00067594), were differentially expressed in fetal and neonatal muscle tissue of goats [24] and that lncR-133a was located on chromosome 16 (Supplementary Figure  S1). The present study evaluated the coding potential of lncR-133a using CPC2 online software (http://cpc2.cbi.pku.edu.cn/, accessed on 12 March 2021) and revealed that the coding potential of lncR-133a was weaker than that of the controls Myod1 and linc-MD1 ( Figure 1a). Moreover, all goat tissues examined (heart, liver, spleen, lung, kidney, and LTL) expressed lncR-133a and expression was highest in the LTL muscle (p < 0.05) ( Figure  1b). In LTL tissue, lncR-133a expression peaked on day 60 of gestation (p < 0.05) ( Figure  1c). The expression level of four marker genes was also measured in said tissues (Supplementary Figure S2a,b), and the expression levels of myogenesis markers (Pax7, MyoD, MyoG, MyHC) during MuSC differentiation were quantified (Supplementary Figure S2c). The expression pattern of these markers during MuSC differentiation agreed with a previous study [27] and demonstrated that lncR-133a expression could be quantified accurately. The expression levels of lncR-133a during goat MuSC differentiation were also measured in vitro (growth medium [GM] and differentiation medium [DM] for 1, 3, 5, and 7 d). The results indicated that lncR-133a was upregulated in the early stage of differentiation and reached its peak in MuSCs differentiated for 3 d (p < 0.05) followed by a gradual decrease (Figure 1d).
Changes in mRNA and protein expression of muscle differentiation marker genes (MyoG and MyoD) and myotube formation as a function of lncR-133a levels were assessed. The results showed that lncR-133a overexpression significantly decreased the mRNA and protein abundance of MyoG and MyoD (p < 0.05; Figure 2b,e), whereas lncR-133a knockdown had the opposite effect (p < 0.05; Figure 2d,e). To confirm that lncR-133a inhibits MuSC differentiation, immunofluorescence analysis was performed to detect myosin heavy chain positive (MyHC + ) cells transfected with pEGFP-N1 or pEGFP-lncR-133a and cultured in DM for 4 days. Compared with the negative control (NC), lncR-133a overexpression significantly downregulated MyHC expression, whereas lncR-133a knockdown had the opposite effect (Figure 2f,i). Furthermore, lncR-133a overexpression strongly decreased myotube length (Figure 2g) and the fusion index (Figure 2h), whereas lncR-133a knockdown had the opposite effect (Figure 2j,k). These results indicate that lncR-133a suppresses the differentiation of goat MuSCs.

LncR-133a Acts as a Molecular Sponge for miR-133a-3p
LncR-133a is found in the cytoplasm and nucleus (Figure 3a), and its expression in the cytoplasm increases significantly in differentiated myoblasts (Figure 3b). We speculate that lncR-133a may function as a molecular sponge of miRNA in myogenic differentiation. To test this hypothesis, miRNAs targets were predicted using TargetScan [28] and RNAhybrid software [29], and the results showed that lncR-133a contained a potential binding site for miR-133a-3p (Figure 3c). A dual-luciferase reporter system, in which the wild-type sequence or a mutated sequence of lncR-133a was bound to the 3 end of the firefly luciferase gene, was used to confirm whether lncR-133a interacted with miR-133a-3p (Figure 3c). The results revealed that miR-133a-3p suppressed the luciferase activity of the wild-type but not the mutated lncR-133a sequence (Figure 3d), demonstrating that miR-133a-3p binds to lncR-133a during myoblast differentiation by complementary base pairing. In addition, lncR-133a overexpression downregulated miR-133a-3p in a dose-dependent manner, whereas lncR-133a knockdown had the opposite effect (Figure 3e,f). Argonaute 2 (AGO2) immunoprecipitation and biotin-labelled lncR-133a capture followed by realtime PCR confirmed the interaction between miR-133a-3p and lncR-133a (Figure 3g,h).
Our results suggest that lncR-133a binds to and inhibits miR-133a-3p during MuSC differentiation. Therefore, rescue experiments were performed to assess whether the effect of miR-133a-3p on MuSC differentiation was blocked by lncR-133a overexpression. The qRT-PCR and Western blot analysis results indicated that lncR-133a overexpression blocked the ability of miR-133a-3p to increase the mRNA and protein levels of MyoG and MyoD (p < 0.05; Figure 4f,g).

LncR-133a Reverse the Effect of miR-133a-3p on MuSC Differentiation
Our results suggest that lncR-133a binds to and inhibits miR-133a-3p during MuSC differentiation. Therefore, rescue experiments were performed to assess whether the effect of miR-133a-3p on MuSC differentiation was blocked by lncR-133a overexpression. The qRT-PCR and Western blot analysis results indicated that lncR-133a overexpression blocked the ability of miR-133a-3p to increase the mRNA and protein levels of MyoG and MyoD (p < 0.05; Figure 4f,g).

FGFR1 Is a Target Gene of miR-133a-3p and Inhibits Myoblast Differentiation
The genes targeted by miR-133a-3p were identified using TargetScan (http://www. targetscan.org, accessed on 21 June 2021). The results revealed that FGFR1 was the strongest candidate because of its well-established role in C2C12 myoblast differentiation [30]. In addition, RNAhybrid software predicted that the FGFR1 3 UTR had a binding site for miR-133a-3p (Figure 5a). A luciferase reporter assay was conducted to confirm whether miR-133a-3p directly targeted FGFR1, and the results showed that miR-133a-3p targeted the wild-type 3 UTR but not the mutant UTR (Figure 5b). In addition, FGFR1 mRNA and protein expression was downregulated by miR-133a-3p overexpression and upregulated by miR-133a-3p knockdown (Figure 5c,d), suggesting that miR-133a-3p inhibits FGFR1 expression during MuSC differentiation. Furthermore, we evaluated the expression pattern of FGFR1 in goat tissues and cultured MuSCs using qRT-PCR. FGFR1 expression was relatively high in the LTL muscle (p < 0.05) (Figure 5e) and peaked on day 75 of gestation ( Figure 5f). In cultured MuSCs, FGFR1 expression peaked on day 3 of differentiation ( Figure 5g).
To confirm whether lncR-133a affected FGFR1 through miR-133a-3p, the effect of To study the effect of FGFR1 on myoblast differentiation, we constructed the overexpression vector pEGFP-FGFR1 and synthesized a siRNA targeting FGFR1. The results showed that FGFR1 was successfully overexpressed in this vector and was inhibited by si-FGFR1 in MuSCs (Figure 5h,i). Moreover, FGFR1 overexpression significantly decreased the mRNA and protein expression of MyoG and MyoD (p < 0.05), whereas FGFR1 knockdown had the opposite effect (p < 0.05, Figure 5j,k). These data suggest that FGFR1 inhibits myoblast differentiation.

Discussion
Skeletal myogenesis is tightly controlled by a complex network, and accumulating evidence suggests that lncRNAs play an important role in this process [9,31]. A few lncRNAs are known to play a role in goat skeletal myogenesis. This study found that lncR-133a was significantly expressed in skeletal muscle and suppressed the differentiation of goat skeletal muscle satellite cells by sponging miR-133a-3p, consequently increasing FGFR1 expression. Several lncRNAs have been identified in the skeletal muscle of pig [32][33][34], cattle [35][36][37], sheep [38][39][40], chicken [41,42], and goats [43]. Moreover, we previously identified lncRNAs in fetal and postnatal goat skeletal muscle using RNA-seq technology [24]. These data provide a basis for understanding the role of lncRNAs in goat muscle formation.
This study found that lncR-133a was highly expressed in the LTL muscle of embryonic goats, and expression changed during muscle development and MuSC differentiation, suggesting that lncR-133a might be involved in the regulation of muscle development. One of the characteristics of lncRNAs is spatio-temporal changes in expression patterns [12,42,43], consistent with our results. The effect of lncR-133a overexpression and knockdown on MuSC differentiation was assessed in vitro. LncR-133a overexpression suppressed myogenic differentiation, whereas lncR-133a silencing had the opposite effect, consistent with the effects of lncRNA 2310043L19Rik [44] and AK017368 [45]. For instance, lncRNA 2310043L19Rik overexpression inhibited myoblast differentiation but promoted cell proliferation, whereas lncRNA 2310043L19Rik silencing had the opposite effect in vitro [44]. Similarly, lncRNA AK017368 inhibited myoblast differentiation, and AK017368 gain/loss-of-function had the opposite effect [45].
Bioinformatics analysis of the lncR-133a sequence was done to elucidate the molecular mechanisms by which lncR-133a regulates muscle differentiation, and the results showed this RNA contains a potential binding site for miR-133a-3p. Dual-luciferase reporter assays confirmed that lncR-133a is a direct target of miR-133a-3p. The regulatory action of lncRNAs is dictated by their subcellular localization. In this respect, many cytoplasmlocated lncRNAs act as ceRNAs by sponging miRNAs, increasing the expression of target genes. For instance, muscle-specific linc-MD1 reduces the inhibition of MAML1 and MEF2C and induces skeletal muscle differentiation by sponging miR-133 and miR-135, respectively [17]. Therefore, RNA-FISH and subcellular localization analysis of lncR-133a was done in this study, and the results indicated that lncR-133a was localized to the cytoplasm, suggesting that lncR-133a might affect myogenesis by acting as a ceRNA and sponging miR-133a-3p. AGO2-RIP and biotin-labeled lncR-133a capture experiments confirmed that lncR-133a sponged miR-133a-3p during MuSC differentiation. In addition, miR-133a-3p expression was inhibited by lncR-133a, suggesting that lncR-133a interacted with miR-133a-3p.
Several studies have reported that miR-133, one of the most abundant myogenic microRNAs, regulates multiple targets involved in skeletal muscle development. The transcription factor serum response factor, which is required for muscle proliferation and differentiation, was the first target discovered [46]. In addition, miR-133 participates in myoblast proliferation and differentiation by negatively modulating IGF-1R/PI3K/Akt signaling through the inhibition of IGF-1R [47]. Another study has reported that miR-133 inhibits myoblast proliferation and promotes myoblast differentiation by repressing the ERK1/2 pathway via FGFR1 and PP2AC [30]. Furthermore, gga-miR-133a-3p inhibits chicken myoblast proliferation and promotes myoblast differentiation by targeting PRRX1 [48]. In this study, miR-133a-3p significantly increased muscle differentiationrelated gene expression, whereas lncR-133a had the opposite effect. Furthermore, lncR-133a reduced the stimulatory effect of miR-133a-3p on MuSC differentiation, suggesting that lncR-133a sponged miR-133a-3p.
The purpose of this study was to see if FGFR1 was a downstream effector of lncR-133a/miR-133a-3p that mediated the regulation of MuSC differentiation. FGFR1 overexpression promoted myocyte proliferation and delayed differentiation in mice [49]. Conversely, the expression of mutated FGFR1 enhanced myocyte differentiation [49]. In addition, the knockdown of FGFR1 and PP2AC by siRNA promoted C2C12 differentiation [30]. In line with these studies, our findings suggest that FGFR1 inhibits MuSC differentiation in goats. The interaction between FGFR1 and miR-133a-3p was assessed using a dual-luciferase reporter system. Mutations in the predicted miR-133a-3p target site in the 3 UTR of the FGFR1 gene abolished luciferase activity, demonstrating that FGFR1 was a target of miR-133a-3p. Moreover, qRT-PCR results indicated that miR-133a-3p inhibited FGFR1 expression during MuSC differentiation. Further experiments revealed that lncR-133a increased FGFR1 expression by sponging miR-133a-3p. Previous study revealed that miR-133 inhibits myoblast proliferation and promotes myoblast differentiation by repressing the ERK1/2 pathway via FGFR1 and PP2AC [30]. Furthermore, ERK1/2 inhibits myoblast differentiation in C2C12 cells [50]. We found that lncR-133a promoted ERK1/2 phosphorylation, suggesting that lncR-133a might regulate MuSC differentiation by activating the ERK1/2 signaling pathway.

Conclusions
In conclusion, we identified a novel lncR-133a as a sponge for miR-133a-3p to activate FGFR1/ERK1/2 signaling, thus suppressing muscle differentiation (Figure 7). The lncR-133a/miR-133a-3p/FGFR1/ERK1/2 axis may shed light on further understanding the skeletal muscle development mechanism. Furthermore, our findings suggest that lncR-133a is a potential regulatory element for muscle development and myogenesis in goats.

Conclusions
In conclusion, we identified a novel lncR-133a as a sponge for miR-133a-3p to activate FGFR1/ERK1/2 signaling, thus suppressing muscle differentiation (Figure 7). The lncR-133a/miR-133a-3p/FGFR1/ERK1/2 axis may shed light on further understanding the skeletal muscle development mechanism. Furthermore, our findings suggest that lncR-133a is a potential regulatory element for muscle development and myogenesis in goats.  Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes13050818/s1, Figure S1: The sequence information of goat lncR-133a; Figure S2: Expression pattern of myogenesis marker genes (Pax7, MyoD, MyoG, and MyHC). (a) qRT-PCR measured the expression levels of four marker genes in goat different tissues on day 60 of gestation. (b) The expression levels of four marker genes in the developmental LTL muscles aged from E45 to B3. (c) Real-time PCR analysis of four marker genes expression in MuSCs during 7 days of differentiation. Data are means ± standard error of the mean of at least three biological replicates, * p < 0.05; Table S1: Specific primers used in this study. Data Availability Statement: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.