A Novel Circular RNA Generated by FGFR2 Gene Promotes Myoblast Proliferation and Differentiation by Sponging miR-133a-5p and miR-29b-1-5p

It is well known that fibroblast growth factor receptor 2 (FGFR2) interacts with its ligand of fibroblast growth factor (FGF) therefore exerting biological functions on cell proliferation and differentiation. In this study, we first reported that the FGFR2 gene could generate a circular RNA of circFGFR2, which regulates skeletal muscle development by sponging miRNA. In our previous study of circular RNA sequencing, we found that circFGFR2, generated by exon 3–6 of FGFR2 gene, differentially expressed during chicken embryo skeletal muscle development. The purpose of this study was to reveal the real mechanism of how circFGFR2 affects skeletal muscle development in chicken. In this study, cell proliferation was analyzed by both flow cytometry analysis of the cell cycle and 5-ethynyl-2′-deoxyuridine (EdU) assays. Cell differentiation was determined by analysis of the expression of the differentiation marker gene and Myosin heavy chain (MyHC) immunofluorescence. The results of flow cytometry analysis of the cell cycle and EdU assays showed that, overexpression of circFGFR2 accelerated the proliferation of myoblast and QM-7 cells, whereas knockdown of circFGFR2 with siRNA reduced the proliferation of both cells. Meanwhile, overexpression of circFGFR2 accelerated the expression of myogenic differentiation 1 (MYOD), myogenin (MYOG) and the formation of myotubes, and knockdown of circFGFR2 showed contrary effects in myoblasts. Results of luciferase reporter assay and biotin-coupled miRNA pull down assay further showed that circFGFR2 could directly target two binding sites of miR-133a-5p and one binding site of miR-29b-1-5p, and further inhibited the expression and activity of these two miRNAs. In addition, we demonstrated that both miR-133a-5p and miR-29b-1-5p inhibited myoblast proliferation and differentiation, while circFGFR2 could eliminate the inhibition effects of the two miRNAs as indicated by rescue experiments. Altogether, our data revealed that a novel circular RNA of circFGFR2 could promote skeletal muscle proliferation and differentiation by sponging miR-133a-5p and miR-29b-1-5p.


RNA Extraction, cDNA Synthesis and Quantitative Real-Time PCR
The total RNA was extracted from cells by using RNAiso reagent (TaKaRa, Otsu, Japan). The quality and concentration of all obtained RNA samples were determined by 1.5% agarose gel electrophoresis and evaluated for optical density 260/280 ratio by Nanodrop 2000 spectrophotometer (Thermo, Waltham, MA, USA). For mRNA and circFGFR2 expression analysis, cDNA synthesis for mRNA was performed using a PrimeScript RT Reagent Kit (Perfect Real Time) (TaKaRa, Otsu, Japan). The β-actin gene was used as an internal control for quantitative real-time PCR (qRT-PCR) analysis. The reverse transcription reaction for miRNA was performed using ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). The specific Bulge-loop miRNA qRT-PCR Primer for miR-133a-5p, miR-29b-1-5p and U6 were designed by RiboBio (RiboBio, Guangzhou, China). qRT-PCR was performed on a Bio-Rad CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA) using iTaq™ Universal SYBR ® Green Supermix Kit (Bio-Rad, Hercules, CA, USA). Each sample was assayed in triplicate, following the manufacturer's instructions. The specificity of the product was evaluated by the melting curve, Cells 2018, 7, 199 4 of 20 and the quantitative values were obtained from the threshold PCR cycle number (Ct) at which the increase in signal is associated with an exponential growth at which the PCR product starts to be detected. The relative mRNA level in each sample was indicated by 2 −∆∆Ct .

RNA Oligonucleotides and Plasmids Construction
The gga-miR-133a-5p mimic, gga-miR-29b-1-5p mimic and mimic control duplexes, the 3 end biotinylated gga-miR-133a-5p, gga-miR-29b-1-5p and mimic control duplexes, siRNA target against circFGFR2 (si-circFGFR2, 5 -CGATGTTGTCGAGCCGCCA-3 ) and non-specific siRNA negative control were synthesized by RiboBio (Guangzhou, China). For circFGFR2 overexpression plasmids construction, the linear sequences of circFGFR2 was amplified by PCR with primer FGFR2-2, and the cDNA template was synthesized from the RNA of chicken primary myoblast by RT-PCR. Then, the obtained linear sequences were cloned into KpnI and BamHI restriction sites of a circular expression vector-the pCD2.1-ciR vector (Geneseed Biotech, Guangzhou, China) according to the manufacturer's protocol, so as to generate the pCD2.1-circFGFR2 overexpression vector. For pmirGLO dual-luciferase reporter construction: the whole linear sequences of circFGFR2 were cloned into XhoI and SalI restriction sites of pmirGLO vector to generate the wild reporter vector (PGLO-WT reporter vector), which includes the predicted binding sites of miR-133a-5p and miR-29b-1-5p. PGLO-MT1 and PGLO-MT2 were two mutational reporter vectors of miR-133a-5p which were cloned into XhoI and SalI restriction sites of pmirGLO vector by PCR mutagenesis. We changed one of miR-133a-5p binding seed sequences from "CCAG" to "TTGA" in PGLO-MT1, while in PGLO-MT2 we changed another miR-133a-5p binding seed sequence (which included the binding site of miR-29b-1-5p) from "CCAG" to "GTTG". All luciferase reporters were constructed by Hongxun Biotech (Suzhou, China).

Cell Culture
Chicken embryo fibroblast cell line (DF-1) cells were cultured in high-glucose Dulbecco's modified Eagle's medium (Gibico, Grand Island, NY, USA) with 10% (v/v) fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and 0.2% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). Quail muscle cell line (QM-7) cells were cultured in high-glucose M199 medium (Gibco, USA) with 10% (v/v) FBS, 10% tryptose phosphate broth solution (Sigma, Louis, MO, USA) and 0.2% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). Chicken primary myoblasts were isolated from the leg muscles of 11-day embryo age (E11) chickens. Leg tissues were collected from E11 chickens by completely removing skin and bones. Leg muscle was minced into sections of approximately 1 mm with scissors and then digested with 0.25% trypsin (Gibco, Grand Island, NY, USA) at 37 • C in a shaking water bath (90 oscillations/min) for 20 min. Digestions were terminated by adding equal values of complete medium-(RPMI)1640 medium with 20% FBS, 1% nonessential amino acids and 0.2% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). The mixture was filtered through a nylon mesh with 70 mm pores (BD Falcon, Greiner, Germany). The filtered cells were centrifuged at 500× g for 5 min, and maintained in complete medium at 37 • C in a 5% CO 2 , humidified atmosphere. Serial plating was performed to enrich myoblasts and to remove fibroblasts.

5-Ethynyl-2 -Deoxyuridine (EdU) Assays
After cells were transfected for 48 h, myoblasts were exposed to 50 µM 5-ethynyl-2 -deoxyuridine (EdU) (RiboBio, Guangzhou, China) for 2 h at 37 • C. Next, the cells were fixed in 4% paraformaldehyde (PFA) for 30 min and 2 mg/mL glycine solution was used to neutralize the 4% PFA. Cells were, then, permeabilized with 0.5% Triton X-100. Subsequently, 1× Apollo reaction cocktail (RiboBio, Guangzhou, China) was added to the cells and incubated for 30 min. The cells were stained with Hoechst 33342 for 30 min for DNA content analysis. Finally, the EdU-stained cells were visualized under a fluorescence microscope (Nikon, Tokyo, Japan or Leica, Wetzlar, Germany). The analysis of myoblast proliferation (ratio of EdU+ to all myoblasts) was performed using images of randomly selected fields obtained on the fluorescence microscope.

Immunofluorescence
For immunofluorescence, cells were seeded in 24-well plates. After transfection for 48 h, cells were fixed in 4% formaldehyde for 20 min then washed three times with PBS for 5 min. Subsequently, the cells were permeabilized by adding 0.1% Triton X-100 for 5 min and blocked with goat serum for 30 min. After incubation with MyHC (B103; DSHB, Iowa City, IA, USA; 0.5 µg/mL) at 37 • C for 2 h, the Fluorescein (FITC)-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (Bioworld, Minneapolis, MN, USA; 1:200) or FITC (Bioworld, Minneapolis, MN, USA; 1:50) was added and the cells were incubated at room temperature for 1 h. The cell nuclei were stained with 4 ,6-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China; 1:50) for 5 min. Images were obtained with a fluorescence microscope (Leica, Wetzlar, Germany). The area of cells labeled with anti-MyHC was measured by using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the total myotube area was calculated as a percentage of the total image area covered by myotubes.

Biotin-Coupled miRNA Pull Down Assay
Transfection procedure: the 100 nM of 3 end biotinylated miR-133a-5p, miR-29b-1-5p or mimic NC (RiboBio, Guangzhou, China) were transfected into QM-7 cells along with 30 µg circFGFR2 expression vector in T75 cell culture bottle. At 24 h after transfection, the cells were harvested and washed in PBS, then lysed in lysis buffer. A total of 100 µL washed streptavidin magnetic beads were blocked for 2 h and then added to each reaction tube to pull down the biotin-coupled RNA complex. All the tubes were incubated for 4 h on a rotator at a low speed (10 r/min). The beads were washed with lysis buffer five times and RNAiso reagent (TaKaRa, Otsu, Japan) was used to recover RNAs specifically interacting with miRNA. The abundance of circFGFR2 in bound fractions was evaluated by qRT-PCR analysis.

Statistical Analysis
In all panels, results are expressed as the mean ± S.E.M. of three independent experiments. For two group comparison analysis, statistical significance of differences between means was analyzed by unpaired Student's t-test. For multiple comparison analysis, data were analyzed by one-way ANOVA followed by both least significant difference (LSD) and Duncan test through Statistical Package for the Social Sciences software (SPSS 17.0, Chicago, IL, USA). We considered p < 0.05 to be statistically significant. * p < 0.05; ** p < 0.01. NC, negative control.

CircFGFR2 Promotes Myoblast Proliferation
To investigate the role of circFGFR2 in skeletal muscle cell proliferation, we conducted overexpression and knocked down experiments by transfecting circFGFR2 overexpression vector and siRNAs (pCD2.1-circFGFR2 and si-circFGFR2) into chicken primary myoblast and QM-7 cell. The relative expression of circFGFR2 was detected after 48 h post transfection by qRT-PCR. Result showed that both the effect of overexpression and knockdown had reached a significant level in both myoblast and QM-7 cell ( Figure 1A-D), and si-circFGFR2 specifically downregulated the expression of circFGFR2 but not linear mRNA of FGFR2 ( Figure 2B). Furthermore, we detected the proliferation process of both chicken primary myoblast and QM-7 cell by flow cytometry for cell cycle analysis and 5-ethynyl-2 -deoxyuridine (EdU) incorporation assays after transfecting with pCD2.1-circFGFR2/pCD2.1-ciR, or si-circFGFR2/control. Cell cycle analysis showed that overexpression of circFGFR2 increased the cell population in S phase and decreased the cell population in G1/0 and G2/M phases ( Figure 1E) while knockdown of circFGFR2 decreased the cell population in S phase and increased the cell population in G1/0 phase, as observed in chicken primary myoblast ( Figure 1F). Meanwhile, the result of EdU strain assay showed that there were significantly more cells in the pCD2.1-circFGFR2 transfected group than in the control group ( Figure 1G,H), whereas knockdown of circFGFR2 significantly decreased the numbers of EdU strained cells ( Figure 1G,I). These results indicated that circFGFR2 could promote the proliferation rate of chicken primary myoblast. As expected, we obtained similar results in QM-7 cell ( Figure 1J-N). These results suggested that circFGFR2 could significantly promote the proliferation of myoblast and QM-7 cell.

CircFGFR2 Promotes Myoblast Differentiation
Myogenesis is a complex process including myoblast proliferation, differentiation and myotube formation and is controlled by myogenic regulatory factors (MRFs), MYOD, MYOG, myogenic factor 5 (Myf5), and myogenic factor 6 (Myf6, also known as myogenic regulatory factor 4, MRF4). These factors activate muscle-specific genes to coordinate myoblasts to terminally withdraw from the cell cycle and subsequently fuse into multinucleated myotubes [43]. Following proliferation, the initiation of terminal differentiation and fusion begins with the expression of myogenin, which together with MYOD, activates the muscle specific structural and contractile genes to stimulate myoblast differentiation [44]. To address the potential role of circFGFR2 in primary myoblast differentiation, the expression of differentiation marker genes, including MYOG and MYOD were analyzed by qRT-PCR after transfecting with pCD2.1-circFGFR2/pCD2.1-ciR, or si-circFGFR2/control. Result showed that overexpression of circFGFR2 significantly promoted the expression of MYOD and MYOG while knockdown of circFGFR2 significantly inhibited the expression of MYOD and MYOG (Figure 2A,B). It indicated that circFGFR2 may promote chicken primary myoblast differentiation. Subsequently, we induced chicken primary myoblast differentiation in vitro, as soon as the muscle cells started to differentiate into myotubes (the first day of differentiation, DM1), we transfected them with pCD2.1-circFGFR2/pCD2.1-ciR. MyHC immunofluorescence staining was carried out on the differentiated myoblasts after 36 h post transfection (DM3). According to the immunofluorescence staining, we found that the areas of myotubes of pCD2.1-circFGFR2 transfected group were prominently greater than that of the control group ( Figure 2C,D). Conversely, the areas of myotubes in the si-circFGFR2 transfected group were lower than that of the control group ( Figure 2E,F). The result showed that circFGFR2 could promote the formation of myotubes and promote the early differentiation of chicken primary myoblast. In all panels, the results are shown as mean ± S.E.M., and the data are represented by three independent assays. Statistical significance of differences between means was assessed using an unpaired Student's t-test (* p < 0.05; ** p < 0.01).

CircFGFR2 Promotes Myoblast Differentiation
Myogenesis is a complex process including myoblast proliferation, differentiation and myotube formation and is controlled by myogenic regulatory factors (MRFs), MYOD, MYOG, myogenic factor 5 (Myf5), and myogenic factor 6 (Myf6, also known as myogenic regulatory factor 4, MRF4). These factors activate muscle-specific genes to coordinate myoblasts to terminally withdraw from the cell cycle and subsequently fuse into multinucleated myotubes [43]. Following proliferation, the initiation  N) The percentage of EdU-stained chicken primary myoblasts after overexpression or knockdown of circFGFR2 for 48 h. In all panels, the results are shown as mean ± S.E.M., and the data are represented by three independent assays. Statistical significance of differences between means was assessed using an unpaired Student's t-test (* p < 0.05; ** p < 0.01). myoblasts after 36 h post transfection (DM3). According to the immunofluorescence staining, we found that the areas of myotubes of pCD2.1-circFGFR2 transfected group were prominently greater than that of the control group ( Figure 2C,D). Conversely, the areas of myotubes in the si-circFGFR2 transfected group were lower than that of the control group ( Figure 2E,F). The result showed that circFGFR2 could promote the formation of myotubes and promote the early differentiation of chicken primary myoblast.  (E,F) Down-regulation of circFGFR2 suppresses the formation of myotubes. In all panels, data are presented as mean ± S.E.M. of three biological replicates. Statistical significance of differences between means was assessed using an unpaired Student's t-test (* p < 0.05; ** p < 0.01).

CircFGFR2
Interacts with miR-133a-5p and miR-29b-1-5p, and Inhibits the Expression of miR-133a-5p and miR-29b-1-5p in Myoblast Circular RNA has been shown to act as miRNA sponge and circFGFR2 could promote myoblast proliferation and differentiation. We hypothesized that circFGFR2 exerts function by acting as miRNA sponge as well as regulating the expression of miRNA. To screen potential miRNAs that bind to circFGFR2, we used RNAhybrid to conduct the putative combination site between circFGFR2 and miR-133a-5p/miR-29b-1-5p. Interestingly, we found that circFGFR2 has two potential miR-133a-5p binding sites (binding site 1and binding site 2) and one potential miR-29b-1-5p binding site. The potential miR-29b-1-5p binding site shares six of seven nucleotides with the binding site 2 of miR-133a-5p. The mature sequence of miR-133a-5p/miR-29b-1-5p and the predicted binding sites of these two miRNAs are shown in Figure 3A  For two group comparison analysis, statistical significance of differences between means was analyzed by unpaired Student's ttest. For multiple comparison analysis, data were analyzed by one-way ANOVA followed by both least significant difference (LSD) and Duncan test through SPSS software. We considered p < 0.05 to be statistically significant. * p < 0.05; ** p < 0.01. NC, negative control.

MiR-133a-5p and miR-29b-1-5p Inhibit Myoblast Proliferation
As circFGFR2 had an effect on myoblast proliferation, we also confirmed that circFGFR2 could inhibit the expression and activity of miR-133a-5p and miR-29b-1-5p. We speculated that miR-133a-5p and miR-29b-1-5p had a potential effect on myoblast proliferation. To confirm our hypothesis, we (D) A schematic drawing showing the putative binding sites of miR-133a-5p/miR-29b-1-5p associated with circFGFR2. (E,F) Luciferase assay was conducted by co-transfecting wild type or mutant linear sequence of circFGFR2 with miR-133a-5p/miR-29b-1-5p mimic or mimic-NC in DF-1 cells. (G,H) Luciferase assay was conducted by co-transfecting wild type circFGFR2 linear sequence and miR-133a-5p/miR-29b-1-5p mimic or mimic-NC and with circFGFR2 overexpression vector (pCD2.1-circFGFR2) or empty vector (pCD2.1-ciR). (I) Biotin-coupled miRNA pull down assay from the myoblast lysates after transfection with 3 end biotinylated miR-133a-5p, miR-29b-1-5p or mimic NC. The expression level of circFGFR2 was quantified by qRT-PCR, and fold enrichment in the streptavidin captured fractions are plotted. (J,K) qRT-PCR analysis of the relative expression of miR-133a-5p and miR-29b-1-5p after overexpression or inhibition of circFGFR2. In all panels, results are expressed as the mean ± S.E.M. of three independent experiments. For two group comparison analysis, statistical significance of differences between means was analyzed by unpaired Student's t-test. For multiple comparison analysis, data were analyzed by one-way ANOVA followed by both least significant difference (LSD) and Duncan test through SPSS software. We considered p < 0.05 to be statistically significant. * p < 0.05; ** p < 0.01. NC, negative control.

CircFGFR2 Eliminates the Inhibition Effect of miR-133a-5p and miR-29b-1-5p on Myoblast Differentiation
We further performed a rescue experiment to investigate whether the suppressing effects of miR-133a-5p and miR-29b-1-5p on myoblast differentiation could be eliminated by circFGFR2 overexpression. As shown in Figure 7A, the expressions of MYOD and MYOG in miR-133a-5p and circFGFR2 co-transfected group were dramatically elevated compared with the miR-133a-5p transfected group. For miR-29b-1-5p, circFGFR2 also eliminated its repression effect on MYOD and MYOG ( Figure 7B). Further MyHC immunofluorescence showed that overexpression of circFGFR2 eliminated the inhibition on myotube formation at DM3 caused by either miR-133a-5p or miR-29b-1-5p ( Figure 7C-F). Taken together, these results demonstrated that circFGFR2 could eliminate the inhibition effect of miR-133a-5p and miR-29b-1-5p on myoblast differentiation. (E,F) Immunofluorescence analysis of MyHC-staining cells after overexpression miR-133a-5p or miR-29b-1-5p. (G,H) Immunofluorescence analysis of MyHC-staining cells after down-regulation of miR-133a-5p or miR-29b-1-5p. In all panels, results are expressed as the mean ± S.E.M. of three independent experiments, and statistical significance of differences between means was assessed using an unpaired Student's t-test (* p < 0.05; ** p < 0.01). NC, negative control. The myotube area of chicken primary myoblasts after co-transfection with the listed nucleic acids (miR-133a-5p, circFGFR2 overexpression vector and miR-133a-5p, empty overexpression vector and mimic NC, respectively). (E,F) The myotubes area of chicken primary myoblasts after co-transfection with the listed nucleic acids (miR-29b-1-5p, circFGFR2 overexpression vector, and miR-29b-1-5p, empty overexpression vector and mimic NC, respectively). In all panels, results are expressed as the mean ± S.E.M. of three independent experiments, and statistical significance of differences between means were analyzed by one-way ANOVA followed by both least significant difference (LSD) and Duncan test through SPSS software. We considered p < 0.05 to be statistically significant. * p < 0.05; * p < 0.01. NC, negative control. The mRNA expression of MYOD and MYOG of chicken primary myoblasts after co-transfection with the listed nucleic acids (miR-29b-1-5p, circFGFR2 overexpression vector and miR-29b-1-5p, empty overexpression vector and mimic NC, respectively). (C,D) The myotube area of chicken primary myoblasts after co-transfection with the listed nucleic acids (miR-133a-5p, circFGFR2 overexpression vector and miR-133a-5p, empty overexpression vector and mimic NC, respectively). (E,F) The myotubes area of chicken primary myoblasts after co-transfection with the listed nucleic acids (miR-29b-1-5p, circFGFR2 overexpression vector, and miR-29b-1-5p, empty overexpression vector and mimic NC, respectively). In all panels, results are expressed as the mean ± S.E.M. of three independent experiments, and statistical significance of differences between means were analyzed by one-way ANOVA followed by both least significant difference (LSD) and Duncan test through SPSS software. We considered p < 0.05 to be statistically significant. * p < 0.05; * p < 0.01. NC, negative control.

Discussion
In recent years, circular RNAs have been successfully identified in various cell types across different species [7,9]. They have shown features of dynamic and tissue-specific expression, which indicate a distinct function in diverse tissues [45,46]. CircFGFR2 is a highly expressed DGcircRNA among millions of circRNAs during embryonic muscle development according to our previous circRNA sequencing results [22], which indicates that it has a potential effect in regulating skeletal muscle development. Here we primarily confirmed that circFGFR2 has a crucial function on skeletal muscle development. In both chicken primary myoblast and QM-7 cell, cell cycle analysis demonstrated that overexpression of circFGFR2 could significantly increase the cell numbers in S phase and reduce the cell numbers in G0/G1 phase, while downregulation of circFGFR2 showed the opposite effects. In addition, EdU incorporation assay confirmed that circFGFR2 elevated the cell proliferation rate as shown by overexpression and knockdown of circFGFR2. The results strongly supported that circFGFR2 could promote skeletal muscle cell proliferation. Skeletal myogenesis comes after cell cycle termination, which is coordinated by various regulatory transcription factors, including MYOD, MYOG, myogenic factor 5 (Mrf5), the muscle regulatory factor 4 (Mrf4), and myocyte enhancer factor-2 (Mef2) families [47,48]. MYOD and MYOG can regulate most myogenesis-related genes thus facilitating myoblast differentiation into myotubes [49,50]. MyHC is a differentiation marker gene of muscle and forms the backbone of the sarcomere thick filaments [51]. The circFGFR2 exerts a function in skeletal muscle cell proliferation, we detected whether circFGFR2 was also involved in skeletal muscle cell differentiation by monitoring the impact of circFGFR2 on the expression of MYOD and MYOG. As expected, circFGFR2 could promote the expression of MYOD and MYOG. MyHC immunofluorescence suggested that circFGFR2 accelerated the formation of myotubes, which confirmed another important role of circFGFR2 in skeletal muscle cell, i.e., it can facilitate myoblast differentiation.
Circular RNA is known to be a functional molecule transcribed from protein-encoding genes which contain MREs like other mRNAs or lncRNAs [52]. However, circular RNA was capable of escaping from degradation as it has no poly A tail could not be recognized by exonuclease compared with mRNAs or lncRNAs [5]. In addition, the expression level of some circular RNAs were not lower than their linear mRNAs [53]. Based on that advantage, they are efficient to act as ceRNA, which are enriched for stable miRNA binding sites and regulate the activity of miRNA. Bioinformatics technology is universally applicable for the analysis of the binding relationship of ceRNA and miRNA [54]. In this study, using the bioinformatics program RNAhybrid, we found that circFGFR2 had two possible binding sites for miR-133a-5p and one site for miR-29b-1-5p. Subsequently, we confirmed that miR-133a-5p and miR-29b-1-5p were actually combined with the predicated sites of circFGFR2 but not with FGFR2 mRNA as indicated by two dual-luciferase reporter assays. Biotin-coupled miRNA pull down is an efficient method to verify the combined relationship between circular RNA and miRNA [18,19,55]. In this study, biotin-miR-133a-5p and biotin-miR-29b-1-5p were efficient in enriching circFGFR2, and overexpression of circFGFR2 significantly inhibits the expression of miR-133a-5p and miR-29b-1-5p which confirm the interacted relationship between circFGFR2 and miR-133a-5p/miR-29b-1-5p. miR-133a-5p and miR-29b-1-5p belong to two miRNA families, miR-133 and miR-29, respectively. These two families have been well-studied miRNAs, and found to be involved in skeletal muscle cell proliferation and differentiation [27,38,56]. In mouse C2C12 cell line, miR-133 which contain a seed sequence of "UUGGUCC" could promote myoblast differentiation and inhibit cell proliferation, and miR-29 which contains a seed sequence of "AGCACCA" could reduce proliferation and facilitate differentiation [28,56]. The roles of miR-133a-5p and miR-29b-1-5p in avian skeletal muscle development still remain unclear. Here we first reported that miR-133a-5p and miR-29b-1-5p could repress the proliferation and differentiation of skeletal muscle cell. The roles of these two miRNAs were different from the studied miR-133 and miR-29 in mouse. We compared the sequence of miR-133a-5p and miR-29b-1-5p with other miR-133s and miR-29s in both chicken and mouse, and found that the mature sequences of gga-miR-133a-5p and gga-miR-29b-1-5p were different from the studied miR-133 and miR-29. Since the seed sequence was different, and miRNA exerts function by targeting the 3 -UTR of their target genes, it is possibly that the function of gga-miR-133a-5p and gga-miR-29b-1-5p was different from the miR-133 and miR-29 which have been studied in mouse. On the other hand, the roles of gga-miR-133a-5p or gga-miR-29b-1-5p were opposite to the effect of circFGFR2 in myoblast. It is therefore reasonable that circFGFR2 could act as a molecular sponge for miR-133a-5p and miR-29b-1-5p. To confirm this, we further performed rescue experiments and found that circFGFR2 eliminated the inhibition effect of miR-133a-5p and miR-29b-1-5p on myoblast proliferation and differentiation. Considering all of this, we declared that circFGFR2 regulates skeletal muscle cell proliferation and differentiation by inhibiting the expression and activity of miR-133a-5p and miR-29b-1-5p in poultry.

Conclusions
In conclusion, we found that a novel circular RNA of circFGFR2, generated by the FGFR2 gene, could regulate myoblast proliferation and differentiation by acting as a sponge of miR-133a-5p and miR-29b-1-5p in poultry.
Author Contributions: X.C. conceived the study, carried out all experiments, analyzed data, and wrote the paper. H.O. provided essential logistical help. Z.W. and B.C. participated in partial experiments. Q.N. conceived the study, and participated in its design and coordination.