As a source of animal protein, chicken occupies an important share in the market of meat and it has provided the human diet across the globe with large amounts of protein . Improving the quantity and quality of chicken meat has always been the goal of researchers in the past few decades. Many genes closely related to the development of skeletal muscle have also been revealed, such as MSTN , MRFs , MEF2 , etc. In recent years, the research of skeletal muscle has gradually transferred to non-coding RNAs. CircRNA, as one of the major non-coding RNAs, has rich and extremely important functions on a variety of biological processes, including skeletal muscle development [19, 25].
In the study, we collected chicken leg muscles of different embryonic ages for transcriptome sequencing and identified 245 and 440 DE circRNAs in comparison group F14vsF20 and S14vsS20. GO enrichment for the host genes of DE circRNAs was performed in F14vsF20 and S14vsS20 respectively, and some BP terms related to skeletal muscle have been identified including myofibril assembly, actomyosin structure organization and protein autophosphorylation. KEGG enrichment analysis showed that two and seven pathways were significantly enriched in F14vsF20 and S14vsS20 respectively (Fig. 4) and they were all closely related to skeletal muscle development. Fig. 4 showed that red nodes, MYH9, YBX3, IGF1R and EPB41L3 were simultaneously enriched in the significant pathways of the two comparison groups. Pink nodes, CTNNA3, AFDN and CREBBP, were only significantly enriched pathways of F14vsF20 group. All other genes (green nodes) were significantly enriched in pathways of S14vS20 and genes such as FGFR2, ACTN2, COL1A2, CDC42, DOCK1 and MYL3 are closely related to skeletal muscle growth.
The hierarchy of skeletal muscles runs from the top to bottom as muscles, fibre bundles, fibres, myofibrils and sarcomeres . In cross-striated muscle, sarcomeres contained Myosin II (thick) filaments and actin (thin) filaments , which were both involved in the regulation of the contraction of striated muscle . The myosin II subfamily is the largest class of myosins and includes skeletal, cardiac and smooth muscle myosins, as well as non-muscle myosin-2 (NM2) isoforms . The class Ⅱ myosin forms the filaments in muscle and non-muscle cells as a hexameric protein complex, consisting of two myosin heavy chain (MyHC) subunits, two regulatory light chains (RLCs) and two essential light chains (ELCs) . Together, the ELCs and the RLCs are named myosin light chains (MLCs), which are important regulators of actin-myosin interactions. Vertebrates hold three paralog genes (MYH9, MYH10 and MYH14), which are located in different chromosomes and encode three NMHC2 isoforms (NMHC2A, 2B and 2C, respectively) . Before muscle-specific myosin II (MM II) is organized in mature myofibrils to carry out that role, however, non-muscle isoforms of myosin II (NM II) are present in premyofibrils and nascent myofibrils that lead to mature myofibril formation . During embryonic development, from 14 to 20 embryonic ages, we found that MYH9 was found as the host gene of DE circRNAs in the significantly enriched pathways of both F14vsF20 and S14vsS20 (Fig. 5). The results suggest that novel_circ_0002968 and novel_circ_0002969, and the host gene MYH9 may play an important role in myofibril formation. In addition, MYL3 as the host gene of DE novel_circ_0004825, was found to be significantly enriched in group S14vsS20 and it was a member of the myosin light chains (MLCs). Study  has found that MYL3 could bind calcium ions, promote muscle development, and participate in the contraction of striated muscles.
YBX3 is a member of Y-box protein family, which contains a conserved cold shock domain (CSD), enabling these proteins to bind to single-stranded nucleic acids . DNA- and RNA-binding capabilities allow members of this family to perform diverse functions, including regulation of transcription, splicing, translation, and mRNA stability . Studies have confirmed that the post-translational phosphorylation of MSY3 (YBX3) by Akt kinase could rescue down-regulation of myogenin caused by binding of MSY3 in skeletal muscle [35, 36]. IGF1R is an IGF-1 receptor with a transmembrane location that activates PI3K/Akt signaling and possesses tyrosine kinase activity, and its expression is significant in terms of myoblast proliferation and normal muscle mass maintenance . IGF1R, as the host gene of novel_circ_0000087, was significantly enriched in adherens junction and focal adhesion pathways in both F14vsF20 and S14vsS20.
CTNNA3, AFDN and CREBBP were found to be significantly enriched in pathways of F14vsF20. The study of CTNNA3 and AFDN in skeletal muscle has not been reported, but there are many studies on them in tumor cells, mostly related to tumor cell proliferation and migration [38, 39]. The host gene of DE novel_circ_0008559 and novel_circ_0006094 were CTNNA3 and AFDN, respectively. We speculated that they may also regulate the growth of skeletal muscle cells. CREB-binding protein (CREBBP, or in short CBP) is a kind of lysine (K) acetyltransferases (KAT) belonging to the KAT3 family of proteins known to modify histones, as well as non-histone proteins, thereby regulating chromatin accessibility and transcription . Svensson et al.  revealed that CREBBP was required for the control and maintenance of contractile function and transcriptional homeostasis in skeletal muscle of adult mice.
In comparison group S12vsS20, we also identified many host genes of DE circRNAs related to skeletal muscle development. Fibroblast Growth Factor Receptor 2 (FGFR2) was a member of FGFRs, which are a family of receptor tyrosine kinases expressed on the cell membrane that play crucial roles in both developmental and adult cells . FGFR2 has been proved to regulate the myogenesis of skeletal muscle [43, 44]. ACTN2 was highly expressed in muscle where it acted as a major structural component of the contractile apparatus at the Z-line . Sharma et al.  found that col1a2+ muscle progenitor cells contributed to new myofibers in normal muscle growth and also during muscle regeneration. Integrin/FAK pathway is required for C2C12 myoblast differentiation by regulating the expression of MyoD and CDC42 . DOCK1 (also known as Dock180) is a prototypical member of a new family of atypical Rho GTPase activators and Laurin et al.  have identified DOCK1 and DOCK5 as critical regulators of the fusion step during primary myogenesis in mammals.
CircRNAs can serve as miRNA sponges to regulate the expression of mRNA by competing for endogenous RNAs (ceRNAs) mechanisms. Ouyang et al.  found that circSVIL could promote myoblast proliferation and differentiation by sponging miR-203 in chicken. Wei et al.  revealed that circFNDC3AL upregulated BCL9 expression to promote chicken skeletal muscle satellite cells proliferation and differentiation by binding to miR-204. KEGG and GO enrichment analyses were performed for miRNAs targeted by DE circRNAs as a sponge. Cysteine and methionine metabolism, RNA transport and Glycolysis / Gluconeogenesis were found in the top 20 pathways of the two groups. In the GO enrichment results, 55 biological process terms related to skeletal muscle were enriched including skeletal muscle cell differentiation, skeletal muscle satellite cell migration, skeletal muscle tissue regeneration, and so on. We selected the miRNAs in these items and constructed the miRNA-circRNA interaction network (Fig. 6).
CircRNAs generally have more than one miRNA binding site. For example, ciRS-7 contains over 60 target sites for miR-7 and can function as a miR-7 sponge and influence miR-7 target gene expression . In the study, novel_circ_0007646 had the highest degree both in the two groups, and the targeted miRNAs include miR-1625-5p, miR-1680-5p and miR-6570-3p pairs, etc. In addition, one miRNA may also bind to multiple circRNAs. The targeted miRNA with the highest connectivity in the two groups was miR-148b-5p, and the predicted results showed that it could bind to circRNAs such as novel_circ_0008913 and novel_circ_0008908 in the fast-growing group, and circ_0008906 and novel_circ_0008907 in the slow-growing group. In the S14vsS20, we also found that the nodes in miR-1759-3p-novel_circ_0001640 pairs both have a high degree, suggesting their important role in skeletal muscle development.