Proliferation, differentiation, and apoptosis are three essential stages in cell development, and miRNAs can achieve extensive regulation of cellular developmental processes by repressing the expression of target genes. According to our previous RNA-seq results, miRNA-10a-5p was differentially expressed at different periods in chicken myoblasts, revealing a possible association with muscle development. In this study, we concluded that miRNA-10a-5p inhibited chicken myoblasts’ proliferation and differentiation and promoted chicken myoblasts’ apoptosis by directly targeting BCL6, a critical transcription factor involved in muscle development and regeneration. Overexpression of BCL6 significantly facilitated myoblasts’ proliferation and differentiation and suppressed myoblasts’ apoptosis. On the contrary, knockdown of BCL6 significantly repressed myoblasts’ proliferation and differentiation and induced myoblasts’ apoptosis. The results above suggest that miRNA-10a-5p plays a potential role in skeletal muscle growth, development and autophagy by targeting the BCL6 gene. We first revealed the functions of miRNA-10a-5p and BCL6 in the proliferation, differentiation, and apoptosis of chicken myoblasts.

The growth and development of skeletal muscle at embryonic stages are vital and it directly affects the growth performance of chickens. Long non-coding RNA (lncRNA) plays an important role in this process. In the experiment, we collected the leg muscles of fast- and slow-growing Bian chickens both at 14- and 20-day embryo ages (14E and 20E) for RNA-seq. Finally, 292 and 347 differentially expressed (DE) lncRNAs were identified in F14vsF20 and S14vsS20, and 1,295 and 1,560 DE mRNAs were also screened, respectively. Then we constructed lncRNA-mRNA networks for the two groups, respectively, and found that 6 of the top 10 lncRNAs ranked with degree are same. GO analysis showed that 12 of the top 20 terms were same in the two comparison groups and most of them were related to energy metabolisms, such as cellular respiration and aerobic respiration. KEGG enrichment revealed that up to 16 pathways of the top 20 in F14vsF20 were same as that of S14vsS20 and most of them were related to growth, including citrate cycle (TCA cycle) and oxidative phosphorylation. Further analysis showed that there were 602 and 102 same DE mRNAs and DE lncRNAs between the two comparison groups. We then identified 442 lncRNA-mRNA pairs, including 201 mRNAs and 32 lncRNAs. Protein-Protein Interactions (PPI) network was predicted for the 201 mRNAs and three core networks were obtained using the plug-in MCODE of Cytoscape. Then the function of genes in the three core networks was further analyzed with ClueGo and they were mainly enriched in six groups of biological processes. On this basis, combined with KEGG pathways and lncRNA-mRNA networks, we identified several candidate lncRNAs and mRNAs. Among them, lncRNAs mainly include TCONS_00061389, TCONS_00025495, TCONS_00017622, TCONS_00216258 and TCONS_00084223, and mRNAs include PLK1, BUB1, TTK, NDUFS7 NDUFAB1, PDHA1, CDK1, SDHA, ACO2 and MDH1. The results would provide a foundation for further experiments on the role of lncRNAs in the regulation of muscle development. And it could also contribute to further clarify the regulatory mechanism of chicken skeletal muscle.

Skeletal muscle growth has always been the focus of the broiler industry, and circRNAs play a significant role in this process. We collected leg muscles of slow- and fast-growing Bian chicken embryos in the study at 14 (S14 and F14) and 20 (S20 and F20) days for RNA-seq. Finally, 123 and 121 differentially expressed circRNAs (DECs) were identified in S14 vs. F14 and S20 vs. F20, respectively. GO enrichment analysis for DECs obtained important biological process (BP) terms including nicotinate nucleotide biosynthetic process, nicotinate nucleotide salvage, and NAD salvage in S20 vs. F20 and protein mannosylation in S14 vs. F14. KEGG pathway analysis showed Wnt signaling pathway, Tight junction, Ubiquitin mediated proteolysis, and Notch signaling pathway were enriched in the top 20. Based on the GO and KEGG analysis results, we found some significant host genes and circRNAs such as NAPRT and novel_circ_0004547, DVL1 and novel_circ_0003578, JAK2 and novel_circ_0010289, DERA and novel_circ_0003082, etc. Further analysis found 19 co-differentially expressed circRNAs between the two comparison groups. We next constructed a circRNA-miRNA network for them, and some candidate circRNA-miRNA pairs related to skeletal muscle were obtained, such as novel_circ_0002153-miR-12219-5p, novel_circ_0003578-miR-3064-3p, and novel_circ_0010661-miR-12260-3p. These results would help to reveal the mechanism for circRNAs in skeletal muscle and also provide some guidance for the breeding of broilers.

MicroRNAs (miRNAs) are widely involved in the growth and development of skeletal muscle through the negative regulation of target genes. In order to screen out the differentially expressed miRNAs (DEMs) associated with skeletal muscle development of Bian chickens at different embryonic ages, we used the leg muscles of fast-growing and slow-growing Bian chickens at the 14th and 20th embryonic ages (F14, F20, S14 and S20) for RNA-seq. A total of 836 known miRNAs were identified, and 121 novel miRNAs were predicted. In the F14 vs. F20 comparison group, 127 DEMs were screened, targeting a total of 2871 genes, with 61 miRNAs significantly upregulated and 66 miRNAs significantly downregulated. In the S14 vs. S20 comparison group, 131 DEMs were screened, targeting a total of 3236 genes, with 60 miRNAs significantly upregulated and 71 miRNAs significantly downregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the predicted target genes were significantly enriched in 706 GO terms and 6 KEGG pathways in the F14 vs. F20 group and 677 GO terms and 5 KEGG pathways in the S14 vs. S20 group. According to the interaction network analysis, we screened five coexpressed DEMs (gga-miR-146a-3p, gga-miR-2954, gga-miR-34a-5p, gga-miR-1625-5p and gga-miR-18b-3p) with the highest connectivity degree with predicted target genes between the two comparison groups, and five hub genes (HSPA5, PKM2, Notch1, Notch2 and RBPJ) related to muscle development were obtained as well. Subsequently, we further identified nine DEMs (gga-let-7g-3p, gga-miR-490-3p, gga-miR-6660-3p, gga-miR-12223-5p, novel-miR-327, gga-miR-18a-5p, gga-miR-18b-5p, gga-miR-34a-5p and gga-miR-1677-3p) with a targeting relationship to the hub genes, suggesting that they may play important roles in the muscle development of Bian chickens. This study reveals the miRNA differences in skeletal muscle development between 14- and 20-day embryos of Bian chickens from fast- and slow-growing groups and provides a miRNA database for further studies on the molecular mechanisms of the skeletal muscle development in Bian chickens.

The growth and development of skeletal muscle determine the productivity of pigeon meat production, and miRNA plays an important role in the growth and development of this type of muscle. However, there are few reports regarding miRNA regulating the growth and development of skeletal muscle in pigeons. To explore the function of miRNA in regulating the growth and development of pigeon skeletal muscle, we used RNA sequencing technology to study the transcriptome of pigeons at two embryonic stages (E8 and E13) and two growth stages (D1 and D10). A total of 32,527 mRNAs were identified in pigeon skeletal muscles, including 14,378 novel mRNAs and 18,149 known mRNAs. A total of 2362 miRNAs were identified, including 1758 known miRNAs and 624 novel miRNAs. In total, 839 differentially expressed miRNAs (DEmiRNAs) and 11,311 differentially expressed mRNAs (DEGs) were identified. STEM clustering analysis assigned DEmiRNAs to 20 profiles, of which 7 were significantly enriched (p-value < 0.05). These seven significantly enriched profiles can be classified into two categories. The first category represents DEmiRNAs continuously downregulated from the developmental stage to the growth stage of pigeon skeletal muscle, and the second category represents DEmiRNAs with low expression at the development and early growth stage, and significant upregulation at the high growth stage. We then constructed an miRNA–mRNA network based on target relationships between DEmiRNAs and DEGs belonging to the seven significantly enriched profiles. Based on the connectivity degree, 20 hub miRNAs responsible for pigeon skeletal muscle development and growth were identified, including cli-miR-20b-5p, miR-130-y, cli-miR-106-5p, cli-miR-181b-5p, miR-1-z, cli-miR-1a-3p, miR-23-y, cli-miR-30d-5p, miR-1-y, etc. The hub miRNAs involved in the miRNA–mRNA regulatory networks and their expression patterns during the development and growth of pigeon skeletal muscle were visualized. GO and KEGG enrichment analysis found potential biological processes and pathways related to muscle growth and development. Our findings expand the knowledge of miRNA expression in pigeons and provide a database for further investigation of the miRNA–mRNA regulatory mechanism underlying pigeon skeletal muscle development and growth.