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.

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.

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.

The kruppel-like factor (KLF) gene family is a group of transcription factors containing highly conserved zinc-finger motifs, which play a crucial role in cell proliferation and differentiation. Chicken has been widely used as a model animal for analyzing gene function, however, little is known about the function of the KLF gene family in chickens. In this study, we performed genome-wide studies of chicken KLF genes and analyzed their biological and expression characteristics. We identified 13 KLF genes from chickens. Our phylogenetic, motif, and conserved domain analyses indicate that the KLF gene family has remained conserved through evolution. Synteny analysis showed the collinear relationship among KLFs, which indicated that they had related biomolecular functions. Interaction network analysis revealed that KLFs worked with 20 genes in biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that KLF2 was involved in Apelin and Forkhead Box O (FOXO) signaling pathways. Moreover, qPCR showed that 13 KLF genes were expressed in the nine selected tissues and displayed various gene expression patterns in chickens. RNA-seq showed that KLF3 and KLF10 genes were differentially expressed in the normal and high-fat diet fed groups, and KLF4, KLF5, KLF6, KLF7, KLF9, KLF12, and KLF13 genes were differentially expressed between undifferentiated and differentiated chicken preadipocytes. Besides, RNA-seq also showed that KLF genes displayed different expression patterns in muscle at 11 and 16 embryonic days old, and in 1-day-old chickens. These results indicated that the KLF genes were involved in the development of muscle and fat in chickens. Our findings provide some valuable reference points for the subsequent study of the function of KLF genes.

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.

Egg production in chickens is a quantitative trait. The aim of this study was to investigate the effect of promoter methylation of the Zona pellucida 2 (ZP2) gene on egg production. Real-time fluorescence quantification showed that the expression of the ZP2 gene in the ovaries of 300-day-old Jinghai yellow chickens in the high-laying group was significantly higher than that in the low-laying group (p < 0.01). A series of deletion fragments of the ZP2 gene promoter in Jinghai yellow chickens had different promoter activities in DF-1 cells, and the core region of the ZP2 gene promoter was found to be between −1552 and −1348. Four CpG islands in the promoter region of the ZP2 gene were detected by software prediction. The overall degree of methylation of the ZP2-1 amplified fragment was negatively correlated with mRNA expression to some extent (R = −0.197); the overall degree of methylation of the ZP2-2 amplified fragment was also negatively correlated with mRNA expression to some extent (R = −0.264), in which the methylation of methylcytosine (mC)-9, mC-20, and mC-21 sites was significantly negatively correlated with mRNA expression (p < 0.05). In addition, the mC-20 and mC-21 sites are located on the Sp1 transcription factor binding site, and it is speculated that these two sites may be the main sites for regulating transcription. In summary, the methylation sites mC-20 and mC-21 of the ZP2 gene may inhibit the binding of Sp1 and DNA, affect the transcription of the ZP2 gene, and then affect the number of eggs produced by the Jinghai yellow chickens.