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The goal of this study was to perform a systematic transcriptome-wide analysis of mRNA-miRNA interactions and to identify candidates involved in the interplay between miRNAs and mRNAs that regulate chicken muscle growth. We used our previously published mRNA (GSE72424) and miRNA (GSE62971) deep sequencing data from two-tailed samples [i.e., the highest (h) and lowest (l) body weights] of Recessive White Rock (WRR) and Xinghua (XH) chickens to conduct integrative analyses of the miRNA-mRNA interactions involved in chicken skeletal muscle growth. A total of 162, 15, 173, and 27 miRNA-mRNA pairs with negatively correlated expression patterns were identified in miRNA-mRNA networks constructed on the basis of the WRR h vs. XH h , WRR h vs. WRR l , WRR l vs. XH l , and XH h vs. XH l comparisons, respectively. Ingenuity Pathway Analysis revealed that gene networks identified for the WRR h vs. XH h contrast were associated with developmental disorders. Importantly, the WRR h vs. XH h contrast miRNA-mRNA network was enriched in IGF-1 signaling pathway genes, including FOXO3. A dual-luciferase reporter assay showed that FOXO3 was a target of miR-142-5p. Furthermore, miR-142-5p overexpression significantly decreased FOXO3 mRNA levels and promoted the expression of growth-related genes. These data demonstrated that miR-142-5p targets FOXO3 and promotes growth-related gene expression and regulates skeletal muscle growth in chicken. Comprehensive analysis facilitated the identification of miRNAs and target genes that might contribute to the regulation of skeletal muscle development. Our results provide new clues for understanding the molecular basis of chicken growth.

Skeletal muscle differentiation can be regulated by various transcription factors and non-coding RNAs. In our previous work, miR-223 is differentially expressed in the skeletal muscle of chicken with different growth rates, but its role, expression and action mechanism in muscle development still remains unknown. Here, we found that MYOD transcription factor can upregulate miR-223 expression by binding to an E-box region of the gga-miR-223 gene promoter during avian myoblast differentiation. IGF2 and ZEB1 are two target genes of miR-223. The target inhibition of miR-223 on IGF2 and ZEB1 are dynamic from proliferation to differentiation of myoblast. miR-223 inhibits IGF2 expression only in the proliferating myoblast, whereas it inhibits ZEB1 mainly in the differentiating myoblast. The inhibition of IGF2 by miR-223 resulted in the repression of myoblast proliferation. During myoblast differentiation, miR-223 would be upregulated owing to the promoting effect of MYOD, and the upregulation of miR-223 would inhibit ZEB1 to promote myoblast differentiation. These results not only demonstrated that the well-known muscle determination factor MYOD can promote myoblast differentiation by upregulate miR-223 transcription, but also identified that miR-223 can influence myoblast proliferation and differentiation by a dynamic manner regulates the expression of its target genes.

Myogenesis mainly involves several steps including myoblast proliferation, differentiation, apoptosis and fusion. Except for muscle specific regulators, few miRNAs were proved to coordinate this complex process. Here, we reported that miR-16 inhibited myoblast proliferation and promoted myoblast apoptosis by directly targeting Bcl2 and FOXO1. The expression level of miR-16 was significantly decreased in the hypertrophic pectoral muscle compared to the normal pectoral muscle in chicken. In vitro, elevating miR-16 significantly inhibited myoblast proliferation and promoted myoblast apoptosis, resulting in about 11.2% cells arrested in G1 phase and 12.3% apoptotic cells in the early stage. Bioinformatic and biochemical analyses revealed Bcl2 and FOXO1 as direct targets of miR-16. Consist to the effect of miR-16 on myogenesis, specific inhibition of Bcl2 or FOXO1 significantly suppressed myoblast proliferation and induced myoblast apoptosis, indicating that both Bcl2 and FOXO1 contributed to miR-16 regulatory function in myogenesis. Interestingly, FOXO1, as the core target, mediated multiple growth-related pathways induced by miR-16 such as PI3K-AKT-MAPK and PI3K-AKT-mTOR. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) revealed that 234 annotated genes bound by FOXO1 in the early-differentiated myoblasts, which were significantly enriched in myogenic proliferation, death and hypotrophy. Altogether, we proposed that miR-16 acted as a coordinated mediator to suppress myogenesis in avian through the control of myoblast proliferation and apoptosis. These findings have provided a novel mechanism whereby miR-16 represses Bcl2 and FOXO1 expression to maintain myoblast growth and skeletal muscle mass., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

Growth performance is an important economic trait in chicken. MicroRNAs (miRNAs) have been shown to play important roles in various biological processes, but their functions in chicken growth are not yet clear. To investigate the function of miRNAs in chicken growth, breast muscle tissues of the two-tail samples (highest and lowest body weight) from Recessive White Rock (WRR) and Xinghua Chickens (XH) were performed on high throughput small RNA deep sequencing. In this study, a total of 921 miRNAs were identified, including 733 known mature miRNAs and 188 novel miRNAs. There were 200, 279, 257 and 297 differentially expressed miRNAs in the comparisons of WRRh vs. WRRl, WRRh vs. XHh, WRRl vs. XHl, and XHh vs. XHl group, respectively. A total of 22 highly differentially expressed miRNAs (fold change > 2 or < 0.5; p-value < 0.05; q-value < 0.01), which also have abundant expression (read counts > 1000) were found in our comparisons. As far as two analyses (WRRh vs. WRRl, and XHh vs. XHl) are concerned, we found 80 common differentially expressed miRNAs, while 110 miRNAs were found in WRRh vs. XHh and WRRl vs. XHl. Furthermore, 26 common miRNAs were identified among all four comparisons. Four differentially expressed miRNAs (miR-223, miR-16, miR-205a and miR-222b-5p) were validated by quantitative real-time RT-PCR (qRT-PCR). Regulatory networks of interactions among miRNAs and their targets were constructed using integrative miRNA target-prediction and network-analysis. Growth hormone receptor (GHR) was confirmed as a target of miR-146b-3p by dual-luciferase assay and qPCR, indicating that miR-34c, miR-223, miR-146b-3p, miR-21 and miR-205a are key growth-related target genes in the network. These miRNAs are proposed as candidate miRNAs for future studies concerning miRNA-target function on regulation of chicken growth.

Avian leukosis virus subgroup J (ALV-J) can cause several different leukemia-like proliferative diseases in the hemopoietic system of chickens. Here, we investigated the transcriptome profiles and miRNA expression profiles of ALV-J-infected and uninfected chicken spleens to identify the genes and miRNAs related to ALV-J invasion. In total, 252 genes and 167 miRNAs were differentially expressed in ALV-J-infected spleens compared to control uninfected spleens. miR-23b expression was up-regulated in ALV-J-infected spleens compared with the control spleens, and transcriptome analysis revealed that the expression of interferon regulatory factor 1 (IRF1) was down-regulated in ALV-J-infected spleens compared to uninfected spleens. A dual-luciferase reporter assay showed that IRF1 was a direct target of miR-23b. miR-23b overexpression significantly (P = 0.0022) decreased IRF1 mRNA levels and repressed IRF1-3'-UTR reporter activity. In vitro experiments revealed that miR-23b overexpression strengthened ALV-J replication, whereas miR-23b loss of function inhibited ALV-J replication. IRF1 overexpression inhibited ALV-J replication, and IRF1 knockdown enhanced ALV-J replication. Moreover, IRF1 overexpression significantly (P = 0.0014) increased IFN-β expression. In conclusion, these results suggested that miR-23b may play an important role in ALV-J replication by targeting IRF1.