miRNAs are a class of non-coding small RNA molecules with a length of 18–24 nucleotides. They can direct the regulation of the expression levels of certain genes, control cell growth and development, and determine tissue type during cell differentiation by reducing the stability of target genes or inhibiting translation levels to influence cell differentiation, proliferation, and apoptosis. In animal cells, miRNAs, by interacting with a specific sequence of target gene mRNA, inhibit protein synthesis or induce mRNA degradation and post-transcriptionally negatively regulate the expression of target genes [39, 40].
In this study, high-throughput microarray technology was used to analyze miRNA and mRNA expression profiles in the skeletal muscles of 14-day-old embryos and 7-week-old dwarf and normal chickens to identify miRNAs related to skeletal muscle growth and development. In chickens, 499 pre-miRNAs and 544 mature-miRNAs have been reported [41, 42]. In the present study, 124 and 125 miRNAs were detected in the skeletal muscles of 14-day-old embryos from dwarf and normal chickens, respectively. We also detected 115 and 116 miRNAs in the skeletal muscles of 7-week-old dwarf and normal chickens, respectively. Such tissue-specific miRNA expression has been reported in a few previous studies [41, 43–46]. Our data also showed that there is significantly different expression for only a few miRNAs at the same developmental stages in dwarf and normal chickens. However, the expression profiles of a greater number of miRNAs at different developmental stages for dwarf and normal chickens were significantly different. When comparing 7-week-old chickens with 14-day-old embryos, more down-regulated miRNAs than up-regulated miRNAs were detected. This would suggest that down-regulated expression of miRNAs is favorable for muscle growth and development in chickens at 7 weeks. In 7-week-old chickens, as compared with 14-day-old embryos, the expression of let-7b, miR-30a-5p, miR-30b, miR-99a and miR-133b was significantly up-regulated, but miR-16c, miR-92, miR-106, miR-203, miR-451 and miR-454 were significantly down-regulated in both dwarf and normal chickens. Considering that GH and GHR play important roles in chicken growth and development, we focused on observing the miRNAs involved in the regulation of their expression.
Four miRNAs, let-7b, miR-16, miR-16c, and miR-181b, are involved in the regulation of GHR. BLAST analysis confirmed that the target location of let-7b was in the deleted region of GHR 3' UTR. But the target locations of miR-16, miR-16c and miR-181b were distant from the deleted region. We concluded that the regulation of let-7b could be critical to GHR expression. As the deletion mutation in dwarf chickens results in the loss of the ability of let-7b to pair with sequences in its target gene, the regulation of growth and development is affected.
Skeletal muscle growth and development in chickens is fastest at the 7-week-old stage; conversely, the growth and development of skeletal muscle during the embryonic period is relatively slow. Comparing dwarf with normal chickens, there was significantly different mRNA expression for 173 genes in the 7-week-old chickens; however, there was significantly different mRNA expression for only 55 genes in the 14-day-old embryos. For both 14-day-old embryos and 7-week-old chickens, mRNA expression of GHR was up-regulated 3.57- and 5.26-fold, respectively, in dwarf chickens compared with normal chickens. It is suggested that the mRNA corresponding to GHR was inhibited in normal chickens as reported by Wu et al..
Comparing the different developmental stages (Table 2), expression levels of let-7b were significantly up-regulated in both dwarf and normal chickens. GHR expression was up-regulated in dwarf chickens and down-regulated in normal chickens, suggesting that let-7b could play a significant role in inhibiting GHR expression, further promoting the growth and development of skeletal muscle.
The let-7b miRNA is a member of the let-7 family. Deletion, or mutation of the function of let-7, may lead to defects in the transformation of nematodes from their larval to adult stage . Methylation, post-translation modifications, and Lin28 genes regulate the let-7 family. Additionally, the family regulates RAS
CDK2, and other target genes that influence a variety of biological phenomena and physiological processes, especially during biological development, cell proliferation and differentiation, and tumor suppression. There are 13 homologs in the let-7 family in the human genome, clustered into eight sites . These gene clusters are located at fragile sites related to lung cancer, breast cancer, urothelial cancer, and cervical cancer, suggesting that they may act as tumor suppressors. Previous studies of the let-7 family have largely focused on tumor suppression mechanisms , and studies investigating the family’s role in growth and development are rare.
The signaling pathway related to the regulation of the growth and development of skeletal muscle by let-7b-mediated GHR has not been previously reported. GH plays important roles in regulating animal growth and development, and its action on tissues and cells is mediated through its binding with GHR on the cell surface. GHR is activated upon binding of GH to stimulate the growth and metabolism of muscle, bone, and cartilage cells [3, 4]. GH also regulates chicken growth through close binding to its receptor and activating expression of IGF. The amount and action of GHR has direct effect on GH physiological function. In the present study, the mRNA expression of GHR was significantly up-regulated in dwarf chickens compared with normal chickens. The up-regulated mRNA expression of GHR retarded chicken growth, probably owing to a certain compensation mechanism . Our data showed that the retarded growth of dwarf chickens was caused by a deletion in the GHR 3′ UTR inducing loss of the let-7b target site. Through signaling pathway analysis, we found that let-7b regulates the expression of GHR, and further regulates SOCS3 through the JAK-STAT signaling pathway. Studies have shown that SOCS3 can inhibit excessive cell growth and induce apoptosis as part of maintaining cell stability [47, 48]. SOCS3 regulates the growth and development of chickens through three adipocytokine signaling pathways. (1) SOCS3 inhibits the tyrosine in IRS1. By inhibiting the phosphorylation of IRS1, SOCS3 inhibits insulin signaling, thus affecting growth. (2) SOCS3 inhibits LEPR, and up-regulated SOCS3 expression in dwarf chickens may affect the function of leptin. Leptin has a wide range of biological effects, with an important role in the metabolic regulation center of the hypothalamus, which plays a role in suppressing appetite, reducing energy intake, increasing energy expenditure and inhibiting fat synthesis. This helps explain why dwarf chickens are more likely to be obese . (3) SOCS3 inhibits JAK; the JAK-STAT signaling pathway is a recently discovered signal transduction pathway stimulated by cytokines, and is involved in cell proliferation, differentiation, apoptosis, immune regulation, and many other important biological processes.
In the present study, little change in expression of let-7b between dwarf and normal chickens was observed; however, growth was retarded in dwarf chickens. In dwarf chickens, let-7b could not inhibit the expression of GHR. This allows for the gene to be up-regulated as let-7b is unable to pair with GHR gene as its target site is deleted. Data from the microarray and qPCR analyses supported that the above pathway, indicating that the expression of GHR is inhibited by let-7b, and the expression of SOCS3 gene is regulated and stimulated by GHR. Further qPCR data supported that SOCS3 could inhibit the expression of IRS1, LEPR and JAK. The expression of IRS1, LEPR and JAK was significantly down-regulated, expression of genes regulating skeletal muscle growth (MYOD1, MyoG and Myf5) and the insulin pathway (IGF1 and IGF2BP3) were also down-regulated significantly.