Abstract
The sex-linked dwarf chicken is caused by the mutation of growth hormone receptor (GHR) gene and characterized by shorter shanks, lower body weight, smaller muscle fiber diameter and fewer muscle fiber number. However, the precise regulatory pathways that lead to the inhibition of skeletal muscle growth in dwarf chickens still remain unclear. Here we found a let-7b mediated pathway might play important role in the regulation of dwarf chicken skeletal muscle growth. Let-7b has higher expression in the skeletal muscle of dwarf chicken than in normal chicken, and the expression of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which is a translational activator of IGF2, showed opposite expression trend to let-7b. In vitro cellular assays validated that let-7b directly inhibits IGF2BP3 expression through binding to its 3′UTR region, and the protein level but not mRNA level of IGF2 would be reduced in let-7b overexpressed chicken myoblast. Let-7b can inhibit cell proliferation and induce cell cycle arrest in chicken myoblast through let-7b-IGF2BP3-IGF2 signaling pathway. Additionally, let-7b can also regulate skeletal muscle growth through let-7b-GHR-GHR downstream genes pathway, but this pathway is non-existent in dwarf chicken because of the deletion mutation of GHR 3′UTR. Notably, as the loss binding site of GHR for let-7b, let-7b has enhanced its binding and inhibition on IGF2BP3 in dwarf myoblast, suggesting that the miRNA can balance its inhibiting effect through dynamic regulate its binding to target genes. Collectively, these results not only indicate that let-7b can inhibit skeletal muscle growth through let-7b-IGF2BP3-IGF2 signaling pathway, but also show that let-7b regulates myoblast proliferation by inhibiting IGF2BP3 expression in dwarf and normal chickens.
Highlights
MicroRNAs are a class of small conserved noncoding RNA that negatively regulate target gene expression through complementarily binding to the 3′ untranslated region (3′UTR) of mRNAs
Our previous microarray data showed that let-7b expression is significantly higher in the skeletal muscle of 7 week (w) dwarf chickens than in 7 w normal chickens (Figure 1A), and IGF2BP3 mRNA expression is significantly lower in the skeletal muscle of 7 week (w) dwarf chickens than in 7 w normal chickens (Figure 2A)
The Western blot analysis showed that the expression of IGF2BP3 protein in dwarf chicken was significantly lower compared to that in normal chicken (Figures 1E,F)
Summary
MicroRNAs (miRNAs) are a class of small conserved noncoding RNA that negatively regulate target gene expression through complementarily binding to the 3′ untranslated region (3′UTR) of mRNAs. Its roles in skeletal muscle development still remain unclear. Let-7b, a member of the let-7 miRNA family, has been found to inhibit chicken growth by repressing growth hormone receptor (GHR) gene expression (Lin et al, 2012). It can regulate fat synthesis and cell proliferation through GHR-mediated signaling pathways (Lin et al, 2012). Our previous study indicated that let-7b might be involved in the regulation of skeletal muscle development (Luo et al, 2016). Its precise roles and regulatory pathways in skeletal muscle still remain to be explored
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