MicroRNAs-mediated Signaling Pathways Regulate Chicken Myoblast Proliferation And Differentiation

The growth and development of skeletal muscle are the most concerned areas in broiler production, and the improvement of chicken muscle growth rate and meat production capacity are two major aims of broiler practitioners. Sex-linked dwarf chicken(SLD) is caused by a mutation of the growth hormone receptor(GHR) gene with smaller size and fewer muscle mass, and it is an ideal model system for skeletal muscle development research. mi RNAs, Myomaker and c-Myc are all important regulators during skeletal muscle development, and mi RNAs are involved in the expression and function of Myomaker and cMyc. By comparing and analyzing the differentially expressed genes and mi RNAs between normal chicken and SLD chicken, we constructed a mi RNAs-m RNAs interaction network and found some important muscle development-related mi RNAs for further functional analysis and validation assays. Additionally, we also determined the roles of Myomaker and c-Myc in chicken myoblast, and found that some mi RNAs can be either involved in Myomaker and c-Myc expression or participate in the regulation of chicken myoblast proliferation and differentiation. Finally, by analyzing the above mi RNAs and their upstream and downstream genes, we constructed a mi RNAs-mediated signaling pathway that can regulate chicken myoblast proliferation and differentiation. The major results were showed as follow,The developmental process of skeletal muscle in SLD chicken is slower than that in normal chicken at day 14 embryo(E14), and the muscle fiber diameter of SLD chicken is smaller than that of normal chicken at 7 w of age(7w). The functions of the differentially expressed genes(DEGs) between these two breeds are mainly involved in muscle development. The expression of let-7b and mi R-128 and the activity of MAPK pathway are differentially expressed between SLD and normal chickens, suggesting their potential involvement in the GHR-deficient induced muscle mass loss. By analyzing the DEGs and differentially expressed mi RNAs(DEMs) between E14 and 7w in these two breeds, we constructed a muscle development-related mi RNAs-m RNAs interaction network, which might play important roles in chicken skeletal muscle development. mi R-203 and the mi R-17 family in this network are two candidate mi RNAs involved in chicken skeletal muscle development.During chicken skeletal muscle development, E10 to E16 are the active period for muscle cell proliferation and differentiation. mi R-203 up-regulated its expression during this period, and it can either repress chicken myoblast proliferation by inhibiting c-JUN and ΔNP63α expression, or repress chicken myoblast differentiation by inhibiting MEF2 C and TAp63α expression. The formation of skeletal muscle fibre would be finished and turned to the process of muscle fibre hypertrophy after E18, in which the expression of mi R-203 is disappearred. The role of mi R-203 during chicken skeletal muscle development can be compared to “the brake”. mi R-203 represses the cell proliferation and differentiation at the right time, and therefore makes the development of skeletal muscle from the stage of cell proliferation and differentiation to the stage of muscle fibre hypertrophy.The expression of mi R-20a-5p and mi R-20b-5p, which belong to mi R-17 family, is significantly higher at E14 than at 7w in both SLD and normal chickens. mi R-20a-5p and mi R-20b-5p can promote myoblast differentiation but inhibit myoblast proliferation by inhibiting E2F1 expression. The E2F1 transcription factor directly binded to the promoters of the mi R-17~96 and mi R-106a~363 cluster and promoted the transcription of pri-mi R-17~96 and pri-mi R-106a~363, and therefore influenced the expression of mi R-20a-5p and mi R-20b-5p.Myomaker overexpression promoted chicken myoblast fusion, and Myomaker knockdown inhibited myoblast fusion and migration. MYOD and MYOG can bind to the E-box 1 of chicken Myomaker promoter and promote the transcription of Myomaker. mi R-140-3p promoted chicken myoblast proliferation and inhibited myoblast differentiation. mi R-140-3p repressed myoblast fusion by inhibiting its target gene Myomaker.c-Myc promoted chicken myoblast proliferation and inhibited myoblast differentiation. The c-Myc ChIP-seq results performed in chicken proliferating myoblasts and differentiated myotubes showed that c-Myc can bind to the promoter regions of many mi RNAs and lnc RNAs. c-Myc regulate the expression of these target mi RNAs and lnc RNAs, therefore influence chicken myoblast proliferation and differentiation.Myoblast proliferation and differentiation can influence muscle fiber formation and determine muscle growth potential after birth. mi R-203, mi R-140-3p, mi R-20a-5p and mi R-20b-5p are involved in chicken myoblast proliferation and differentiation by directly inhibiting the muscle development-related genes. As an important gene transcription factor, c-Myc is involved in the transcription of many muscle development-related mi RNAs, including mi R-203, mi R-140-3p and mi R-20a-5p. The transcription factor, mi RNAs and their target genes in this study constructed an interaction network that can regulate chicken myoblast proliferation and differentiation. This network not only further complements the molecular regulatory pathways that are involved in poultry skeletal muscle development, but also helps to reveal the molecular regulation process of chicken growth, as well as provides theoretical help for improving the growth potential and growth rate of chicken muscle.