MiR-223 Regulates Poultry Myoblast Proliferation And Differentiation By Inhibiting IGF2 And ZEB1

MicroRNAs(miRNAs) are small non-coding RNA of 20–25 nucleotides that have critical roles in the regulation of gene expression at posttranscriptional level.Numerous studies showed that miRNAsare involved in a series of growth and metabolism of broilers such as proliferation and differentiation of nerve cells and muscle cells, cell migration, sexual maturation, immune response. Here we identified that miR-223 as key molecule can regulate growth performance of broilers according to RNA-seq results performed in breast muscle between White Rock(WRR) and Xinghua Chickens(XH). However, the funtion of miR-223 in chicken myoblast proliferation and differentiation still poorly understood. In this study, flow cytometry, cell counting assay, immunofluorescence, dual-luciferase, quantitative real-time PCR(q RT-PCR) and western blot were used to investigate the expression, regulation and biological function of miR-223. The main results were as follows:(1) miR-223 expression in the muscle from day 10 to day20 of chick embryo(E10 to E20) were detected by q RT-PCR. Results showed that miR-223 level was increased continuously and reached the peak at E13 in breast muscle but E14 in leg muscle, then gradually declined. Mir-223 expression in muscles at E13 to E17 was significantly higher than that at the rest of embryonic ages, indicated that miR-223 might be important for muscle growth during chick embryonic development.(2) Myoblasts differentiation experiment in vitrodemonstrated that miR-223 expression was significantlyincrease during myoblasts differentiation process(P< 0.01).(3) Mi R-223 overexpression or inhibition in poultry myoblasts were tested by Flow cytometry, cell counting assay, q RT-PCR and immunofluorescence.Results showed that the biological functions of miR-223 in poultry myoblast were repressed cell proliferation and enhanced cell differentiation.(4) IGF2 and ZEB1 were confirmed to be the target gene of miR-223 by dual-luciferase reperter gene assay. Further q RT-PCR and western blot showed that both IGF2 and ZEB1 in poultry myoblasts were negatively regulated by miR-223.(5) IGF2 can promote the proliferation and differentiation process of poultry myoblasts(P< 0.01). ZEB1 knockdown has no significant influence in myoblast proliferation but dramaticallypromoted myoblastdifferentiation(P< 0.01).In conclusion, these results demonstrated that IGF2 is the functional target of miR-223 for the suppression of poultry myoblasts proliferation, and ZEB1 is another miR-223 target gene which is responsible for the improvement of myoblast differentiation.