Figure 7

MYOD regulates miR-223 transcription by binding to the E-box 1 region. (a) The location of the two E-boxes in the 1932-kb upstream region of the gga-miR-223 gene TSS. (b) Identification of the core region in the gga-miR-223 gene promoter by luciferase reporter assays. (c) ChIP-qPCR analysis using anti-MYOG, anti-MYOD or chicken IgG antibodies, and the values showed that MYOD could bind to the R1 region of the chicken gga-miR-223 gene in myoblasts. A region from the GAPDH gene was amplified as a NC to verify the specificity of the enrichment. (d) MYOD overexpression promoted the relative luciferase activity of the pGL3-1932 reporter in DF-1 cell. (e) MYOD overexpression upregulated miR-223 expression in DF-1 cell. (f) Relative expressions of miR-223 and its downstream genes after transfection of si-MYOD in chicken primary myoblast. (g) CCK-8 assay indicated that MYOG loss-of-function significantly reduced myoblast proliferation in chicken primary myoblast. Results are shown as the mean±S.E.M. of three independent experiments. Independent sample t-test was used to analysis the statistical differences between groups. *P<0.05; **P<0.01