A Novel YY1-miR-1 Regulatory Circuit in Skeletal Myogenesis Revealed by Genome-Wide Prediction of YY1-miRNA Network
Figure 7
miR-1 inhibits YY1 expression through targeting its 3′UTR.
(A) Predicted target site of miR-1 in the 3′UTR of mouse YY1. (B) A wild type (WT) luciferase reporter plasmid was generated by fusing a ∼500 bp fragment of the YY1 3′UTR encompassing the miR-1 binding site downstream of the luciferase (Luc) reporter gene. The mutant plasmid was generated by mutating the miR-1 binding site from ACAUUCU to GGGCCUU. WT or Mutant reporter construct was transfected into C2C12 cells with indicated miRNA oligos and Renilla luciferase reporter plasmid. Luciferase activities were determined at 48 h post-transfection and normalized to Renilla readings. Relative Luciferase Unit (RLU) is shown with respect to wild type and NC transfection where luciferase activities were set to a value of 1. The data represent the average of three independent experiments ± S.D. (C) Upper: C2C12 myoblasts were transfected with either NC or miR-1 oligos. Total RNAs were used to detect YY1 expression level with GAPDH as normalization. Expression folds are shown with respect to negative control where YY1 levels were set to a value of 1. Quantitative values are represented as mean ± S.D. Lower: YY1 protein was then probed in extracts from cells 48 hr after transfection. Blots were stripped and reprobed for α-Tubulin as the loading control. The p value was determined by Student's T-test: *p<0.05, **p<0.01, ***p<0.001.