Figure 3

NKX2-1 binds to miRNA’s regulatory regions and regulates their transcriptional activity. (A) Scheme representing 1 kb of the 5′ flanking region of each miRNA. The core of the NKX2-1 consensus binding site (CAAG/CTTG) is represented as a green circle. (B) Chromatin immunoprecipitation assays using NKX2-1 antibody or IgG control followed by qPCR analysis of the 5′ flanking regions of miR-200c, miR-221 and miR-1195. Data represents percentage of the input in log scale. (*) p ≤ 0.05. (C) Binding of NKX2-1 to miR-200c 5′ flanking region in E11.5 and E19.5 mouse lung previously determined by ChIP-chip analysis [12]. Data represents binding signal (y axis) for each probe on the promoter tiling array (x axis) corresponding to the 5′ flanking region of the pre-miR-200c. miR-221 and miR-1195 5′ flanking regions were not represented in the array. Red, E11.5; blue, E19.5; black line, transcript. N = 3 (D) Promoter-luciferase analysis of the transcriptional activity of the 1 kb 5′ flanking regions of different miRNAs in MLE15 transduced with NKx2-1 shRNA or control. Data represents Firefly luciferase normalized to Renilla luciferase. Values are relative to the 0-Luciferase (pGL3) control vector in each cell line. (*) p < 0.05 in 0-Luc vs. miR-200c –Luc; (**) p < 0.05 in miR-200c-Luc + scrambled vs. miR-200c.