Figure 2.

IKZF1 directly regulates the expression of MDIG and MYC in HCC cells. (a) Various lengths of human MDIG gene promoter plasmids containing the luciferase gene were constructed and transfected into Huh7, MHCC-97H and MHCC-97L cells. After 24 h, luciferase activities were measured and normalized to the activity of the pGL3 basic vector (Vector) transfected into cells. The negative regulatory element is localized in the -206/-171 bp region. (b) The promoter activities in the -206 bp region were inhibited after transfection of Huh7, MHCC-97H and MHCC-97L cells with IKZF1. (c) Putative transcription factor IKZF1 binding sites on the MDIG promoter are indicated. (d) The plasmid pWPXL-IKZF1 and the MDIG luciferase reporter vectors (wild-type or mutant IKZF1 binding sites, in the -206/-171 bp region) were co-transfected into Huh7, MHCC-97H and MHCC-97L cells. The relative luciferase activities were determined by a reporter gene assay. IKZF1 rescued MDIG promoter activity when the predicted binding site was mutated. (e, f) CHIP analysis showed increased binding of IKZF1, respectively, with MDIG (e) and MYC (f) promoter region, rabbit IgG was used as negative control. Quantitative analysis of CHIP data was performed on the bound fraction, and IgG was used as a negative control. The graph of (a), (b), (c), (d), (e) and (f) are shown as mean±s.d. of n=3 independent experiments. Unpaired Student’s t-tests were used for statistical analysis (*P<0.05, **P<0.01, ***P<0.001)