FIGURE 1.

CREB1 is a direct target gene of miR-433. A, transient transfection assays. HeLa cells were cotransfected with pTarget (Control), pTarget-miR-433 (miR-433), and 3′-UTRs of the indicated genes. Luciferase activity (Luc act) was normalized to β-gal activity (gal). *, p < 0.01. B, left, sequence alignment of miR-433 and the 3′-UTR of CREB1 in humans and mice. The seed match region is indicated in red. mut, mutant. Middle and right, Western blotting was performed to detect endogenous hCREB1 (middle) and mCREB1 (right) protein expression in human HeLa and mouse Hepa1 cells that were transfected with pTarget-miR-433 (2 μg) or control pTarget (2 μg). C, transient transfection assays. HeLa cells were cotransfected with the wild-type (wt; 25 ng) or mutant (mut; 25 ng) hCREB1 3′-UTR, pTarget-miR-433 (150 ng), or control pTarget (150 ng). The luciferase activity assay was performed, and the activity was normalized to β-gal activity. D, Western blotting was performed to determine endogenous hCREB1 proteins in Huh7 and HepG2 cells that were transfected with pTarget-miR-433 (2 μg) or control pTarget (2 μg). E, Western blotting was performed to determine endogenous hCREB1 and phosphorylated hCREB1 (p-hCREB1) proteins in MHCC97H cells that were transfected with pTarget, pTarget-miR-433, anti-miR control (anti-con), or anti-miR-433. β-Actin served as an internal control. F, qPCR analysis of miR-433 expression. G, left, Western blotting (WB) was performed to determine endogenous mCREB1 and phosphorylated mCREB1 (p-mCREB1) proteins in the livers of wild-type (WT) and Shp^−/− (non-tumor (NT)) mice and in Shp^−/− liver tumors (T). Right, qPCR analysis of miR-433 expression and Creb1 mRNA. Error bars represent S.E.