FIGURE 5.

HMGA2 is a novel target of miR-33a in mouse and human lung cancer cells. A, diagram depicting the 3′-UTR of the human HMGA2 gene. Locations of predicted miRNA binding sites for let-7 (black), miR-33a (gray), and miR-32 (white) are marked with arrows, and seed site locations are listed below the arrows. B, NCI-H1299 cells were transfected with the HMGA2 3′-UTR reporter and either a scrambled control oligonucleotide (Scr) or an miR-33a mimic (miR-33a). Luciferase activity was read 48 h post-transfection (n = 3). C, HMGA2 3′-UTR is not a target for miR-32. NCI-H1299 cells were cotransfected with an miR-32 mimic or a negative control oligonucleotide (NC). At the same time, the transfections include a wild-type HMGA2 3′-UTR reporter construct (Wt) or an HMGA2 3′-UTR reporter with a deleted miR-32 binding site (ΔmiR-32). After 48 h, Renilla and firefly luciferase activities were assayed. Although miR-32 resulted in a slight inhibition of the HMGA2 reporter, deletion of the sole predicted miR-32 binding site did not cause a derepression of the HMGA2 reporter. RLU, relative luminescence units. D, quantification of endogenous HMGA2 mRNA after transfection of NCI-H1299 cells with a scrambled control oligonucleotide or an miR-33a mimic. HGMA2 expression levels were normalized to GAPDH (n = 3). E, Western blot analysis confirmed a reduced expression of HMGA2 in the human NCI-H1299 cells transfected with miR-33a mimic. F, the Hmga2 expression was knocked down in the murine 394T4-shTtf-1 cells transfected with an miR-33a mimic. *, p < 0.05; **, p < 0.01; ***, p < 0.001.