Figure 3. IKBKB is a direct target of miR-16 in breast cancer cells.

A. Schematic description of interaction between IKBKB and miR-16. B. MDA-MB-231 and MCF-7 were transfected with 50 nM miR-NC or miR-16 mimics. Cell lysates were prepared for western blotting with an antibody against IKBKB 48 h after transfection. β-actin was used as a loading control. The gray density was quantified using the ImageJ software and normalized to β-actin. C. 0, 25, 50, 100 nM miR-16 mimics were transfected into MDA-MB-231 and MCF-7 cells, and then the expression of IKBKB was detected by western blotting assay as (B) performed. D. MDA-MB-231 and MCF7 were transfected with 100 nM anti-miR-NC or miR-16 inhibitor, and then similar experiments as in (B) were performed. E. MDA-MB-231 and MCF-7 were transfected with 50 nM miR-NC or miR-16 mimics. Total cellular RNA was collected and IKBKB mRNA levels were measured by qRT-PCR 24 h after transfection. The relative mRNA levels of IKBKB were shown in the bar diagram from three independent experiments and normalized to GAPDH. F. MDA-MB-231 and MCF-7 were transfected with 100 nM anti-miR-NC or miR-16 inhibitor, and similar experiments as in (E) were performed and analyzed. G. MDA-MB-231 and MCF-7 were co-transfected with pMIR-IKBKB-3′UTR-wt or pMIR-IKBKB-3′UTR-mut and 50 nM miR-16 mimics using Lipofectamine 3000 reagent. Luciferase activity was measured 48 h after transfection. The pRL-TK vector was used as an internal control. The results were expressed as relative luciferase activity (firefly luc/renilla luc). H. MDA-MB-231 and MCF-7 cells were co-transfected with pMIR-IKBKB-3′UTR-wt or pMIR-IKBKB-3′UTR-mut and 100 nM miR-16 inhibitor using Lipofectamine 3000 reagent, and similar experiments as in (G) were performed and analyzed. Columns, means of three independent experiments; bars, S.E. *, p<0.05, **, p<0.01.