Figure 3. NRF1 bound to and activated the pre-miR-140 promoter. (A) The expression levels of pre-miR-140 was detected by qPCR in MDA-MB-231 cells treated with simvastatin(1-3µM) for 24h. (B) The location of NRF1-binding sites in the pre-miR-140 proximal promoter region was predicted by the JASPAR CORE database. (C) The relative miRNA expression levels of pre-miR-140 in MDA-MB-231 cells transfected with NRF1 over-expressing plasmid compared with empty plasmid. (D) Sequential deletion and mutation analyses identified NRF1-responsive regions in the pre-miR-140 proximal promoter region. pGL3-P2, pGL3-P3 and pGL3-P4 represented the deletion, and pGL3-M1, pGL3-M2 and pGL3-M1/2 represented the mutation. Serially truncated and mutated pre-miR-140 promoter vectors were co-transfected with NRF1 over-expressing plasmid or empty plasmid into MDA-MB-231 cells, and the relative luciferase activities were determined. (E) Effect of simvastatin on pre-miR-140 promoter driven luciferase activity. MDA-MB-231 cells were transfected with pGL3-P1 or pGL3-M1/2 plasmids, along with 3µM simvastatin or DMSO. (F) MDA-MB-231 cells were cultured and treated with 3µM simvastatin or NC(DMSO) for 24h and NRF1 protein was measured by Western blot. β-actin served as a control. The p-values were calculated using standard Student t-tests. Error bars represent mean±SEM of three individual experiments. ** P ≤ 0.01.