Figure 4

NF-κB1 is a direct target of miR-9. (A) The predicted miR-9 binding site on NF-κB1 mRNA 3'UTR is shown. (B) Deletion mutation at the miR-9 "seed region" binding site on the NF-κB1 mRNA 3'UTR is shown. Four nucleotides were deleted on the mutated 3'UTR. (C) MGC803 cells were transfected with the wild type of EGFP- NF-κB1 3'UTR (Wt. UTR) or mutated EGFP- NF-κB1 3'UTR (Mut. UTR) reporter vector as well as pri-miR-9 or control vector. Although pri-miR-9 suppressed the EGFP fluorescence intensity of EGFP- NF-κB1 3'UTR, mutation of the miR-9 binding site abolished the effect of miR-9 on the EGFP fluorescent intensity. (D) MGC803 cells were transfected with pri-miR-9 or control vector, and the NF-κB1 protein (P105 and P50) expression level was evaluated by Western blot. GAPDH protein was regarded as endogenous normalizer and the relative NF-κB1 protein quantity is shown. (E) MGC803 cells were transfected with pri-miR-9 or control vector, and expression of NF-κB1 mRNA was measured by quantitative RT-PCR. β-actin mRNA was regarded as an endogenous normalizer and the relative NF-κB1 mRNA expression level is shown. (*P < 0.05)