NF1 is a target gene of miR-103a-3. (A) Sequence alignment of miR-103a-3p with the 3′-UTR of NF1 gene. The red labeled nucleotides represent point mutations in the 3′-UTR sequence. miR-103a-3p negatively regulates NF1 expression at both the (B) protein and (C) mRNA levels. A549 cells were transfected with miR-103a-3p mimic or inhibitor. After 72 h of transfection, cells were subjected to reverse transcription-quantitative PCR and western blot analysis. (D) 3′-UTR luciferase reporter assay for NF1. Each miR-103a-3p binding site of NF1 3′-UTR was cloned into luciferase reporter constructs and transfected into A549 cells with miR-103a-3p mimic or control oligonucleotides. The luciferase assay results revealed that the signal was significantly decreased following transfection with the third site of the NF1 3′-UTR. (E) The third NF1 3′-UTR binding site inhibits the expression of firefly luciferin. The indicated luciferin plasmid was transfected with miR-103a-3p mimic or control oligonucleotides. *P<0.05 and **P<0.01 with comparisons indicated by lines. NF1, neurofibromin 1; miR, microRNA; 3′-UTR, 3′-untranslated region; NC, negative control; mut, mutant.