Fig 6. miR-302b inhibited the IGFBP2-mediated signaling pathway through NFIA regulation.

Overexpression of the miR-302b mimic reduced IGFBP2 mRNA (A) and protein levels (B). After overnight culture U87-MG cells were transfected with the indicated dose of the miR-302b mimic or 50 nM scrambled miRNA mimic (Ctrl) for 24 h, relative mRNA and protein levels of IGFBP2 were measured using real-time PCR and immunoblotting assays. Data are expressed as the mean ± SD of three independent experiments. After analyzing by one way ANOVA with Tukey—Kramer multiple comparison test, different letters above bars denote samples that were significantly different (p < 0.05) compared with control. (C) miR-302b dose dependently reduced IGFBP2 promoter activity. After overnight culture U87-MG cells were cotransfected with the indicated dose of the miR-302b mimic, 50 nM scrambled miRNA mimic, and 500 ng of pGL3-IGFBP2 prom for 24 h, luciferase activity was measured. Cells were also cotransfected with pNL1.1.TK[Nluc/TK] plasmids (5 ng), and the NanoLucR luciferase value was used as an internal control. (D) ChIP revealed that miR-302b could inhibit the binding of NFIA to the IGFBP2 promoter. Data are expressed as the mean ± SD of three independent experiments. After analyzing by one way ANOVA with Tukey—Kramer multiple comparison test, different letters above bars denote samples that were significantly different (p < 0.05) compared with control. (E) Effects of NFIA on the miR-302b-inhibited IGFBP2 signaling pathway. After overnight culture U87-MG cells were cotransfected with 50 nM miR-302b mimic, 50 nM scrambled miRNA mimic, 1 μg of NFIA OE plasmids, empty pcDNA3 vectors, NFIA shRNA, or scrambled shRNA for 24 h, relative protein levels of IGFBP2 signaling downstream regulators were measured using immunoblotting assays.