Figure 5

Validation of miRNA target-predicted genes. (A) qRT-PCR analysis performed on 1W and 3W differentiated hNSCs cultured on 2D and 3D substrates and expressed as fold change compared with proliferative control. Statistical analysis showed significant difference compared with control; ±SDMs, *P < 0.05, **P < 0.001, ***P < 0.005, Student t test. (B) SOX5 and NR4A3 protein quantification performed by Western blot on 1W and 3W differentiated hNSCs cultured on both 2D or 3D substrates and proliferative control. (C) Dual luciferase report assay. Measurement of the relative luciferase activity of SOX5 and NR4A3 3′-UTR constructs transfected with hsa-miR-96, and hsa-miR-7 and 17, respectively. Data are expressed as mean values ± SDMs and are shown as percentage of control (cells transfected with either SOX5 or NR4A3 3′-UTR constructs and control microRNA). Each bar represents values from three independent experiments, measured in triplicate. The relative activity of firefly luciferase expression was normalized to renilla luciferase activity. Data were analyzed with Student t test, ***P < 0.005. (D) MiRNA KEGG pathway analysis results obtained by using DIANA Lab.