Figure 4

Validation of human SOCS3 as a direct miR-19b target.(a), Quantitative real-time PCR analysis of miR-19b expression in Caco2 cells and HT29 cells transfected with pre-miR-19b, anti-miR-19b or ncRNA. Left: Caco2 cells. Right: HT29 cells. Data are presented as the mean±SEM from three independent experiments (Caco2 group: ^***p = 0.000092, ncRNA vs. pre-miR-19b, ^***p = 0.000031, ncRNA vs. anti-miR-19b; HT29 group: ^***p = 0.000253, ncRNA vs. pre-miR-19b,^**p = 0.003, ncRNA vs. anti-miR-19b). (b), Immunoblot for endogenous SOCS3 in Caco2 cells and HT29 cells transfected with pre-miR-19b, anti-miR-19b or ncRNA. Left: Caco2 cells. Right: HT29 cells. α-Tubulin was used as a loading control. Top panel: representative image; Bottom panel: quantitative analysis. The original whole-blot pictures are available in Supplementary Fig. S6. Results are presented as the mean±SEM from three independent experiments (Caco2 group: ^**p = 0.0014, ncRNA vs. pre-miR-19b,^***p = 0.00036,ncRNA vs. anti-miR-19b; HT29 group: ^*p = 0.038, ncRNA vs. pre-miR-19b,^**p = 0.007, ncRNA vs. anti-miR-19b). (c), Quantitative real-time PCR analysis of SOCS3 expression in Caco2 cells and HT29 cells treated with pre-miR-19b, anti-miR-19b or ncRNA. Left: Caco2 cells. Right: HT29 cells. The y-axis shows relative SOCS3 mRNA levels normalized to GAPDH. The results are presented as the mean±SEM from three independent experiments (p > 0.05 vs. ncRNA). (d), Direct recognition of the SOCS3 3’-UTR by miR-19b. Firefly luciferase reporters containing wild-type (WT) or mutant (MT) miR-19b binding sites in the SOCS3 3’-UTR were co-transfected into Caco2 cells and HT29 cells with scrambled ncRNA, pre-miR-19b or anti-miR-19b. Twenty-four hours after transfection, a luciferase assay was performed. Firefly luciferase values were normalized to β-galactosidase activity and plotted as relative luciferase activity. The results were calculated as the ratio of firefly luciferase activity in miR-19b-transfected cells to the luciferase activity in control cells. Left: Caco2 cells. Right: HT29 cells. The results are presented as the mean±SEM from three independent experiments (Caco2 group: ^*p = 0.013, pre-scramble vs. pre-miR-19b,^***p = 0.0000, anti-scramble vs. anti-miR-19b; HT29 group:^***p = 0.000021, pre-scramble vs. pre-miR-19b,^***p = 0.000019, anti-scramble vs. anti-miR-19b).