Figure 4.

Inhibitory effects of TuD RNA on endogenous miR-21 activity. TuD RNA expression plasmid vectors or LNA/DNA antisense oligonucleotides were transiently transfected into PA-1 (A) or HCT-116 (B) cells together with the Renilla luciferase miR-21 reporter (miR-21-RL) (open bars) or the untargeted control Renilla luciferase reporter (UT-RL) (black bars) as well as the Firefly luciferase reporter (FL) as a transfection control. After performing a dual luciferase assay, the expression levels were normalized to the ratio of the activity of miR-21-RL to that of FL in TuD-NC vector-transfected PA-1 (A) or HCT-116 (B) cells and are represented by the mean ± SEM (n = 3). (C) Comparison of the inhibitory effects of TuD RNA and a conventional miRNA inhibitory vector. A dual luciferase assay was performed 72 h after transfection in HCT-116 cells. The ratio of miR-21-RL/FL to UT-RL/FL are represented by the mean ± SEM (n = 3) as the expression levels. (D) The levels of mature miR-21 in RNA from HCT-116 cells transfected with TuD-miR21 expression vectors determined by quantitative real time RT-PCR. The miR-21 expression levels were normalized to those of HCT-116 cells transfected with TuD-NC expression vector and are represented by the mean ± SEM (n = 3). U6 snRNA was served as an endogenous control. (E) The levels of pre- and mature miR-21 in the same RNA samples as (D) determined by northern blotting. Y4 scRNA was served as a loading control.