Figure 4

MiR-454-3p negatively regulates BTG1 expression. (A) Construction of a vector with either the wild-type sequence of the miR-454-3p binding site within the 3′-UTR of BTG1 (Wt BTG1 3′-UTR) or a mutated seed sequence (Mut BTG1 3′-UTR). The black box shows the predicted miR-454-3p binding site. The seed sequence is shown in red. (B) Luciferase reporter assay results are shown at 48 h following co-transfection of 786-O cells with the Wt BTG1 or Mut BTG1 vectors together with miR-454-3p mimic (miR-454-3p) or nonsense small RNA oligonucleotides (negative control, NC). Results (means ± SE) are representative for three independent experiments. (C) Western blotting was performed at 48 h after transfection with miR-454-3p or NC oligonucleotides. GAPDH was used as loading control. Fold changes of protein levels represent the average of three separate blots. (D) BTG1 expression is regulated by miR-454-3p at the mRNA level. QRT-PCR was conducted to quantify the expression level of BTG1 mRNA at 48 h after 786-O cells were transfected with miR-454-3p or NC oligonucleotides. (E) MiR-454-3p and BTG1 mRNA levels were monitored by qRT-PCR at the indicated time points after irradiation with 5 Gy of X-rays. U6 and GAPDH were used as controls. (F) SKA2 mRNA levels were monitored by qRT-PCR at the indicated times after irradiation with 5 Gy of X-rays. GAPDH was used as a control. The data (means ± SE) are representative for at least four independent experiments with similar results.