Figure 5

The loss of the retinoblastoma tumor suppressor ( RB ) expression plays a role in Smurf2 downregulation in triple-negative breast cancer (TNBC) cells, via upregulation of miR-15, miR-16 and miR-128. (A) Immunoblotting for RB protein in the indicated TNBC cell lines. (B) Increased expression of Smurf2 by forced expression of RB in RB-null BT549 cells. Cells were transfected with an expression vector for a green fluorescence protein (GFP)-RB fusion protein, a vector for GFP, or an empty vector (pcDNA3.1). Levels of Smurf2 and RB were determined at 42 h post-transfection by immunoblotting. The bar graph indicates relative levels for Smurf2 protein normalized by those for α-tubulin, shown as means + SEM from three experiments. The asterisk indicates statistical significance (p < 0.05). (C) Decreased expression of miR-15a, miR-15b, miR-16 and miR-128 in BT549 cells transfected with the GFP-RB vector. Levels of the indicated microRNAs were determined by real time PCR. Data are normalized by signals for RNU6-2 as controls, and presented as relative expression levels to that in cells transfected with an empty vector as 1.0. The bars indicate S.E.M. from three experiments, and the asterisk indicates statistical significance (p < 0.05).