FIGURE 4.

miR-191-5p down-regulates SOX4 and p53 levels. (A) Diagram showing wild-type/mutated miR-191-5p binding site in the SOX4 3′ UTR. (B) Luciferase activity of the SOX4 wild-type/mutated miR-191-5p binding site bearing constructs was measured in response to pc or p53 overexpression. (C) SOX4 levels were measured at the protein level in MCF7 cells expressing pc-191 or Anti-191 along with their controls. The results show that miR-191-5p overexpression down-regulates SOX4 at the protein level. β-Actin was used for normalization of Western blotting data. (D,E) To evaluate the effect of SOX4 on p53 protein stability in breast cancer, MCF7 cells were transfected with SOX4 or control vector (pc), and 48 h post-transfection, p53 transcript as well as protein levels were measured by qRT-PCR and Western blotting. The real-time graph was plotted using $2^{-\mathrm{\Delta \Delta }{\mathrm{C}}_{\mathrm{t}}}$ formula. (F,G) The transcript and protein levels of p53 and p21 were evaluated by qRT-PCR and Western blotting in response to miR-191-5p overexpression or inhibition in MCF7 cells. The real-time graph was plotted using $2^{-\mathrm{\Delta \Delta }{\mathrm{C}}_{\mathrm{t}}}$ formula. (H) p53 protein level was determined by Western blotting in MCF7 cells transfected with pc-191 alone or in combination with SOX4. The graphical data points represent mean ± SD of at least three independent experiments. (*) P < 0.05, (**) P < 0.01. Error bars denote ±SD.