Fig. 4.

Induction of miR-4295 by ISO bound to USP28 3′UTR and inhibited USP28 protein translation in human BC cells. a USP28 3′-UTR luciferase reporters were co-transfected with pRL-TK into T24T cells; stable transfectants were exposed to 20 μM ISO for the indicated time periods to assess the ISO inhibition of USP28 3′UTR luciferase activity. *Significant difference (p < 0.05). b Expression levels of predicted miRNA were evaluated using quantitative real-time PCR. T24T cells were treated with medium-containing either vehicle or 20 μM ISO. c, e Over-expression of miR-137 and miR-4295 in T24T cells. *Significant difference (p < 0.05). d, f miR-4295, but not miR-137, inhibited USP28 and CD44 protein expression. g Inhibition of USP28 3′UTR activity by ectopic expression of miR-4295 in T24T cells. *Significant difference (p < 0.05). h miR-4295-binding site was crucial for ISO inhibition of USP28 3′UTR luciferase activity. *Significant difference (p < 0.05)