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. 2016 Jul 18;7(35):56540–56557. doi: 10.18632/oncotarget.10645

Figure 6. miR-4295 was able to directly bind to p63α 3′UTR resulting in blockage of p63α protein translation and subsequently promoting EGF-induced malignant transformation of human bladder epithelial cells.

Figure 6

A. Real-time PCR was used to identify the expression level of miR-4295 in UROtsa cells; B. p63α 3′UTR luciferase reporters were co-transfected together with pRL-TK into UROtsa(Vector) and UROtsa(miR-4295) cells, respectively. Twenty-four hours post transfection, the transfectants were extracted for measurement of the luciferase activity, while TK was used as internal control. The results were presented as p63α 3′UTR activity relative to vector control transfectant, and each bar indicates mean±SD from three independent experiments. The symbol (*) shows a significant difference (P< 0.05); C. UROtsa(Vector) and UROtsa(miR-4295) were transfected with either p63α 3′UTR luciferase reporter or mutant of p63α 3′UTR luciferase reporter that has miR-4295 binding site mutation, Twenty-four hours post transfection, the transfectants were extracted to evaluate the luciferase activity, with TK used as an internal control. The results were presented as p63α 3′UTR activity relative to control vector transfectant. Each bar indicates mean±SD from three independent experiments. The symbol (*) shows a significant difference (P<0.05); D. The cell extracts obtained from UROtsa(Vector) and UROtsa(miR-4295) cells were subjected to Western blot to determine expression of p63α and XIAP, while α-Tubulin was used as a protein loading control; E. and F. UROtsa(Vector) and UROtsa(miR-4295) transfectants were subjected to anchorage-independent growth in the presence or absence of EGF as indicated. Representative images of the colonies of indicated cells were captured under microscopy following 3 week incubation period (E); the number of colonies was counted under microscopy and the results were presented as colonies per 50,000 cells from three independent experiments. The asterisk (*) indicates a significant increase as compared with the medium control, and the symbol (♣) indicates a significant inhibition in comparison to UROtsa(Vector) cells (p<0.05) (F).