Skip to main content
. 2016 Apr 15;7(23):34112–34130. doi: 10.18632/oncotarget.8746

Figure 4. miR-302d was specific activated by p100 and directly inhibited Cyclin D1 protein translation.

Figure 4

(A–D) cyclin d1 3′UTR luciferase reporter was transiently transfected into the indicated cells and luciferase activity of each transfectant was evaluated. The results were presented as relative cyclin d1 3′-UTR activity. The symbol (*) indicates a significant difference (p < 0.05). (E–H) The levels of indicated microRNAs were evaluated by quantitative real-time PCR. The symbol (*) indicates a significant difference as compared with control cells as indicated (p < 0.05). (I) p100−/− cells were stably transfected with construct of miR-302/367 and the miRNA expression levels were determined by real-time PCR. The symbol (*) indicates a significant increase in comparison to the scramble control transfectant (p < 0.05). (J) The cyclin d1 3′-UTR luciferase reporter was transiently transfected into p100−/−(Vector) or p100(miR-302) cells. Luciferase activity of each transfectant was evaluated and the results were presented as relative cyclin d1 3′-UTR activity. The symbol (*) indicates a significant decrease as compared with that in vector transfectant (p < 0.05). (K) UMUC3(shp100) cells were stably transfected with construct of miR-302/367. The miR-302d expression was determined by real-time PCR and the symbol (*) indicates a significant increase as compared with control vector transfectant (p < 0.05). (L and M) The cell extracts as indicated were subjected to Western Blot and GAPDH was used as a protein loading control. (N) Flow-cytometry analysis of cell cycle alteration was carried out as indicated. (O) Schematic of the construction of the cyclin d1 mRNA 3′-UTR luciferase reporter and its mutants were aligned with miR-302d. (P) Wild-type and mutant of cyclin d1 3′-UTR luciferase reporters were co-transfected with pRL-TK into p100+/+ and p100−/− cells, respectively. Luciferase activity of each transfectant was evaluated and the results were presented as relative cyclin d1 3′-UTR activity. The symbol (*) indicates a significant decrease in cyclin d1 3′-UTR activity as compared with that in WT cyclin d1 3′-UTR reporter transfectant (p < 0.05).