Figure 4

USP25 expression is downregulated by miR-200c directly targeting of the 3’-UTR of USP25. (A) USP25 was found for the potential regulatory targets of miR-200c by considering the up-regulated genes from the gene chip and using prediction tools, including miRNAMap, miRanda, and PicTar. (B) The USP25 mRNA levels were determined by real-time PCR analysis after transfection with the miR-200c mimics or negative control in SPC-A-1sci cells or after transfection with the miR-200c inhibitor or negative control in SPC-A-1cells. β-actin served as an internal control. (C) The putative miR-200c binding site in the USP25 3’-UTR. (D) Luciferase activity assays for luciferase reporters with wild-type or mutant USP25 3’-UTR were performed after co-transfected with miR-200c mimics or miR-control in 293T cells. The luciferase activity of each sample was normalized to Renilla luciferase activity. (E) USP25 protein levels were determined by western blot analyses after transfection with the miR-200c mimics or negative control in SPC-A-1sci cells or after transfection with the miR-200c inhibitor or negative control in SPC-A-1 cells. β-actin served as an internal control. The results are representative of at least three independent experiments. Statistical analysis was performed using Student’s t- test. Error bars represent S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001.