Stem Cell Feature in EMT Cells Figure 3. Gene expression profiling of stem cell markers in PC3 PDGF-D cells with EMT signatures. (A) The expression of genes at the mRNA levels for Oct4, Nanog, Sox family genes and Lin28B in PC3 Neo and PC3 PDGF-D cells were determined by using real time RT-PCR. (B) The results from Western blot showed that the expressions of Oct4 Sox2, Nanog, Stat3 and Lin28B were significantly increased in PC3 PDGF-D cells. (C) Real time RT-PCR was used to quantify the mRNA expression of EED, EZH2 and Suz12a. Relative mRNA levels were normalized to GAPDH. (D) The results from Western blot showed that the expression of EZH2 was significantly increased in PC3 PDGF-D cells. (E) The mRNA levels of Notch signaling factors in PC3 Neo and PC3 PDGF-D cells were determined by using real time RT-PCR. (F) The results from Western blot showed that the expressions of Notch and Notch ligands were significantly increased in PC3 PDGF-D cells. GAPDH was used for protein loading control. (N: PC3 Neo cells, D: PC3 PDGF-D cells,p10: passage 10). doi:10.1371/journal.pone.0012445.g003 These results suggest that miR-200b and miR-200c but not miR- 200a inhibited self-renewal of PC3 PDGF-D cells by controlling target gene expressions of miR-200b and miR-200c. Thus, we assessed the expression of the putative targets of miR-200b and miR-200c such as Notch1, Bmi1 and lin28B in PC3 PDGF-D cells transfected with miR-200 family because miR-200b and miR-200c have three or one putative binding sites in the 39UTR of Lin28B, Bmi1 mRNA (Fig. S3) and Notch1 mRNA. The miR-200b and miR-200c transfection dramatically reduced the expression of Notch1 and lin28B, but had no effect on the expression of Bmi1 (Fig. 4C). The expression of ZEB1 was found to be down-regulated by forced expression of miR-200b and miR-200c (Fig. 4C), which was consistent with the reversal of EMT phenotype (Fig 4A). We also found that miR-200c expression was repressed in ARCaPM cells compared with ARCaPE cells (Fig. 4D). Moreover, re- expression of miR-200c in ARCaPM cells by transfection with pre- miR-200c precursors markedly reduced the prostasphere-forming capacity compared to transfection with control miRNA (Fig. 4E). These results were consistent with down-regulation of Notch1 expression and reversal of EMT phenotype as characterized by increased expression of E-cadherin and decreased expression of ZEB1 as well as cellular morphology change in ARCaPM cells transfected with pre-miR-200c precursors (Fig. 4F and Fig. 4G). Down-regulation of Notch1 by miR-200 was partially responsible for the inhibition of colonogenic and prostasphere-forming ability of PC3 PDGF-D cells The miR-200b and miR-200c has one binding sites in the 39UTR of Notch1 (Fig. 5A). To determine whether Notch1 is the direct target of miR-200b and miR-200c, we analyzed the activity of 39 UTR of Notch1 in PC3 Neo and PC3 PDGF-D cells transfected with 39UTR of Notch1 luciferase plasmid as well as PC3 PDGF-D PLoS ONE | www.plosone.org 5 August 2010 | Volume 5 | Issue 8 | e12445