Stem Cell Feature in EMT Cells Figure 6. Lin28B-mediated repression of let-7 was partially responsible for regulating self-renewal in PC3 PDGF-D cells. (A) The levels for let-7 family in PC3 Neo and PC3 PDGF-D cells were determined by using real time RT-PCR. (B) Real time RT-PCR was used to quantify the expression of let-7 in PC3 PDGF-D cells transfected with Lin28B siRNA for 3 days. (C) Photomicrographs showing prostaspheres generated from PC3 PDGF-D cells transfected with pre-let-7 or combination of pre-let-7 and Lin28B siRNA 14 days after transfection (bar:100 mm). (D) The cells were collected for prostasphere-forming assay after 14-day transfection. Let-7a, let-7b, let-7d and combination of Let-7a, let-7b, let-7d as well as co- transfection with let-7 and Lin28B siRNA decreased the number of prostaspheres. (E) The results from Western blot showed the expression of Lin28B, Sox2, Nanog, and Oct4 in PC3 PDGF-D cells transfected with pre-let-7 or co-transfected with pre-let-7 and Lin28B siRNA after 9-day transfection. *, p,0.05 compared to control; **, p,0.01 compared to control. (Con: control, a: pre-let-7a, abd: combination of pre-let-7a, pre-let-7a, pre-let-7d, al: combination of pre-let-7a and Lin28B siRNA, Lin28B-si: Lin28B siRNA, Neo: PC3 Neo cells, PDGF-D: PC3 PDGF-D cells). doi:10.1371/journal.pone.0012445.g006 PDGF-D cells with EMT phenotype. In order to assess the mechanistic link of these molecules (Sox2, Nanog, Oct4 and Lin28B) with the self-renewal capacity of PC3 PDGF-D cells, we knocked down the expression of Sox2, Nanog, Oct4 and Lin28B by siRNA transfection using Sox2, Nanog, Oct4 and Lin28B specific siRNA. We found that knock-down of Sox2, Nanog, Oct4 and Lin28B significantly repressed prostasphere-forming ability (Fig. 7A). Furthermore, PC3 PDGF-D cells transfected with Sox2, Nanog, Oct4 and Lin28B siRNA showed significantly increased numbers of prostaspheres in smaller size (30–50 mm) compared to that transfected with control siRNA concomitant with decreased numbers of prostaspheres with .50 mm in diameter (Fig. 7B). These results were consistent with protein expression results as shown in Fig. 7C, suggesting that Sox2, Nanog, Oct4 and Lin28B are required for self-renewal of PC3 PDGF-D cells. The cells with EMT phenotype promoted tumorigenicity In our previous studies, we showed that the tumors from PC3 PDGF-D cells grew much faster compared to that from PC3 Neo cells in an animal model of experimental bone metastasis [26]. For our current animal model studies, PC3 Neo and PC3 PDGF-D cells were injected subcutaneously into the right and left flanks of each mouse and compared tumor growth rate between PC3 Neo and PC3 PDGF-D cells. We found that the volume of tumors induced by PC3 PDGF-D cells was significantly larger than that of PC3 Neo cells (Fig. 8A). To reveal whether tumor cells in vivo have the signatures in the expression of genes similar to in vitro experiments, we assessed the expression of selected genes related to EMT and stem cell markers. We found down-regulation of E- cadherin and up-regulation of mesenchymal markers such as vimentin and ZEB1 in tumor tissues derived from PC3 PDGF-D cells compared with PC3 Neo control cells. Concomitantly, Lin28B, Sox2, Nanog and Oct4 as well as Notch1 expression was also significantly increased in tumors derived from PC3 PDGF-D cells, which was consistent with increased PDGF-D expression (Fig. 8B). Immunohistochemical analysis showed that the tumors derived from PC3 PDGF-D cells contained much intense staining for vimentin and Notch1 and less intense staining for E-cadherin (Fig. 8C). These results suggest that the cells with EMT phenotype promoted tumorigenicity in vivo. PLoS ONE | www.plosone.org 8 August 2010 | Volume 5 | Issue 8 | e12445