Stem Cell Feature in EMT Cells Figure 8. The cells with EMT phenotype promoted tumor growth. (A) Tumor growth curve showing that over-expression of PDGF-D could promote tumor growth in SCID mice much faster than PC3 Neo cells. (B) Western blot analysis of tumor lysates showing the expression of EMT and stem cell makers. (C) H&E evaluation of the tumors from both the groups showed high grade carcinoma associated with tumor apoptosis and necrosis. The results from the immunostaining showed much intense staining for vimentin and Notch1 and less intense staining for E-cadherin. (D) Regulatory model showing mechanistic link between EMT and stem cells: EMT induced by different factors characterized by increased expression of ZEB1, which causes loss of miR-200 family. Loss of miR-200b and miR-200c lead to increased ZEB1, Notch1 and Lin28B expression, resulting in decreased expression of let-7. Downregulation of let-7 leads to the up-regulation of Sox2, Nanog and Oct4, which together with Notch1 and Lin28B contribute to stem cell signatures. (*, p ,0.05, **, p,0.01 compared to PC3 Neo, E-cad: E-cadherin). doi:10.1371/journal.pone.0012445.g008 [28,40,41]. In order to investigate whether miR-200 family could contribute to the generation of cancer stem-like cell characteristics in ARCaPM and PC3 PDGF-D cells by regulating the expression of Nanog, Oct4 and Sox2, Lin28B and other stem cell-associated makers, we have searched targets of miR-200 family in www. microRNA.org. We found that miR-200b and miR-200c have three binding sites at the 39UTR of Lin28B mRNA, one in Notch1, Sox2 and Bmi1. Interestingly, miR-200b and miR-200c were significantly decreased in PC3 PDGF-D cells and miR-200c was down-regulated in ARCaPM cells. Therefore, we analyzed the expression of Lin28B, Notch1 and Bmi1, and performed the sphere-forming assay using ARCaPM and PC3 PDGF-D cells transfected with miR-200. We found that re-expression of miR-200b and miR-200c dramatically reduced the sphere-forming ability concomitant with decreased expression of Lin28B and Notch1 in PC3 PDGF-D cells. The miR- 200c also dramatically reduced the sphere-forming ability concom- itant with decreased expression of Notch1 in ARCaPM cells compared to its isogenic ARCaPE cells. Lin28B, a Lin28 homolog, was significantly repressed by re- expression of miR-200b and miR-200c in our cell model. It has been shown to selectively block the processing of pri-let-7 miRNAs in embryonic cells, resulting in decreased mature let-7 [19]. In the present study, we found that Lin28B was significantly increased and let-7 family was strongly repressed in PC3 PDGF-D cells. Transfection of Lin28B siRNA enhanced let-7 expression, suggesting increased Lin28B was responsible for the loss of let-7 in PC3 PDGF-D cells. The let-7 was known to promote differentiation of embryonic cells, reduced expression of let-7 was associated with increased self-renewal ability in breast cancer cells [18]. Thus, we hypothesized that let-7 could regulate self- renewal by regulating Nanog, Oct4, Sox2 and Lin28B expression in PC3 PDGF-D cells. In order to prove our hypothesis, PC3 PDGF-D cells were transfected with let-7, and we found that let-7a and let-7b could reduce the sphere-forming capacity concomitant with decreased expression of Sox2 and Nanog, These results were consistent with the data showing that let-7 significantly inhibited PLoS ONE | www.plosone.org 10 August 2010 | Volume 5 | Issue 8 | e12445