Stem Cell Feature in EMT Cells Figure 5. Inactivation of Notch1 repressed the colonogenic and prostasphere-forming capacity. (A) Conserved, predicted binding sites for the seed sequences of miR-200b and mir-200c in the 39UTR of Notch1 mRNA. (B) MiR-200b and miR-200c inhibited the Notch1 39UTR luciferase activity. **, p,0.01 compared to Neo control cells; #, p,0.01 compared to PDGF-D control cells. (C) DAPT treatment, a c-secretase inhibitor, reduced the clonogenic ability in PC3 PDGF-D cells (upper panel).Western Blot showing that DAPT treatment reduced active form of Notch1 in dose- dependent manner (lower panel). (D) and (E) showing that transfection of Notch1 siRNA after 9 days repressed the prostasphere-forming capacity in PC3 PDGF-D cells (Bar:100 mm), which is consistent with downregulation of Notch1 expression (F). **, p,0.01 compared to control. (Neo: PC3 Neo cells, PDGF-D: PC3 PDGF-D cells, Con: control, 200a: pre-miR-200a, 200b: pre-miR-200b, 200c: pre-miR-200c). doi:10.1371/journal.pone.0012445.g005 PC3 PDGF-D cells (Fig. 5C, upper panel), which was consistent with Western blot data showing that DAPT treatment inhibited active form of Notch1 (Fig. 5C, lower panel). Moreover, transfection of Notch1 siRNA dramatically repressed the prostasphere-forming ability of PC3 PDGF-D cells (Fig. 5D and 5E), which was consistent with down-regulation of Notch1 protein expression (Fig. 5F). These results suggest that miR-200 mediated down-regulation of Notch1 was partially responsible for self-renewal capacity and colonogenic growth of EMT-like cells having stem cell features. Lin28B inhibited production of let-7, regulating self- renewal by controlling the expression of Sox2, Nanog and Oct4 Our results presented above showed that the miR-200b and miR-200c inhibited lin28B expression (Fig. 4C), which is known to bind to primary let-7 and pre-let-7 RNA and inhibits the accumulation of mature let-7 miRNA [19]. The results from our miRNA microarray data showed that all the members of let-7 family were significantly reduced in PC3 PDGF-D cells (Table S3). These results were confirmed by real time RT-PCR assay (Fig. 6A). Knock-down of Lin28B by siRNA strongly increased mature let-7 expressions (Fig. 6B); suggesting lin28B regulated mature let-7 expression in PC3 PDGF-D cells. It is well known that let-7 family plays a critical role in regulating self-renewal in embryonic stem cells and breast cancer stem-like cells [18,37]. To determine whether let-7 could control self-renewal of PC3 PDGF-D cells, we performed the sphere-forming assay. Re-expression of selected let- 7 family such as let-7a, let-7b, let-7d or combination of let-7a, let- 7b and let-7d markedly inhibited prostasphere-forming capability (Fig. 6C and 6D) and simultaneously reduced the size of prostaspheres (Fig. 6C, S4A and S4B). Since Lin28B inhibits accumulation of mature let-7 by binding to pre-let-7 RNA and thereby blocking the processing of pre-let-7 RNA, PC3 PDGF-D cells with high level of Lin28B were co-transfected with pre-let-7 and Lin28B siRNA. We found that prostasphere numbers from cells co-transfected with pre-let-7 and Lin28B siRNA were less than that of transfection with pre-let-7 alone (Fig. 6D); however, except for let-7d, there was no difference in prostasphere size between pre-let-7 alone and co-transfection with pre-let-7 and Lin28B siRNA (Fig. 6C, S4A and S4B). These results were consistent with the data from Western blot analysis showing that the re-expression of let-7 significantly inhibited Sox2 and Nanog expression in PC3 PDGF-D cells by transfection of pre-let-7a, pre- let-7b or co-transfection of pre-let-7a, pre-let-7b or pre-let-7d with Lin28B siRNA. Transfection of pre-let-7d alone was able to inhibit the expression of Lin28B and Oct4 but had marginal effects on Sox2 and Nanog expression (Fig. 6E). Sox2, Nanog, Oct4 and Lin28B were required for self- renewal of PC3 PDGF-D cells We have shown that let-7 family regulated self-renewal and inhibited Sox2, Nanog, Oct4 and Lin28B expression in PC3 PLoS ONE | www.plosone.org 7 August 2010 | Volume 5 | Issue 8 | e12445