Table 1.
Characteristics of the 129 patients who underwent radical prostatectomy (RP) and the 11 patients in the BR-DIM intervention group from whom normal and tumor specimens were obtained.
Figure 1.
Loss of the let-7 family inversely correlated with increased expression of EZH2 in PCa tissue specimens compared to adjacent normal prostate tissues.
(A) Microarray profiling was done for assessing the expression of miRNA using total RNA extracted from three PCa tissue specimens and matched adjacent normal prostate tissues. The results showed that let-7a, let-7b, let-7c and let-7d was highly expressed in prostate tissues and their expression was lost in human PCa tissue specimens (* p<0.05, ** p<0.01). (B) The results from real time-RT-PCR confirmed that the levels of let-7b, let-7c and let-7d were significantly down-regulated in tumors with higher Gleason grade. (7a: let-7a; N: normal; TG6: tumor tissues from patients with Gleason grade 6. n = 39 for normal, n = 44 for TG6, n = 52 for TG7, n = 33 for TG8, 9). (C) A significant up-regulation of the expression of EZH2 was observed in PCa tissue specimens with Gleason grade 7 (n = 46) and higher (n = 33), but not in PCa specimens with Gleason grade 6 (n = 39). (D) EZH2 levels were inversely correlated with let-7b and let-7c expressions in tumor specimens with Gleason grade 7.
Figure 2.
Let-7 regulated EZH2 expression, and inhibited clonogenic growth capacity of PCa cell lines.
(A) Expression of EZH2 was found to be higher in PCa cell lines compared with immortalized prostate epithelial cell lines: PZ-HPV-7 and RWPE-1 (upper panel) and transfection of let-7 precursors inhibited EZH2 expression in PC3 and PC3 PDGF-D cells 3 days after transfection (middle and lower panel). (B) let-7 family members repressed EZH2 3′UTR luciferase activity in PC3 PDGF-D cells (lower levels of let-7 family in these cells) co-transfected with let-7 and EZH2 3′UTR luciferase plasmid. (C) let-7 binding sites in the 3′UTR of EZH2 mRNA were shown. (D) Transfection of let-7 precursors significantly reduced clonogenic growth capacity of PC3 PDGF-D cells (Con: control, 7a: pre-let-7a, ** p<0.01).
Figure 3.
BR-DIM treatment increased let-7 and consequently reduced EZH2 expression.
(A) Total RNA was isolated from LNCaP, C4-2B and PC3 cells treated with 25 µM BR-DIM for 24 h and the results from real time RT-PCR showing that the expression of let-7 was increased following BR-DIM treatment compared to untreated control (c: DMSO control). (B) Levels of EZH2 mRNA were repressed by BR-DIM treatment in a dose dependent meaner. (C and D) The cell lysates were prepared from cells treated with BR-DIM for 48 h and Western blot showing the protein levels of EZH2, which was down-regulated by BR-DIM treatment (PC3 PD: PC3 PDGF-D cells, *, p<0.05; **, p<0.01).
Figure 4.
BR-DIM intervention in PCa patients resulted in the increased expression of let-7 family and consequently inhibited EZH2 expression in tumor tissues.
RNA was obtained from BR-DIM intervention clinical trial samples where BR-DIM was given to patients for 2–4 weeks prior to surgery. RNA was also obtained from formalin-fixed paraffin-embedded (FFPE) tissue specimens of PCa patients with matched tumor Gleason grade, tumor stage and patient age as control group. The expression of miRNAs and mRNA was assessed using real time RT-PCR. Relative miRNA and mRNA levels were normalized to RNU1A1 and beta-actin, respectively. (A) BR-DIM intervention led to the increased trend in levels of Let-7a expression. (B and C) let-7b, let-7c and let-7d were significantly up-regulated by BR-DIM intervention. (D) EZH2 expression was down-regulated by BR-DIM intervention.
Figure 5.
BR-DIM treatment inhibited clonogenic growth and prostasphere-forming ability.
(A) Single cell suspensions of C4-2B and PC3 PDGF-D cells were plated in ultra low adherent wells of 6-well plate at 2000 cells/well in DMEM/F12 supplemented with B27 and N2 and with 10, 25 µM BR-DIM and incubated for 6 days. Prostasphere numbers were reduced by BR-DIM treatment (upper panel). Prostaspheres were photographed and the results showed that 10 and 25 µM BR-DIM significant reduced the size of prostaspheres (lower panel). (B) C4-2B and PC3 PDGF-D cells were seeded in 100 mm dishes at 2000 cells/dish, after 24 h incubation, the cells were treated with 10 or 25 µM BR-DIM for 72 h and then the culture medium was changed with fresh media without BR-DIM for every 3 days. After 2 weeks, the colonies were stained and photographed. (C and D) Soft agar assay showed that BR-DIM treatment reduced the size (left panel) and the numbers of colonies of C4-2B cells (right panel; **, p<0.01).