Analysis of miR-101 and miR-26a on Ezh2 3'-UTR in reporter assay, and endogenous Ezh2 expression. A. In wild type (wt) reporter, a 493-bp fragment containing the last 50 bps of Ezh2 coding region and the first 443 bps of Ezh2 3'-UTR embracing the predicted miR-101 and miR-26a target sites is subcloned downstream of GLuc driven by PGK promoter. The four reporter constructs contain mutations in Ezh2 3'-UTR at miR-101 target sites: m(45-66), m(101-121) and m(45-66&101-121); and at miR-26a target site: m(236-257). B. Increasing miR-101 and miR-26a (0, 50 and 100 nM, compensated with miR-cont to 100 nM, if necessary) were cotransfected with wt reporter presented in "A" and the SEAP-expressing plasmid into PC-3 cells (triplicated). GLuc activity was determined and normalized by SEAP activity (see Materials and Methods for details). C. One hundred nM of miR-cont or miR-101 was cotransfected with 50 ng of the indicated reporter constructs and the SEAP-expressing plasmid. GLuc activity was measured and normalized as described above. D. The experiment was performed as "C" using miR-26a and reporter plasmids as labeled. E. PC-3 cells were transfected with miR-cont, miR-101, or miR-26a (120 nM). Ezh2 and β-actin expression was determined by Western blot. Relative Ezh2 levels normalized by β-actin are indicated. F. Ezh2 mRNA levels (normalized to GAPDH) in microRNA-transfected cells analyzed by Real-Time RT-PCR. The asterisk: p ≤ 0.05. G and H. GLuc activity measurement (triplicated) and Western blot were performed as C, D and E in DU-145 (G) and LNCaP (H) cells.