Figure 3.

miR-106b and miR-32 inhibit expression of E2F1 and Bim, respectively, by a 3’UTR-mediated mechanism. (A,C) LNCaP and PC-3 human prostate cancer cells were transfected with either microRNA precursor (miR-106b or miR-32) or antisense microRNA (antisense miR-106b or antisense miR-32), or their respective vector controls, scrambled precursor microRNA (Scrambled-P) and scrambled antisense microRNA (Scrambled-A). Protein extracts were prepared 48 hours after transfection and protein expression was examined by Western blot analysis. To obtain relative intensity values, E2F1 and Bim expression were normalized to β-actin. (B) pGL3 luciferase reporter constructs containing either the wild-type or mutant 3’UTR target sequence of miR-106b in the E2F1 gene were co-transfected into LNCaP cells with either precursor microRNA negative control or miR-106b precursor (each n = 3; mean ± standard deviation). For comparison, cells were also transfected with the pGL3 control vector that did not contain the 3’UTR. After 24 hours, luciferase activity was determined in the cell extracts. In the presence of the wild-type E2F1 3’UTR, transfection with precursor miR-106b lead to a significant inhibition of the luciferase reporter when compared with the vector control (P = 0.045, two-sided t-test). This inhibition was not observed if the reporter construct contained a mutant 3’UTR target sequence of miR-106b. (D) pGL3 luciferase reporter constructs containing either the wild-type or mutant 3’UTR target sequence of miR-32 in the BCL2L11 (Bim) gene were co-transfected into LNCaP cells with either precursor microRNA negative control or miR-32 precursor (each n = 3). In the presence of the wild-type BCL2L11 3’UTR, transfection with miR-32 lead to a significant inhibition of the luciferase reporter when compared with the vector control (P = 0.003, two-sided t-test). This inhibition was attenuated if the reporter construct contained a mutant 3’UTR target sequence of miR-32.