Figure 4
miR-214 targets PTK6 and inhibits its expression. (A) The binding sites of miR-214 in the 3′UTR region of PTK6 were analyzed by TargetScan (http://www.targetscan.org/vert_72/) and presented (top). We mutated the binding nucleotides of miR-214 to the 3′UTR region of PTK6 to generate a mutant miR-214 mimic. Constructs of the wild-type and mutant miR-214 mimics are shown (bottom). (B) Luciferase reporter assay: PC3 cells were first transfected with one µg of PTK6-3′UTR-Luciferase reporter construct for 18 h and then transfected with NC, wild-type miR-214, or mutant miR-214 mimic for an additional 24 h. Transfected cells were lysed, and luciferase activity was then measured. **P < 0.005 compared with NC or wild-type miR-214 mimic. (C) RWPE-2, PC3, DU145, and MDA-PCa-2b cells were transfected with NC or miR-214 mimic. After 48 h, relative PTK6 expression was analyzed by RT/qPCR. ***P < 0.0005 compared with NC-transfected cells. (D) RWPE-2, PC3, DU145, and MDA-PCa-2b cells were transfected with NC or miR-214 mimic. After 48 h, PTK6 protein levels were analyzed by western blotting (top) and quantified using ImageJ (bottom). ***P < 0.0005 compared with NC-transfected cells. (E) PC3 cells were transfected with NC or miR-214 mimic for 48 h, and then PTK6 expression (green) was visualized by immunofluorescence as described in the materials and methods section. (F) RWPE-2, PC3, DU145, and MDA-PCa-2b cells were transfected with PTK6 plasmid or empty vector plasmid, and expression of PTK6 protein levels were analyzed by western blotting (upper panel). Effect of PTK6 overexpression on cell viability of prostate cancer cells was measured by MTT assay and plotted (lower panel). The data represent mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test was used to determine statistical significance for RT/qPCR and western blot analysis.
