Additional file 3: of miR-221-5p regulates proliferation and migration in human prostate cancer cells and reduces tumor growth in vivo

Figure S2. miR-221-5p exerts tumor suppressive function on PCa cell lines in vitro. (a) Cell cycle of wild-type PC-3 M-Pro4luc2 and C4–2 cells was analysed by PI staining at 0 h, 16 h and 48 h after release starvation. The frequency of cells in G1, S and G2/M phase was quantified by Dean-Jett-Fox model. Results of n = 2 technical replicates were analysed by unpaired, two-tailed t-test. * p < 0.05, ** p < 0.01. (b) PC-3 M-Pro4luc2 cells were transfected with 10 nM miR-221-5p or 10 nM scrambled. The next day, cells were transfected with increasing concentrations of anti-miR-221-5p (10 nM, 20 nM, 30 nM, 60 nM and 120 nM) or 10 nM anti-scrambled. miR-221-5p expression was assessed 48 h later and LOG difference (LOG(2-ΔΔCt)) to scrambled/anti-scrambled co-transfection control calculated. Data of n = 3 technical replicates are represented and were analysed by two-way ANOVA with Sidak’s multiple comparison test. **** p < 0.0001 miR-221-5p/anti-miR-221-5p compared to scrambled/anti-scrambled control, #### p < 0.0001 scrambled/anti-miR-221-5p compared to scrambled/anti-scrambled control, p < 0.0001 miR-221-5p/anti-miR-221-5p compared to scrambled/anti-miR-221-5p. (c) miR-221-5p overexpression in C4–2 cells 72 h post transfection with miR-221-5p or scrambled control. LOG difference to scrambled was calculated as LOG(2-ΔΔCt). Data of one representative experiment are shown and were analysed by unpaired, two-tailed t-test. **** p < 0.0001. (d) Proliferation of miR-221-5p and scrambled overexpressing C4–2 cells. Images were taken 72 h post transfection. Proliferation was measured by MTS at four time points (0 h, 24 h, 48 h and 72 h). Data of n = 3 independent experiments are shown as fold change normalised to T’0 h and were analysed by two-way ANOVA with repeated measures by both factors with Sidak’s multiple comparison test. *** p < 0.001, **** p < 0.0001. (e) Clonogenicity assay of C4–2 cells transfected with miR-221-5p or scrambled. All technical replicates of three independent experiments (n = 3) were pooled and fold change to scrambled was calculated. Data were analysed by unpaired, two-tailed t-test. **** p < 0.0001. (PDF 245 kb)