miR-221 Augments TRAIL-Mediated Apoptosis in Prostate Cancer Cells by Inducing Endogenous TRAIL Expression and Targeting the Functional Repressors SOCS3 and PIK3R1

Figure 1

Overexpression of miR-221 augmented TRAIL effects in terms of cell viability and apoptosis induction in PCa cells. (a) TRAIL administration (final concentration of 10 ng/ml) led to a significantly lower proportion of proliferating PC3 and DU145 cells. In contrast, LNCaP cells and nonmalignant RWPE cells did not show a significant change in viability after TRAIL administration. Relative cell viability was normalised to the viability of untreated control cell lines, which was arbitrarily set as 100% (b, c). Transient transfection with pre-miR-221 significantly augmented TRAIL sensitivity in terms of cell viability in (b) PC3 and (c) DU145 cells (120 h p. t.). (d) Higher doses of TRAIL (50 ng/ml) led to a decrease in cell viability of LNCaP cells. Overexpression of miR-221 caused increased cell viability. In combination, pre-miR-221 transfected LNCaP cells were significantly less susceptible towards TRAIL effects compared to control transfections. (b, c, d) Relative cell viability was normalised to the viability of control cells, which was arbitrarily set as 100%. Control cells (Ctr) were transfected with a scrambled, non targeting pre-miRNA. (e) miR-221 overexpression significantly augmented Caspase 3/7 activity in PC3 cells. 10 ng/ml TRAIL was administered 24 h p. t. Experiments took place 48 h p. t. Caspase positivity was normalized to the Caspase activity of control cells, which was arbitrarily set as 100%. Control cells (Ctr) were transfected with a scrambled, non targeting pre-miRNA . (a, b, c, d, e) Data represent mean + SD from five independent experiments, *; **. (b, c, d, e) ANOVA with Bonferroni post hoc testing. (f) Western blotting experiments confirmed higher expression levels of cleaved PARP1 in pre-miR-221 transfected PC3 cells compared to pre-miR-control transfections (48 h p. t.). TRAIL treatment in doses of 10 ng/ml and 50 ng/ml caused higher expression levels of cleaved PARP1 in pre-miR-Control and pre-miR-221 transfected PC3 cells compared to untreated cells–with higher levels in TRAIL-treated cells overexpressing miR-221.

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