Figure 3.

Elevated expression of miR-519a-3p induces resistance toward apoptosis induction. (a) RTCA (real-time cell analyzer) viability assay of MCF10A cells transfected with microRNA control or miR-519a-3p. Transfected cells were seeded in E-16 plates and were then treated with TRAIL (at t=36 h) or FasL (t=18 h). TRAIL (250 ng/ml; left panel) and FasL (10 μg/ml; right panel) treatment induced a decrease in cell index (as proxy for cell viability) in miRNA control-transfected but not miR-519a-3p-transfected MCF10A cells (n=6). (b) TRAIL- and FasL-induced apoptosis was reduced upon overexpression of miR-519a-3p in MCF10A cells using Annexin V and 7-AAD. MCF10A were transfected with miR-519a-3p or miRNA control for 48 h and then treated with 60 ng/ml TRAIL, 5 μg/ml FasL or medium control for additional 24 h. Shown is the analysis of early (Annexin V positive and 7-AAD negative) and late (Annexin V positive and 7-AAD positive) apoptosis (n=3). (c) TRAIL (60 ng/ml)-induced DNA fragmentation was reduced by miR-519a-3p in MCF10A and MDA-MB-231 cells, whereas FasL (5 μg/ml)-induced DNA fragmentation was reduced by miR-519a-3p only in MCF10A. (d) Activation of caspase-3/7 activity in MCF10A, HCC1143 and MDA-MB-231 cells by TRAIL and FasL (except in MDA-MB-231) was strongly reduced by miR-519a-3p (n=8). Data are expressed as mean±S.D.; *P<0.05, **P<0.01, ***P<0.001. All P-values are based on analysis of miRNA control versus miR-519a-3p