Figure 4.

MiR-519a-3p inhibits NK cell-mediated cytotoxicity via caspase-7, MICA and ULBP2. (a) MCF10A cells were treated with granzyme B (GrzB) and perforin for 6 h and caspase-3/7 activity was measured. MiR-519a-3p reduced granzyme B/perforin-induced apoptosis induction (n=5). (b) Granzyme B/perforin-induced apoptosis was reduced by silencing of CASP7 but not of CASP8 in MCF10A cells. Cells were transfected with siTRAIL-R2, siCASP7, siCASP8 and miRNA control for 48 h and then treated with granzyme B/perforin for 6 h and active caspase-3/7 was measured (n=8). (c) Luciferase reporter assays were performed using psiCHECK constructs in MCF-7 cells. Luciferase activity was reduced in the presence of MICA and ULBP2 3′UTRs after miR-519a-3p transfection compared with miRNA control. The signal was rescued after mutating the respective binding sites for miRNA-519a-3p (n=5). (d) FACS analysis results showing downregulation of MICA and ULBP2 in breast cancer cells. Cells were transfected with miRNA control or miR-519a-3p for 48 h and cells were stained for MICA and ULBP2. Median fluorescence intensity for isotype control (gray), miRNA control (black) and miR-519a-3p (red) is depicted in each histogram. Shown is one representative FACS plot of at least three experimental repeats. (e) ⁵¹Cr release assay of MCF10A, MDA-MB-468 and HCC1143 cells after transfection with miR-519a-3p or miRNA control and subsequent co-culture with primary human NK cells in indicated ratios. Lysis of MCF10A, MDA-MB-468 and HCC1143 was measured in a 4 h ⁵¹Cr release assay (n=3). Data are expressed as mean+S.D.; **P<0.01, ***P<0.001. All P-values are based on analysis of miRNA control versus miR-519a-3p or siRNA control versus siCASP7, siCASP8 and siTRAILR2