miR-4759 enhances the cytotoxicity of PBMCs to TNBC cells via PD-L1 down-regulation in co-culture model. (A) MDA-MB-231 and BT-549 cells were transfected with miR-4759 or miR-Scr. PBMCs were subsequently co-cultured with miR-treated TNBC cells in IncuCyte for 48 h. The apoptotic cell population was determined with green fluorescent signals. (B) The relative apoptotic population of miR-4759 treated cells was compared with miR-Scr group. The results represent the mean ± SD. from three independent experiments. Statistical significance was tested by t-test. *, p < 0.05. (C) Co-culture media were assayed for IFN-γ, Granzyme B, TNF-α and IL-10 by cytokine ELISA assay. The results represent the mean ± SD. from three independent experiments. Statistical significance was tested by t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (D) TNBC cells with or without stable overexpression of PD-L1 were transfected with miR-4759 or miR-Scr. The expression levels of PD-L1 were analysed by western blotting. One representative experiment of three experiments is shown. (E) TNBC cells with or without stable overexpression of PD-L1 were transfected with miR-4759 or miR-Scr. PBMCs were subsequently co-cultured with miR-treated TNBC cells in IncuCyte for 24 h. Apoptotic cells were determined with green fluorescent signals. (F) The relative apoptotic population of miR-4759 treated cells was compared with miR-Scr group. The results represent the mean ± SD. from three independent experiments. Statistical significance was tested by t-test. *, p < 0.05, **, p < 0.01. (G) MDA-MB-231 and BT-549 cells were transfected with miR-4759/miR-Scr or treated with control IgG/anti-PD-L1. PBMCs were subsequently co-cultured with treated TNBC cells in IncuCyte for 48 h. The apoptotic cell population was determined with green fluorescent signals. (H) The relative apoptotic population of different groups was compared with the control group. The results represent the mean ± SD. from three independent experiments. Statistical significance was tested by t-test. *, p < 0.05, **, p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)