We next examined the effect of miRNA-mediated NKG2DL downregulation on NK cell recognition. BCap37 cells were transfected with control miRNA (Ctrl) or a miR-20a mimic or inhibitor. NK cells separated from the buffy coat of healthy donors were used in cytotoxicity assays at various effector-to-target (E:T) ratios. Transfection with a miR-20a mimic caused a remarkable reduction in the cytotoxicity activity at different E:T ratios. However, treatment with miR-20a inhibitor could effectively reverse these inhibitory effects (Figure 4a). These results implied that silencing NKG2DL-targeting miRNA could enhance NK cell-mediated cytotoxicity. Importantly, these differences between various treatments were abolished after NKG2D in NK cells was blocked with an anti-NKG2D monoclonal antibody (mAb) (Figure 4c). These results confirmed that the miRNA-mediated NK cell cytotoxicity was mediated through the NKG2D–NKG2DL pathway. The results were then verified using another human BC cell line (MDA-MB-231) as a target cell (Figure 4b) or LAK cells expanded from healthy donors as an effector cell (Supplementary Figure 2f).