To further investigate the effects of NKG2DL-targeting miRNAs on BC immunogenicity in vivo, we conducted a short-term lung clearance assay using C57BL/6 mice (Figure 5a). Although human NKG2DLs are different from mouse NKG2DLs,29 it is feasible to detect the activity of NKG2D-associated functions on human malignant cells in an immunocompetent mouse model.30 BCap37 cells treated with miR-20a mimic (which decreased NKG2DLs expression) were less susceptible to immune cell targeting. Thus, fewer cells were killed and more cells were detected in the harvested lungs than in those from the group transfected with control miRNA (Ctrl group) (fluorescence intensity (FI), mean±S.D. 143.1±10.53%). On the other hand, when NKG2DLs were increased with a miR-20a inhibitor, fewer labeled BCap37 cells were detected in the lungs compared with the Ctrl group (33.7±6.2%, Figure 5b top and 5c left). These results were confirmed using another BC cell line (MDA-MB-231) (mimic versus Ctrl 216.9±18.9%, inhibitor versus Ctrl 58.2±5.5%, Figure 5b bottom and 5c right). Importantly, the differences between different groups were abolished when NKG2Ds were blocked in vivo with an anti-NKG2D mAb in the BC cells (Figure 5d). The differences were reduced when NK cells were depleted with an anti-NK1.1 mAb in the BC cells. Interestingly, in the anti-NK1.1 group, BCap37 cells showed an increase of detection after treated with a miR-20a inhibitor, which required further observation. Taken together, these results confirmed that the miRNA-mediated regulation of immune recognition was through the NKG2D–NKG2DL pathway.