We found that ULBP2 had no predicted binding sites but was still effectively regulated by the tested miRNAs in different breast cell lines (Figures 2e–g and Supplementary Figure 2b and c). Both ERK and AKT are known to be involved in the regulation of ULBP2 expression.24, 25, 26 In addition, members of the miR-17-92 cluster target and inhibit the MAPK/ERK signaling pathway,27, 28 and miR-20a directly targets MAPK1 (ERK2) and inhibits its expression in BC cells (Wengong Si et al. unpublished data). For these reasons, the expression and activation status of the MAPK/ERK pathway were studied in untreated and miRNA-treated BCap37 cells. A western blotting assay showed that compared with the control treatment, the miR-20a mimic effectively inhibited the expression of p-ERK1/2 (mainly p-ERK2) and total ERK1/2. Moreover, the miR-20a inhibitor enhanced the expression of p-ERK1/2 and total ERK1/2, whereas neither the miR-20a mimic nor inhibitor affected AKT and p-AKT expression (Figure 3c). This miRNA-mediated downregulation of ERK2 was typically associated with a decrease in ERK2 (MAPK1) mRNA transcripts (Figure 3d). We confirmed these results by assessing the effects of miR-20b, miR-93 and miR-106b on ERK2 protein and mRNA expression in BCap37 cells. (Supplementary Figure d and e). To clarify the role of the MAPK/ERK signaling pathway in ULBP2 regulation, we silenced ERK2 expression by siRNA or increased constitutive ERK2 expression with ERK2-pcDNA transfection in BCap37 cells (Figure 3f). Flow cytometry analysis showed that the inhibition of ERK2 effectively downregulated the expression of ULBP2 but not MICA/B, whereas the overexpression of ERK2 increased the ULBP2 expression (Figure 3g). Moreover, ERK2 overexpression could reverse miR-20a-mediated ULBP2 downregulation in BCap37 cells (Figure 3e). These findings suggest that downregulated ULBP2 expression in miRNA-treated BC cells results from the MAPK/ERK signaling pathway inhibition.