Figure 3 | Cell Death & Disease

Figure 3

From: Silencing NKG2D ligand-targeting miRNAs enhances natural killer cell-mediated cytotoxicity in breast cancer

Figure 3

The mechanisms under miRNA-mediated MICA/B and ULBP2 downregulation. (a) Schematic representation of the miR-20a and miR-20b predicted binding sites in the 3’-UTRs of MICA/B mRNAs and 3’-UTR-mutated alignments. (b) Relative luciferase activity was assessed after transfecting the indicated reporter plasmids (MICA/B-WT 3’-UTR or MICA/B-mut 3’-UTR) into BCap37 cells with mimics of miR-20a, miR-20b or control miRNA (Ctrl). (c) and (d) BC cells were pre-exposed to lipo2000 only (Ctrl), a miR-20a mimic or inhibitor for 48 h. (c) Representative images of the western blotting assay. MiR-20a inversely regulated the expression of p-ERK1/2 (mainly p-ERK2) and ERK1/2 in BCap37 cells. (d) Quantitative PCR assay. MiR-20a inversely regulated the mRNA expression of ERK2 (MAPK1). (e) BCap37 cells were exposed to lipo2000 only (Ctrl), miR-20a mimic (miR-20a) or co-transfection of ERK2-pcDNA and miR-20a mimic (ERK2+miR-20a) for 48 h. Flow cytometry revealed that ERK2 overexpression could reverse miR-20a-mediated ULBP2 downregulation. (f) Representatives images of the western blotting assay. BCap37 cells were exposed to a pcDNA vector or control siRNA (Ctrl), ERK2-pcDNA (ERK) or ERK2-siRNA (siERK). (g) BCap37 cells were treated as shown in (f), and the flow cytometry analysis revealed that ERK2 effectively regulated the expression of ULBP2 but did not affected MICA/B. Error bars represent the S.D. obtained from three independent experiments. *P<0.05, **P<0.01, *** P<0.001

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