Fig. 6

MiR-138–2 enhances DBZ-mediated inhibition of Notch pathway. a CCK-8 assay. 786–0 (left) and CAKI-1 (right) cells were exposed to the indicated concentrations of DBZ. At the indicated time points after treatment, cell viability was examined by CCK-8 assay (n = 3). b Immunoblotting. 786–0 (left) and CAKI-1 (right) cells were treated with the vehicle or with DBZ (at a final concentration of 10 μM/ml). After treatment, whole cell lysates were prepared and analyzed by immunoblotting. β-tubulin was used as a loading control. c qPCR. 786–0 (left) and CAKI-1 (right) cells were treated as in (b). After treatment, total RNA was isolated and analyzed for pre-miR-138–2, miR-138-5p and miR-138–2-3p by qPCR (n = 3). d CCK-8 assay. 786–0 (upper) and CAKI-1 (lower) cells were transfected with or without miR-138–2 expression plasmid. After transfection, cells were treated with DBZ (5 μM/ml) or left untreated. At the indicated time periods after treatment, cell viability was assessed by CCK-8 assay (n = 3). e Migration assay. 786–0 (left) and CAKI-1 (right) cells stably overexpressing NOTCH1 or HES1 were transiently transfected with or without miR-138–2 expression plasmid. Cells were then subjected to transwell assay (n = 3). f CCK-8 assay. 786–0 (left) and CAKI-1 (right) cells stably overexpressing NOTCH1 or HES1 were treated as in (e). At the indicated time points after transfection, cell viability was examined by CCK-8 assay (n = 3). g Proposed model for miR-138–2 modulates the NOTCH signaling pathway in Normal cell (left) and HES1-miR-138–2-NOTCH1 positive feedback loop in Tumor cell (right). In normal cells, the mature miR-138-5p and miR-138–2-3p inhibited the mRNA expression of MAML1, APH1AH and NOTCH1, respectively, to prevent excessive cell proliferation and migration. However, in tumor cells, over-activated HES1 transcription inhibits miR-138–2 gene, enabling tumor to activate the NOTCH1-HES1 axis without control, promoting tumor proliferation and metastasis