Figure 4
miR-491-5p by targeting EGFR downregulates AKT and MAPK pathways leading to BIM induction and dephosphorylation in IGROV1-R10 cells. (a) IGROV1-R10 cells were transfected with miR-491-5p or miR-Ctrl for 72 h. Total cell extracts were assessed for levels of both phosphorylated AKT (p-AKT) and ERK (p-ERK), both total AKT and ERK and EGFR by western blotting. β-actin level is shown as loading control. (b) Schematic representation of 3′-UTR EGFR mRNA with its predicted binding sites of hsa-miR-491-5p. Luciferase reporter constructs containing human EGFR 3′-UTR wild-type (WT) or mutant deletion of putative binding site for miR-491-5p were transfected into SKOV3 cells concomitantly with miR-491-5p or the control miRNA (miR-Ctrl). Luciferase assays were carried out 24 h after transfection. Firefly luciferase activity was normalized to renilla luciferase. Data are presented as mean±S.D. of three independent experiments. (c) IGROV1-R10 cells were treated with cetuximab (100 μg/ml) for 72 h and protein extracts were analysed by western blot with indicated antibodies. (d and e) IGROV1-R10 cells were transfected with siRNA against BCL-XL (siRNA-BCL-xL) or siRNA control (siRNA-Ctrl) followed by treatment with DMSO or with 7.5 μM LY294002 (PI3K inhibitor) or 2 μM CI-1040 (MEK inhibitor) or both pharmacological inhibitors (LY294002+CI-1040) for 48 h. (d) Representative cell morphology and DNA content histograms obtained by flow cytometry are shown. The percentage of cells in sub-G1 phase was indicated for each condition. Similar results were obtained in three independent experiments. (e) Protein lysates were immobloted with the indicated total and phospho-specific antibodies. β-actin level is shown as loading control. (f) IGROV1-R10 cells were transfected with siRNA against BCL-XL (siRNA-BCL-XL) or siRNA control followed by a treatment with LY294002+CI-1040 for 48 h. Whole-cell lysates were analysed by western blotting for PARP (native and cleaved forms) and caspase-3 (pro and cleaved (active) forms)
