Fig 2: miR-503 target identification in MCF-7 and MCF-7/ADR cells. Luciferase reporter assays confirmed that eIF4G was a direct target of miR-503 in (A) MCF-7 and (B) MCF-7/ADR cells (*P<0.05). miR, microRNA; eIF4G, eukaryotic translation initiation factor 4-γ 1; UTR, untranslated region; NC, negative control.

Fig 3: Requirement for the eIF4F complex. (A) Schematic cartoon of the eIF4F complex, which is composed of eIF4E, eIF4G and eIF4A. The interactions with the PIC, cap analog, L protease and hippuristanol are depicted on the figure. (B) RRL was treated with cap analog (1 µM) or with water (ctrl) and then programmed with capped and polyadenylated globin-Renilla and SARS-CoV-2 leader or 5′UTRORF1ab-Renilla mRNAs. Products of expression were quantified by luciferase assays and normalized to the control condition (set at 100%). (C–F) Untreated RRL was pre-incubated alone (ctrl) or in presence of different amounts of L protease (µL) as indicated, and then programmed with (D) globin-Renilla, (E) EMCV IRES-Renilla, (F) SARS-CoV-2 leader or 5′UTRORF1ab-Renilla mRNAs. Translation products were (C) resolved by a 7.5% SDS-PAGE and then transferred to a PVDF membrane for Western blotting analysis against eIF4G, or (D–F) analyzed by luciferase assay and normalized to the control condition (set at 100%). (G) RRL pre-treated with DMSO or increasing amounts (µM) of hippuristanol (hipp.) was programmed for 30 min with globin-Renilla, HCV-IRES Renilla, SARS-CoV-2 leader or ORF-1ab 5′UTR-Renilla mRNAs. Expression products were analyzed by luciferase assay and normalized to the control condition (set at 100%). Values shown are the mean (+/− S.D.) for three independent experiments.

Fig 4: miR-503 negatively regulates the expression of ABCB1, ABCC1 and ABCG2, and increases MCF-7/ADR sensitivity to ADM, TAX and TAM. (A) After 48 h, fluorescence microscopy was used to detect transfection efficiency, and the transfection efficiency of the miR-503 and miR-503 inhibitor was >35%. (B) The expression of eIF4G mRNA was downregulated, while the expression levels of ABCB1, ABCC1 and ABCG2 mRNA in the miR-503 mimic-transfected MCF-7/ADR cells were not changed in comparison with the control cells and untreated cells, as determined by reverse transcription-quantitative polymerase chain reaction (**P<0.01 vs. negative microRNA or mock group). (C) miR-503 negatively regulated the expression of eIF4G, ABCB1, ABCC1 and ABCG2, as determined by western blot assay. (D) miR-503 inhibited drug resistance, as determined by MTT assay (*P<0.05, **P<0.01). (E) Cell cycle distributions were analyzed with flow cytometry, MCF-7/ADR cells transfected with miR-503 were arrested in G0/G1 phases. (F) miR-503 increased the percentage of apoptosis cells treated with anticancer drugs (20 µM ADM, 10 µM TAM and 5 µM TAX). ABCB1, ABC sub-family B member 1; ABCC1, ABC sub-family C member 1; ABCG2, ABC sub-family G member 2; ADM, Adriamycin; TAM, tamoxifen; TAX, taxol; miR, microRNA; eIF4G, eukaryotic translation initiation factor 4-γ 1; NC, negative control.

Fig 5: eIF4G siRNA negatively regulated the expression of ABCB1, ABCC1 and ABCG2, and increased MCF-7/ADR sensitivity to ADM, TAX and TAM. (A) Transfection efficiency of eIF4G siRNA and NC siRNA. After 48 h, fluorescence microscopy was used to detect transfection efficiency, and the transfection efficiency was >35%. (B) eIF4G siRNA negatively regulated the expression of ABCB1, ABCC1 and ABCG2, as assessed by western blot assay. (C) The effect of eIF4G siRNA on mRNA. (D) eIF4G siRNA reduced cell viability compared with siRNA NC or untransfected cultures, as determined by MTT assay (*P<0.05, **P<0.01). (E) Cell cycle distributions were analyzed by flow cytometry, and MCF-7/ADR cells transfected with eIF4G siRNA were arrested in G0/G1 phases. (F) eIF4G siRNA increased the percentage of apoptotic cells treated with anticancer drugs (20 µM ADM, 10 µM TAM and 5 µM TAX). eIF4G, eukaryotic translation initiation factor 4-γ 1; siRNA, small interfering RNA; ABCB1, ABC sub-family B member 1; ABCC1, ABC sub-family C member 1; ABCG2, ABC sub-family G member 2; ADM, Adriamycin; TAM, tamoxifen; TAX, taxol; NC, negative control.