Figure 4.

miR-22-3p expression suppresses eEF2K expression in TNBC cells by binding directly to the 3′ UTR of eEF2K mRNA

(A) Prediction of binding sites on the 3′ UTR region of eEF2K mRNA that is bound by miR-22-3p by using four different algorithms (TargetScan, miRDB, DIANA Tools, and microRNA.org). (B) Protein levels of eEF2K upon ectopic miR-22-3p expression in MDA-MB-231 cells. The MDA-MB-231 cells were transfected with the miR-22-3p mimic or control miRNA, and eEF2K protein levels were analyzed using western blotting 72 h after transfection. β-actin was used as a loading control. Band intensities were quantified using densitometry. (C) miR-22-3p transfection decreased eEF2K mRNA expression in MDA-MB-231 cells. The relative eEF2K mRNA expression levels in these MDA-MB-231 cells were analyzed using qRT-PCR with specific primers 48 h after transfection with miR-22-3p or a control miRNA. Data were normalized to the expression of β-actin and are presented as mean (±SD) from three independent experiments. ∗∗p < 0.001. (D) Two predicted miR-22-3p-binding sites in the 3′ UTR of human wild-type eEF2K mRNA and their sequences. Mutations that were introduced in the seed miR-22-3p-binding sequence of the full-length eEF2K 3′ UTR are shown in red. (E) Luciferase reporter assay results showing that miR-22-3p directly targets the eEF2K 3′ UTR-luciferase reporter pEZX-MT06 (with wild-type or mutant [MT] miR-22-3p-binding sites) in cells incubated with a miR-22-3p mimic for 48 h before analysis. The firefly luciferase activity of the reporter was normalized to the internal Renilla luciferase activity. The data are presented as mean (±SD) from three independent experiments. ∗∗∗∗p < 0.0001.