Figure 4.

The miRNA miR-877 downregulated the expression of eEF2K by targeting the 3′-UTR of eEF2K. (A) Sequence alignment of the wild-type and mutant eEF2K 3′-UTR, indicating the potential binding sites for miR-877. (B) Luciferase reporter assays revealed a reduction in reporter activity following the transfection of the wild-type eEF2K 3′-UTR reporter construct into the 786-O and ACHN RCC cells overexpressing miR-877. The eEF2K 3′-UTR mutant and control constructs did not exhibit any effect on reporter activity. A Renilla luciferase construct was co-transfected into the cells as an internal control. The normalized luciferase activity of the control construct in each experiment was set to 1. The data represent the mean ± standard error. *P<0.01 vs. control. The (C) mRNA and (D) protein levels of eEF2K were examined by RT-qPCR and western blot analysis, respectively, in RCC 786-O and ACHN cells overexpressing miR-877. The mRNA data were normalized to the levels of GAPDH, which was used as a loading control in western blot analysis. *P<0.01 vs. control miRNA. miR, microRNA; eEF2K, eukaryotic elongation factor-2 kinase; UTR, untranslated region; RT-qPCR, reverse transcription-polymerase quantitative chain reaction; NC, negative control; WT, wild-type; mut, mutant.