Figure 3.

miR-195 inhibits AEG-1 expression by targeting its 3′-UTR. (A) Putative binding sequence between the miR-195 and wild-type (wt-AEG-1) or mutant-type (mut-AEG-1) 3′-UTR of AEG-1. Mutations were generated in the complementary site that binds to the seed region of miR-195. (B) miR-195 inhibited the activity of a luciferase reporter that contained the wild-type 3′-UTR of AEG-1 (wt-AEG-1). miR-NC or miR-195 mimic was co-transfected with luciferase reporter plasmid that contained either the wild-type (wt) or mutant (mut) 3′-UTR of AEG-1. β-gal signal was used as an internal control to calibrate the differences in the transfection and harvest efficiencies. The luciferase activity of each sample was normalized to the β-gal signal. *P<0.05. (C) AEG-1 mRNA was downregulated in Huh7 cells transfected with miR-195 mimic. mRNA relative expression was measured by reverse transcription-quantitative polymerase chain reaction at 48 h after transfection of miR-NC or miR-195 mimic. The average mRNA expression from miR-NC group is designated as 1. *P<0.05 vs. miR-NC. (D) Expression of miR-195 reduced the protein levels of AEG-1 in Huh7 cells. HCC cells that were transfected with miR-NC or miR-195 mimic for 48 h were analyzed via western blot assay. GAPDH was used as an internal control. miR-195, microRNA-195; AEG-1, astrocyte elevated gene 1; 3′-UTR, 3′ untranslated region; wt-, wild type; mut-, mutant.