Figure 2

miR-425 directly targets PTEN. (A) Reporter assay in HEK293 cells and AGS cells transfected with miR-425 and constructs carrying luciferase cDNA fused to the 3’ UTRs of predicted candidate targets (mean ± SEM). (B) Effects of anti-miR-425 on the luciferase activity of luciferase constructs fused with the 3’ UTRs of PTEN in HEK293 cells and NCI-N87 cells (mean ± SEM). (C, D) Reporter assay in HEK293 cells and AGS cells transfected with luciferase constructs carrying PTEN 3’ UTRs with mutations in the miR-425-binding sites (mean ± SEM). (E) HEK293 cells and AGS cells were transfected with a luciferase construct carrying the wild type PTEN 3’ UTR (LUC-WT) or a construct carrying a mutated PTEN 3’ UTR (LUC-Mut). After 24 h, the cells were treated with IL-1β, and luciferase activity was quantified 24 h after treatment. (F) Western blot and RT-PCR showing PTEN protein levels and mRNA levels in AGS cells after 48 h of miR-425 transfection. (G) AGS cells were transfected with anti-miR-425, and 24 h later, the cells were either left untreated or treated with IL-1β and subsequently harvested 24 h after treatment. Whole cell lysates were immunoblotted with PTEN antibodies. (H) NCI-N87 cells were transfected with anti-miR-425 and harvested 24 h after treatment. Whole cell lysates were immunoblotted with PTEN antibodies. (I) AGS cells were transfected with a plasmid carrying only the open reading frame sequence of PTEN (PTEN-CDS). After 24 h, the cells were treated with IL-1β or miR-425. Whole cell lysates were subjected to immunoblotting after 24 h of treatment. (*p < 0.05; **p < 0.01; ***p < 0.001).