Figure 5.

miR-718 targets PTEN for degradation. A, Schematic of the seed sequence of miR-718 within the 3′ UTR of PTEN. B, the PTEN-3′ UTR-Luc reporter construct was co-transfected with miR-718 expression vector, miR-146 expression vector, or miRNA negative control (Neg. Con) into 293T cells. After 48 h, 293T cells were lysed, and the normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) was calculated. Data shown represent the mean ± S.D. of triplicate determinations and are representative of three independent experiments with similar results. C, whole cell extracts from Con miR and miR-718 macrophages stimulated with 100 ng/ml LPS were analyzed for PTEN, total Akt, and phosphorylated Akt (P-Akt-Ser⁴⁷³) protein expression by immunoblotting. Med, medium. D, Con miR and miR-718 macrophages stimulated with 100 ng/ml LPS for 2 h were fixed, permeabilized, and stained with PTEN antibody and DAPI (Nucleus) and subjected to confocal microscopy. E, Con miR and miR-718 macrophages stimulated with 100 ng/ml LPS for 2 h were fixed, permeabilized, and stained with phosphorylated Akt (P-Akt) antibody and DAPI (Nucleus) and subjected to confocal microscopy. In D and E, images are representative of at least 10 fields of view and three independent experiments. Scale bars = 20 μm. F, immortalized Con miR, miR-718, Irak1/miR-718-dKO, and TLR4-KO macrophages were pretreated with 10 μm Akt inhibitor IV and then stimulated with LPS for 2 h. TNFα production was measured by ELISA. The data shown represent the mean ± S.D. of triplicate determinations and are representative of three independent experiments with similar results. *, p < 0.05.