Fig. 6
MiR-25-3p targets PHLPP2 and evokes oncogenic AKT-p70S6K signaling. a Relative reporter gene activity of psiCHECK2 vector bearing PHLPP2 3′-UTR fraction 1 (left panel) or fraction 2 (right panel) in PDAC cells co-transfected with increasing amounts (1, 5, and 10 pmol) of miR-25-3p mimic. b Relative reporter gene activity of constructs bearing PHLPP2 3′-UTR fraction 1 (left panel) or fraction 2 (right panel) in PDAC cells co-transfected with 5 pmol of miR-25-3p mimic or its inhibitor. c Reporter gene activity of psiCHECK2 vector bearing PHLPP2 3′-UTR fraction 1 (left panel) or fraction 2 (right panel) or their mutant counterparts in PDAC cells in the presence of 5 pmol miR-25-3p mimic. Results in (a), (b), and (c) represent means ± S.D. from three experiments and each had six replicates. d Stable overexpression or knockdown of miR-25-3p on the levels of PHLPP2 RNA (left panel) and protein (right panel). Results of mRNA represent means ± S.D. from three independent experiments. e RNA immunoprecipitation assays show significantly increased association with Ago2 of both miR-25-3p (left panel) and PHLPP2 mRNA (right panel) in PDAC cells overexpressing miR-25-3p. Results represent means ± S.D. from three independent experiments. f MiR-25-3p evokes AKT-p70S6K signaling via PHLPP2. Knockdown of PHLPP2 substantially enhanced AKT and p70S6K phosphorylation activation in PDAC cells. g Overexpression or knockdown of miR-25-3p had substantial effects on PHLPP2 expression and phosphorylation of its downstream signaling modules. h Knockdown of PHLPP2 expression substantially rescued the activation of AKT and p70S6K suppressed by miR-25-3p knockdown in PDAC cells. Ctrl control, KD knockdown. i Knockdown of PHLPP2 expression significantly reversed the effects of miR-25-3p knockdown on PDAC cell proliferation. Results represent mean ± S.D. from three independent experiments. j Knockdown of PHLPP2 expression significantly reversed the effects of miR-25-3p knockdown on PDAC cell migration and invasion. Left panel shows representative images of transwell assays and right panel shows quantitative statistics. Results are mean ± S.D. from three random fields. All statistic analyses in this figure are Student t test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the corresponding control
