Fig. 2

miR-32-5p targets PTEN and activates the PI3K/Akt pathway. a miR-32-5p and its putative binding sites in the 3’-UTR of PTEN. Mutant miR-32-5p binding sites were generated in the complementary site for the seed region of miR-32-5p. b Effects of miR-32-5p on luciferase activity in Bel7402, Bel/5-FU, and HEK293T cells carrying the WT and MuT 3’-UTR of PTEN. n = three independent experiments, *p < 0.05 by Student’s t-test. c The expression of miR-32-5p and PTEN mRNA according to the increasing dose of miR-32-5p mimics and inhibitor by real-time PCR. The miR-32-5p level is normalized to the corresponding inner control U6, the PTEN level is calculated using the corresponding internal control GAPDH and the miRs/U6 or PTEN/GAPDH in Bel7402 cells is set as 1.0-fold. n = three independent experiments, *p < 0.05, **p < 0.01 by Student’s t-test. d, e The expression of PTEN, phosphorylation of Akt, P70S6K, and mTOR according to the dose of miR-32-5p mimics and inhibitor by Western blots; the up- or down-regulation of PTEN replicates the effects of miR-32-5p inhibitor or mimics, respectively, and PTEN-expressing vector or siPTEN reverses the expression of PTEN and phosphorylation of Akt, P70S6K and mTOR in the cells transfected miR-32-5p mimics or inhibitor, respectively. miR-32-5p mimics and PTEN-targeting siRNA rescue the expression of p-Akt, p-P70S6K and p-mTOR after WM treatment. The relative expression of PTEN is normalized to the level of the corresponding internal control β-actin, and the relative expression of p-Akt, p-P70S6K and p-mTOR is normalized to the levels of Akt, P70S6K, and mTOR, respectively. WT, wild-type; MuT, mutant; p-Akt, phosphorylated Akt; p-P70S6K, phosphorylated P70S6K; p-mTOR, phosphorylated mTOR; siPTEN, PTEN siRNA; WM, Wortmannin