Fig. 4

MiR-371b-5p utilizes PTEN to orchestrate the PI3K/Akt signaling in hiPSC-ATIICs. a The predicted RNA duplexes of miR-371b-5p target site in the 3′UTR of PTEN (top panel). The underlined nucleotides (UUUGA) in the target site (top panel) were replaced with GGGUC in the mutant 3′UTR construct (bottom panel). b Schematic structure of dual-luciferase miRNA target reporter, in which wt or mutant PTEN 3′UTR was inserted into the Pmel and XbaI sites downstream of firefly luciferase (Luc2). c Normalized firefly luciferase activity using the dual-luciferase miRNA target reporter with a wt or mutant PTEN 3′UTR in hiPSC-ATIICs co-transfected with miR-371b-5p mimic. d QRT-PCR analysis of mRNA expression of PTEN in hiPSC-ATIICs transfected with various doses of miR-371b-5p mimic as indicated. The expression levels were normalized to 18 s and presented as percentages of control (left panel). PTEN protein expression of each group of miR-371b-5p mimic-transfected hiPSC-ATIICs was analyzed by Western blot using GAPDH as an internal control (right panel). e The miR-371b-5p mimic-transfected hiPSC-ATIICs were incubated for 24 hours with and without Rosiglitazone (20 μM). The protein extracts were prepared for Western blot analysis of PTEN, Akt/pAkt, GSK3β/pGSK3β, FOXO3/pFOXO3, and FOXO1/pFOXO1 with GAPDH as control (left panel), and the number of viable cells in the cultures was counted 2 days after transfection (right panel). ATIICs alveolar epithelial type II cells