Fig. 4.

MiR-193b directly targets FASN, and the inhibition of miR193b reduces metformin-mediated reduction of FASN. a MiR-193b is predicted to target a sequence in the 3′UTR of FASN. b Cells were transfected with transfection reagent alone (mock), negative control mimic, miR-193b mimic, a 193b antagomiR (α193), or combination antagomiR and mimic. Twenty-four hours later, reporter vector containing the FASN 3′UTR downstream of the luciferase gene or an empty vector (EV) and renilla luciferase vector (as a loading control) were transfected. Dual luciferase reporter assay was performed 24 h later. Shown are relative luciferase units (luciferase divided by the renilla values) normalized to the mock condition. For FASN 3′UTR compared to EV, *P < 0.05, two-way ANOVA with Dunnett’s multiple comparisons test. c MDA-MB-468 (left) and BT-549 (right) cells stably expressing a miR-193b antagonist (miR-193b-Zip) or a scrambled control (miR-Scr-Zip) were seeded in 5 mM glucose prior to 5 mM metformin treatment for 24 h. Immunoblot analysis on whole cell lysates was performed for FASN, P-ACC, ACC, P-ACLY, ACLY, AMPK, and IGFIR, with β-actin shown as a loading control. Data are representative of three independent experiments