Fig. 3. PTEN is negatively regulated by miR-140-3p target binding in HSC-T6 cells. (A) Potential target sites (red) on PTEN 3′UTR were predicted on TargetScan software and mutated. (B) Luciferase reporter assay was conducted to identify the binding sites. Relative luciferase activity was significantly reduced in cells transfected with PTEN wild type (wt) 3′UTR and miR-140-3p mimic (miR-140-3p). (C) RIP were performed. Dramatically high expression of PTEN were obtained from Ago2, separately. (D) Effect of miR-140-3p on expression of PTEN in cells. Relative expression levels of PTEN were evaluated using Western blotting, and the gray intensity was analyzed. All experiments were performed in triplicate, ^*p<0.05. RIP, RNA immunoprecipitation; PTEN, phosphatase and tensin homolog deleted on chromosome.