Figure 4.

PTEN is a direct target of miR-26a. (A) Putative binding site of miR-26a and PTEN with (B) mut and wt 3′ UTRs. (C) Luciferase assay of 293 cells co-transfected with firefly luciferase constructs containing the PTEN wt or mut 3′-UTRs and miR-26a mimics, mimics NC, miR-26a inhibitor or inhibitor NC, as indicated (n=3). Data are presented as the mean ± standard deviation of three independent experiments. ^**P<0.01, vs. mimics NC, ^##P<0.01, vs. inhibitor NC. (D) Protein expression of PTEN following transfection with miR-26a mimic or miR-26a inhibitor was measured by western blot analysis. (E) VSMCs were incubated with 50, 100, 200 or 400 µM H₂O₂ for 6 h, and the mRNA expression of PTEN was determined by RT-qPCR analysis. (F) VSMCs were transfected with miR-26a mimics for 2 h, followed by treatment with 200 µM H₂O₂ for 6 h, and the mRNA expression of PTEN was determined by RT-qPCR analysis. Data are presented as the mean ± standard deviation of three independent experiments. ^*P<0.05 and ^**P< 0.01, vs. control group; ^##P<0.01, vs. H₂O₂ + mimics NC group. VSMCs, vascular smooth muscle cells; H₂O₂, hydrogen peroxide; miR, microRNA; PTEN, phosphatase and tensin homolog; wt, wild-type; mut, mutant; reverse transcription-quantitative polymerase chain reaction analysis; NC, negative control.