Figure 4: MiR-125b-5p regulates Keap1 at posttranscriptional level.

(a) In silico analyses by TargetScan and PicTar predicts 3′-UTR of KEAP1 as a target of miR-125b-5p. (b) Western blot analysis of KEAP1 in mice treated with AAV-Ttr-miR-125b-5p or control AAV. Vinculin (VCL) was used as a loading control. Quantification of western blotting is shown in the right panel. *P<0.05, two-tailed Student’s t-test. (c) Quantitative reverse transcriptase–PCR (qRT–PCR) analysis shows that Keap1 mRNA levels did not change significantly. Not significant (NS), two-tailed Student’s t-test. (d) Luciferase reporter assay confirms the binding of miR-125b-5p with 3′-UTR of Keap1. Primary hepatocytes transfected with miR-125b-5p mimic have lower relative luciferase units (RLUs), whereas hepatocytes transfected with miR-125b-5p inhibitor showed higher RLUs than the control miRNA scramble transfection. *P<0.05, **P<0.01, one-way analysis of variance (ANOVA). (e) Mutated two nucleotides in mouse Keap1 3′-UTR are indicated in red. (f) Luciferase reporter assay using mutated 3′-UTR of Keap1 demonstrates unchanged luciferase activity. Not significant (NS), one-way ANOVA. (g) Western blot analysis of NRF2 in mice treated with AAV-Ttr-miR-125b-5p or control AAV. α-Tubulin was used as a loading control. Quantification of western blotting is shown in the right panel. *P<0.05, two-tailed Student’s t-test. (h) qRT–PCR analysis of Ugt1a6, Gclc, Nqo1 and Gsta2 expression in mice treated with AAV-Ttr-miR-125b-5p or control AAV. *P<0.05, two-tailed Student’s t-test. Data are presented as mean±s.e.m. from at least three independent experiments.