Figure 6: MiR-125b-5p protects human hepatocytes against ALF. | Nature Communications

Figure 6: MiR-125b-5p protects human hepatocytes against ALF.

From: MicroRNA-125b-5p mimic inhibits acute liver failure

Figure 6

(a) Western blotting shows reduction of KEAP1 protein levels in primary human hepatocytes transfected with miR-125b-5p mimic. (b) Quantification of western blotting shown in a. *P<0.05, two-tailed Student’s t-test. (c) Detection of KEAP1 mRNA levels by quantitative reverse transcriptase–PCR (qRT–PCR) in primary human hepatocytes transfected with miR-125b-5p mimic. (d) Luciferase reporter assay confirms the binding of miR-125b-5p with 3′-UTR of human KEAP1. Primary human hepatocytes transfected with miR-125b-5p mimic show lower relative luciferase units (RLUs), whereas those transfected with miR-125b-5p inhibitor showed higher RLUs than the scramble miRNA transfection. *P<0.05, one-way analysis of variance (ANOVA). (e) Mutated two nucleotides in human 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 primary human hepatocytes transfected with miR-125b-5p mimic. Vinculin was used as a loading control. (h) Quantification of western blotting is shown in g. *P<0.05, two-tailed Student’s t-test. (i) qRT–PCR analysis of UGT1A6, GCLC, NQO1 and GSTA2 expression in primary human hepatocytes transfected with miR-125b-5p mimic or inhibitor. *P<0.05, **P<0.01, two-tailed Student’s t-test. (j) GSH/GSSG assay and (k) TUNEL assay revealed reduced hepatocyte damage in miR-125b-5p mimic-treated human hepatocytes after APAP-induced hepatic toxicity. Scale bars, 200 μm. *P<0.05, one-way ANOVA. (l) Quantification of TUNEL staining shown in k. *P<0.05, one-way ANOVA. (m) Caspase-3/7 activity assay and (n) TUNEL assay showed less apoptotic cells in miR-125b-5p mimic-treated human hepatocytes after FAS-induced hepatic damage. Scale bars, 200 μm. *P<0.05, **P<0.01, one-way ANOVA. (o) Quantification of TUNEL staining shown in n. *P<0.05, **P<0.01, one-way ANOVA. Data are presented as mean±s.e.m. from at least three independent experiments.

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