Fig 1: LPS/GalN-induced ALF is attenuated in antagomir-21-treated mice. (A) Relative expression levels of miR-21 in liver tissues from various groups were analyzed by quantitative PCR. (B) Serum ALT and AST levels were measured by ELISA in each group. (C) Representative photomicrographs of hematoxylin and eosin-stained sections of liver tissue from each group (magnification, ×50). (D) TargetScan database demonstrated that KLF6 was a potential target of miR-21 with one putative miR-21-5p seed match site. (E) Protein expression levels of KLF6 were evaluated by western blotting of the liver tissues from each groups. (F) Serum IL-23 levels from various groups were measured by ELISA. Protein expression levels of (G) IL-23, and (H) LC3II/LC3I and p62 were evaluated by western blotting in liver tissues from various groups. **P<0.01. ALT, alanine transaminase; ALF, acute liver failure; AST, aspartate transaminase; GalN, D-galactosamine; IL, interleukin; LPS, lipopolysaccharide; miR, microRNA; NC, negative control.
Fig 2: miR-21 promotes the expression of IL-23 via inhibiting KLF6, which promotes autophagy. miR-21 promoted the expression of IL-23 by targeting KLF6 in THP-1-derived macrophages, which regulates autophagy, thus promoting inflammatory storms, and subsequent severe hepatocellular necrosis and acute liver failure. IL, interleukin; KLF6, Krüppel-like-factor-6; miR, microRNA.
Fig 3: miR-21 promotes the expression of IL-23 and inhibits the expression of KLF6. (A) miR-21 mimics, miR-21 mimics NC, miR-21 inhibitor and miRNA inhibitor NC were constructed and transfected into THP-1-derived macrophages; the expression levels of miR-21 were confirmed by qPCR. (B) IL-23 protein expression levels were analyzed in THP-1-derived macrophages post-transfection by western blotting. (C) Levels of IL-23 in the cell supernatants were evaluated by ELISA. (D) Reporter constructs (pGL3-KLF6) with putative WT or MUT 3′-UTRs downstream of the firefly luciferase were cloned and cotransfected into cells with miR-21 mimics NC or miR-21 mimics, and relative luciferase activity was evaluated in the various groups. The relative expression levels of KLF6 were evaluated by (E) qPCR and (F) western blotting in various groups. **P<0.01. IL, interleukin; KLF6, Krüppel-like-factor-6; miR, microRNA; NC, negative control; MUT, mutant; ns, not significant; qPCR, quantitative PCR; WT, wild-type.
Fig 4: KLF6 promotes autophagy, and KLF6 blocks the miR-21-induced increase in IL-23 levels. (A) KLF6 OE plasmid and NC plasmid were transfected into THP-1-derived macrophages, and the expression levels of KLF6 were confirmed by western blotting. (B) Relative protein expression levels of KLF6 were evaluated by western blotting in THP-1-derived macrophages post-transfection. (C) Western blot analysis of LC3II/I, p62, ATG7 and Beclin 1 expression in macrophages post-transfection. (D) IL-23 expression was evaluated in THP-1-derived macrophages post-transfection. (E) ELISA evaluated the expression of IL-23 in the supernatant post-transfection. *P<0.05, **P<0.01, ***P<0.001. IL, interleukin; KLF6, Krüppel-like-factor-6; miR, microRNA; NC, negative control; OE, overexpression.
Supplier Page from Sino Biological, Inc. for Human KLF6 Gene ORF cDNA clone expression plasmid, C-GFPSpark tag