Figure 6.

The profibrotic role of Fendrr in liver fibrosis depends on its interaction with STAT2.A, schematic diagram of domain structures of WT STAT2 and STAT2 mutants. B, the CC domain of STAT2 is required for its interaction with Fendrr. AML12 cells were transfected with pCI-STAT2, pCI-STAT2-Mut1, pCI-STAT2-Mut2, and pCI-STAT2-Mut3, respectively. The interaction between Fendrr and STAT2 mutant was examined by RNA pull-down using the biotinylated Fendrr and Western blot. C and D, ectopic expression of STAT2-Mut1 reduced IL-6 expression in hepatocytes. AML12-fendrr cells were transfected with pCI-STAT2 or pCI-STAT2-Mut1 and cultured for 48 h. The expression of STAT2, STAT2-Mut1, and IL-6 were detected by qRT-PCR (C) and Western blot (D), respectively. E, ELISA assay of IL-6 in the medium of AML12-Fendrr cells transfected with pCI-STAT2 or pCI-STAT2-Mut1. F, overexpression of STAT2 mutant protect the mice from CCl₄-induces liver fibrosis. Mice were injected with oil (oil, n = 5), CCl₄ (CCl₄, n = 7), CCl₄ in combination with injection of control lentivirus (CCl4+Ctrl, n = 7), and CCl₄ in combination with injection of lv-STAT2-Mut1 (CCl4+ STAT2-Mut1, n = 7). The injection of lentivirus was started at 2 days before the first CCl₄ injection and last for 4 weeks at a frequency of twice per week. The extent of liver fibrosis was evaluated by H&E and sirius red staining, and immunostaining of collagen I and α-SMA. The scale bar represents 100 μm. G, quantification of the sirius red positive area. H, quantification of Col I staining area. I, quantification of α-SMA staining area. J, quantitative evaluation of hepatic hydroxyproline. The hydroxyproline contents are expressed as μg/mg wet liver weight. K, serum IL-6 was assayed by ELISA. L, assessment of serum ALT levels. M, hepatic Tgfb1, Pdgfa, Col1a1, and Timp1 mRNA expression were examined by qRT-PCR. The results are shown as fold change compared with oil group mice. H, data are the mean ± SD of at least three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and ns, no significant change. α-SMA, smooth muscle α-actin; ALT, alanine aminotransferase; CC, coiled-coil; Col I, collagen I; IL-6, interleukin 6; qRT-PCR, quantitative real-time PCR; TIMP, tissue inhibitors of metalloproteinase.