Figure 9: Dyregulated Mϕ miR-142-5p and miR-130a-3p enhance liver fibrosis.

(a–f) Mice were intravenously injected with LNA-modified miR-142-5p ASO, miR-130a-3p mimic or both every 3 days after CCL₄ challenge. Mice were sacrificed after 6 weeks (mean±s.e.m., n=8 mice/group; *P<0.05, **P<0.01 by two-tailed Student’s t-test). (a) Representative images of Sirius Red staining and α-SMA immunochemical staining of mouse liver sections. Inserts show higher magnification. Scale bar, 50 μm. (b) Expression of miR-142-5p and miR-130a-3p in hepatic macrophages was evaluated by qRT–PCR. (c) SOCS1 level, STAT6 phosphorylation and PPARγ level in hepatic macrophages were determined by western blot analysis (n=3). (d) Expression of CD206 in hepatic macrophages was evaluated by flow cytometry analysis. (e) Expression of CCL17, FIZZ1 and TGF-β1 in hepatic macrophages was evaluated by qRT–PCR. (f) Hydroxyproline in the livers of mice was measured by the Sircol assay (g) C57BL/6 wild-type or Ccr2^−/− mice were gavaged with CCL₄ every 5 days for 6 weeks.1 × 10⁶ bone marrow-derived monocytes were treated with IL-13 for 24 h, and injected intravenously into CCL₄-treated Ccr2^−/− mice either at weeks 0, 1, and 2 (0–2 weeks) or at weeks 3, 4 and 5 (3–5 weeks) of treatment. Hydroxyproline was measured in the livers 6 weeks after CCL₄ challenge (mean±s.e.m., n=8 mice/group P<0.05, **P<0.01, ***P<0.001 by two-tailed Student’s t-test). (h) Bone marrow-derived monocytes were transduced with control, miR-142-5p ASO or miR-130a-3p mimics or both. After 24 h, the monocytes were stimulated with IL-13 for 24 h and injected intravenously into CCL₄-treated Ccr2^−/− mice at weeks 3–5 of treatment. (left) Schematics of experimental designs. (right) Mice were killed 6 weeks after CCL₄ challenge and hydroxyproline was measured in the livers. (Mean±s.e.m., n=8 mice/group P<0.05, **P<0.01, ***P<0.001 by two-tailed Student’s t-test.) (i) Representative H&E staining, FISH for miR-142-5p, miR-130a-3p or scramble control and co-immunostaining for CD68 in normal and cirrhotic human liver tissues. Scale bars, 50 μm. Quantification of miR-142-5p⁺ and miR-130a-3p⁺macrophages (normal liver group, n=24; cirrhotic liver group, n=39, mean±s.e.m., **P<0.01, ***P<0.001 by two-tailed Student’s t-test.).