Efficacy of miR-21 silencing in preventing collagen accumulation after injury and treating muscular dystrophy by reversing fibrosis. (A) Prevention of fibrosis. TA muscles of WT mice were lacerated, and an antagomir for miR-21 (Ant-miR-21), a mimic for miR-21 (Mimic-miR-21), or Scramble oligomiR (Scramble; used as a negative control) was injected daily for 3 d starting at day 16 after laceration (before significant collagen deposition, which peaks at 21 d), and muscles were collected at day 21 after laceration. Sirius red staining on muscles of treated mice is shown. Bar, 25 µm. (B) Collagen protein accumulation and miR-21 expression were quantified in noninjured and 21-d lacerated muscles of WT mice; similarly, fibronectin was analyzed by immunofluorescence with a specific antibody (see representative panels in Fig. S1 E), and the positively stained areas were quantified by image analysis (*, P < 0.001; means ± SEM; n = 5 for each group). (C) Therapy for fibrosis. Sirius red staining of gastrocnemius muscle from 24-mo-old mdx mice (aged mdx) after administration of anti–mir-21 (or Scramble) every other day for 1 mo before collection of the muscles (left) and from 3-mo-old mice (young mdx) after administration of Mimic-miR-21 or Scramble with the same protocol (right) is shown. Bar, 25 µm. (D) As in B, collagen accumulation and miR-21 expression were quantified; similarly, fibronectin was analyzed by immunohistochemistry (see representative panels in Fig. S1 F) and quantified (*, P < 0.001; means ± SEM; n = 5 for each group). (E) The mRNA expression levels of Coll I, TIMP-1, and FN1 in the muscles of mice described in D are shown (*, P < 0.001 vs. WT mice; means ± SEM; n = 5 for each group). (F) H/E staining of gastrocnemius muscle sections from 24-mo-old mdx mice treated with Ant-miR-21 (or Scramble) for 1 mo. Bar, 50 µm.