Figure 1. Dysregulated miR-21 expression in dystrophic muscle of DMD patients and mdx mice is associated with fibrotic outcome. (A) A representative example of increased collagen deposition (fibrosis), as detected by Sirius red staining of TA muscle of WT mice examined at 21 d after tissue injury induced by laceration. Bar, 25 µm. (B) Quantification of fibrosis (collagen levels) and miR-21 expression in muscles of WT mice before and at the indicated time points after laceration (*, P < 0.001 vs. 0 d; means ± SEM; n = 5 for each group). (C) miR-21 expression levels were analyzed in the diaphragm muscle of 0.5-, 1-, 3-, 8-, 12-, and 24-mo-old mdx mice by quantitative PCR. mdx mice values were normalized with respect to values of aged-matched WT mice (*, P < 0.001 vs. 0.5 mo; means ± SEM; n = 5 for each group). (D) A representative example of Sirius red staining of diaphragm muscle sections of 3-mo-old WT mice and of mdx mice at 3, 8, and 24 mo of age. Bar, 25 µm. (E) Collagen content was analyzed in the diaphragm muscle at the indicated ages in mdx and WT mice of the same ages (*, P < 0.001 vs. WT; means ± SEM; n = 4 for each group). (F) mRNA from diaphragms of mdx mice (and WT mice) of 3 and 12 mo of age was analyzed by quantitative PCR. The expression levels of fibrosis/ECM-related genes, collagen I (Coll I), and TIMP-1 are shown. Results are expressed as fold induction overexpression levels in WT mice (*, P < 0.01; means ± SEM; n = 4 for each group). (G) Quantification of fibroblasts after detection by immunohistochemistry using antibodies against FSP-1 and TCF-4 in the diaphragm of WT and mdx mice. Data obtained from both markers were averaged and expressed as a fold increase in mdx versus WT muscles (*, P < 0.001; means ± SEM; n = 4 for each group). (H) Serial sections of mdx diaphragms were subjected to ISH with a specific probe for miR-21 expression or a scrambled control probe (ISH in WT samples is shown in Fig. S1 D), immunostaining using antibodies specific for TCF-4, Sirius red staining for collagen detection, and H/E. A blood vessel is outlined (dashed line) in all pictures as a reference. Squares indicate the insets shown at the right at higher magnifications. Bars, 25 µm. (I) A representative example of Sirius red staining in sections of muscle biopsies from DMD patients and healthy control subjects. Bar, 25 µm. (J) Collagen accumulation and miR-21 expression were analyzed in muscle biopsies of DMD patients and healthy controls (*, P < 0.01; means ± SEM; n = 7 patients 6–12 yr of age; n = 6 healthy 9–15 yr of age).
Figure 1.

Dysregulated miR-21 expression in dystrophic muscle of DMD patients and mdx mice is associated with fibrotic outcome. (A) A representative example of increased collagen deposition (fibrosis), as detected by Sirius red staining of TA muscle of WT mice examined at 21 d after tissue injury induced by laceration. Bar, 25 µm. (B) Quantification of fibrosis (collagen levels) and miR-21 expression in muscles of WT mice before and at the indicated time points after laceration (*, P < 0.001 vs. 0 d; means ± SEM; n = 5 for each group). (C) miR-21 expression levels were analyzed in the diaphragm muscle of 0.5-, 1-, 3-, 8-, 12-, and 24-mo-old mdx mice by quantitative PCR. mdx mice values were normalized with respect to values of aged-matched WT mice (*, P < 0.001 vs. 0.5 mo; means ± SEM; n = 5 for each group). (D) A representative example of Sirius red staining of diaphragm muscle sections of 3-mo-old WT mice and of mdx mice at 3, 8, and 24 mo of age. Bar, 25 µm. (E) Collagen content was analyzed in the diaphragm muscle at the indicated ages in mdx and WT mice of the same ages (*, P < 0.001 vs. WT; means ± SEM; n = 4 for each group). (F) mRNA from diaphragms of mdx mice (and WT mice) of 3 and 12 mo of age was analyzed by quantitative PCR. The expression levels of fibrosis/ECM-related genes, collagen I (Coll I), and TIMP-1 are shown. Results are expressed as fold induction overexpression levels in WT mice (*, P < 0.01; means ± SEM; n = 4 for each group). (G) Quantification of fibroblasts after detection by immunohistochemistry using antibodies against FSP-1 and TCF-4 in the diaphragm of WT and mdx mice. Data obtained from both markers were averaged and expressed as a fold increase in mdx versus WT muscles (*, P < 0.001; means ± SEM; n = 4 for each group). (H) Serial sections of mdx diaphragms were subjected to ISH with a specific probe for miR-21 expression or a scrambled control probe (ISH in WT samples is shown in Fig. S1 D), immunostaining using antibodies specific for TCF-4, Sirius red staining for collagen detection, and H/E. A blood vessel is outlined (dashed line) in all pictures as a reference. Squares indicate the insets shown at the right at higher magnifications. Bars, 25 µm. (I) A representative example of Sirius red staining in sections of muscle biopsies from DMD patients and healthy control subjects. Bar, 25 µm. (J) Collagen accumulation and miR-21 expression were analyzed in muscle biopsies of DMD patients and healthy controls (*, P < 0.01; means ± SEM; n = 7 patients 6–12 yr of age; n = 6 healthy 9–15 yr of age).

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