Overexpression of miR669a delays skeletal muscle regeneration in ctx-injured TA muscles. TA muscles of 7-d-old Swiss mice were ctx injured, injected intramuscularly with AAV2/9-nLacZ-miR669a2× (miR669a), and analyzed 7, 14, and 21 d after injury. AAV2/9-nLacZ-miRdsRed2× (miRdsRED) was used as a negative control. (A, B, D, and E) X-gal reaction on miRdsRed (A) and miR669a (D) treated muscle sections. Laminin expression in miRdsRed (B)- and miR669a (E)-treated muscle sections. miR669a-treated muscles showed smaller fibers compared with control muscles. (C, C′, F, and F′) Laminin and β-galactosidase expression on miRdsRed (C and C′)- and miR669a (F and F′)-injected muscle sections. Muscle fibers with central position of β-galactosidase–positive nuclei were predominantly found in miR669a-treated muscles (F′). (G) MyHC, MyoD, and Pax7 expression analysis on protein extracts from miRdsRed- and miR669a-injected muscle. (H) Relative quantification for MyHC, MyoD, and Pax7 expression in miRdsRed- and miR669a-treated muscles. P-values are shown (*, P < 0.01; **, P < 0.05). (I) Morphometric analysis of muscle fibers areas at 7, 14, and 21 d after injury in miRdsRed- and miR669a-injected muscles. It is remarkable that the averaged area of miR669a-treated muscle fibers is extremely reduced (300–500 µm²) compared with the control muscle fibers (1,000–1,500 µm²). Four mice per each group of treatment were independently and statistically analyzed using Wilcoxon-Matt-Whitney test (P < 0.001). Error bars show means ± SEM. Bars: (A, B, D, and E) 50 µm; (C, C′, F, and F′) 100 µm.