Figure 5

Ad-miR-20a delays skeletal muscle regeneration after the CTX injury. C57BL/6 mice were injected in their tibialis anterior muscles with CTX. Adenoviruses expressing miR-20a (Ad-miR-20a) or control (Ad-miR-NC) were injected into the tibialis anterior muscles 10 days prior to the CTX injection. The tibialis anterior muscles were collected on 0, 1, 3, 5 and 10 days post-CTX injury. (a) Quantitative PCR shows that the injection of Ad-miR-20a increases miR-20a in the mouse tibialis anterior muscles 10 days after the adenoviruses injection. qRT-PCR of miR-20a normalized to SNORNA234 is shown relative to control adenovirus-injected samples. The error bars depict the means±S.D. of the samples from six mice. *P<0.05. (b, left) Injection of Ad-miR-20a in the tibialis anterior muscles downregulates the ENH1 protein level as detected by western blot on post-CTX injury day 0 and 5 (top panel). M1, mouse 1; M2, mouse 2; M3, mouse 3. Beta-tubulin served as the loading control. (Right) The ENH1 proteins were quantified, and the data were expressed relative to the control adenovirus-injected (Ad-miR-NC) samples. The data are presented as the means±S.D. of the samples from five mice. **P<0.01 and ***P<0.001. (c) H&E images of the tibialis anterior muscle cross-sections of mice injected with Ad-miR-NC or Ad-miR-20a on 5 and 10 days post-CTX injury. Arrows indicate the regenerating myofibers. Scale bar, 200 μm. (d, left) Immunostaining of Ki67 in the tibialis anterior muscle on day 3 after the CTX injury. Scale bar, 200 μm. (Right) Quantification of Ki67-positive cells from 10 random fields per sample. The error bars depict the means±S.D. of the samples from six mice. *P<0.05. (e, left) Western blot for PCNA in the tibialis anterior muscles on day 1 after the CTX injury. (Right) Quantification of PCNA protein levels. Ad-miR-20a increased the protein levels of PCNA in the tibialis anterior muscle. The results were normalized to beta-tubulin and expressed relative to the control. The error bars depict the means±S.D. of samples from five mice. *P<0.05. (f) Ad-miR-20a downregulated the protein expression of myogenin in the tibialis anterior muscles on day 3 post-CTX injury. Western blot (left) and the quantification (right) of myogenin in the tibialis anterior muscles. The rest is as in e. (g) Immunostaining of MHC in regenerating fibers on day 5 post-CTX injury. Scale bar, 200 μm. (h) Analysis of the cross-sectional areas of regenerating fibers on day 5 post injury using the ImageJ software. Only myofibers containing centralized nuclei were measured. The error bars depict the means±S.D. of samples from five mice. (i) Average area of the cross-sections of regenerating fibers on day 5 post-CTX injury. Approximately eight random fields were captured, and >1500 fibers were measured per sample. The error bars depict the means±S.D. of samples from five mice. **P<0.01