Figure 3.

Aging of skeletal muscle-specific Dicer knockout mouse. Mice were treated with either vehicle (wild-type, WT) or tamoxifen (knockout, KO) at four months of age and then allowed to age for an additional 22 months. (A) Dicer expression was significantly decreased by 80% in skeletal muscle of KO mice compared to WT mice at 26 months of age. (B) Sequencing of a cloned fragment (exon 22–25) of Dicer mRNA isolated from skeletal muscle revealed deletion of floxed exon 23 and 24 in KO sample (0.75 kb fragment) whereas WT sample contained full-length fragment of ~1.5 kb. Trace from sequencing showed joining of exon 22 and 25 resulting from the deletion of exons 23 and 24 in KO sample. (C) qPCR analysis showed myomiR (miR-1, -133a and -206) and non-myomiR (miR-16, let-7a and let-7b) expression remained significantly lower with aging in skeletal muscle of KO mice compared to WT mice. (D) Northern blot analysis showed miR-1 expression was significantly lower by ~50% in skeletal muscle of KO mice compared to WT mice, consistent with qPCR results presented in panel C. (E) qPCR analysis showed a two-fold increase in both miR-1 primary miRNA (pri-miR-1A or -1B) transcript levels in skeletal muscle of KO mice compared to WT mice indicating an inability to effectively process the pri-miRNA transcript. Values are presented as the mean ± SEM (n = 8–9) with expression normalized to WT. Asterisk denotes significant difference between WT and KO groups (p ≤ 0.05).