Figure 6

Screening and characterization of candidate miRNAs with dynamic expression patterns during muscle regeneration. A combinatorial screen was used to identify miRNAs from potential target genes that uniquely change expression in wild type satellite cells following a muscle injury. Gene expression changes of ≥ two-fold, P ≤ 0.01 occurring in wild type satellite cells 48 h post-muscle injury compared to freshly isolated satellite cells that changed ≤ two.0-fold, P ≥ 0.9 in syndecan-4 null cells yielded 47 genes (Table 6) that were subjected to the flow schematic to identify potential miRNAs (A). The union from both algorithms yielded 12 candidate miRNAs, six of which were detectable by RNase protection assay in cultured satellite cells or the satellite cell derived MM14 cell line in growth or differentiation conditions (Table 7). These six miRNAs are expressed in uninjured skeletal muscle (B) and four of the six change expression dynamically during skeletal muscle regeneration at 12 h (C, F), 48 h (D, F) and 5d (E, F) post-muscle injury. The same four micro RNAs (miR-16, miR-93, miR-106b, and miR-124) exhibit dynamic changes in relative expression when comparing activated satellite cells (G, J) to proliferating satellite cells (H, J) and quiescent satellite cells (I, J). All qPCR data was normalized to U6 RNA. miRNA levels in uninjured TA muscle were set to 1 (y-axis). Values above the y-axis indicate higher miRNA expression than in uninjured TA muscle and values below the y-axis indicate lower miRNA expression than in uninjured TA muscle (C-J). Graphs B-E and G-I are log scale and values are mean ± SEM (n = 3). Graphs F and J are average fold difference as compared to relative expression in B.