Figure 3

Generation and validation of a weighted cumulative context score for type 2 diabetes miRNAs. (a) Target genes with a more negative cumulative context score (CCS) are, on average, expressed at a lower level than non-targeted genes (Additional file 2). To determine which genes are most targeted when there is a shift in global miRNA expression, the distribution of CCS was adjusted on a gene by gene basis for the magnitude of up-/down-modulation of [miRNA] - wCCS. As can be seen, despite the vast number of potential predicted targets (Figure S5A in Additional file 1), few target genes have highly scoring wCCSs. There were 279 genes in the first quartile predicted to be up-regulated (reduced regulation by miRNAs) and 355 in the first quartile predicted to be down-regulated (increased regulation by miRNAs). The composition of these lists was validated using pathway and ontology analysis (b). Consistent with the global Affymetrix analysis (Figure S2 in Additional file 1) the mRNA of developmental related first quartile wCCS genes was identical between patients and controls. This was true regardless of whether the gene should be up-regulated (BDNF, BCL2L11(BIM), HOXA3, HOXC8, HOXA7 and HOXB7), down-regulated (HOXC4), or unchanged (CDC42 and PTBP1). This indicates miRNA are operating to block protein translation. Error bars = s.e.m. (c) Proteins highly ranked for being up-regulated were selected and protein expression was analyzed in skeletal muscle biopsies from normal glucose tolerant controls (NGT; n = 6) and subjects with T2D (DM; n = 6). From a second set of subjects, satellite cells were isolated from skeletal muscle biopsies from normal glucose tolerant controls (NGT) (n = 5 to 6) and subjects with T2D (DM; n = 5 to 6). The satellite cells were harvested in a proliferative state or as differentiated into myotubes. Protein expression was analyzed by using western blotting and specific antibodies towards the protein targets. HOXA3 (top left) was detected as a 30 kDa band, significantly up-regulated in muscle from subjects with T2D (P = 0.006). BCL2L11 (BIM; top middle) was detected as a band around 25 kDa, significantly up-regulated in muscle from subjects with T2D (P = 0.014). HOXC8 (top right) was detected as a band around 36 kDa and demonstrated a clear trend for up-regulation (P = 0.07). BDNF (bottom) was detected as a band at 14 kDa, up-regulated in proliferating satellite cells derived from subjects with T2D where it is typically expressed (p = 0.014) but was not expressed in differentiated satellite cells or adult muscle. * = P value < 0.05; ** = P value < 0.001.