Figure 2.

miR-431 regulates SMAD4 expression by directly interacting with the Smad4 3′ UTR. (A) Schematic depicting the sites of putative interaction of down-regulated miRNAs within the 3′ UTR of the Smad4 mRNA. (B) The effect of M-miR-431 on the activity of a luciferase-Smad4 3′ UTR reporter construct. (**) P < 0.01. (C) Effect of miR-431 on the activity of luciferase reporters bearing either a wild-type Smad4 3′ UTR or a mutant (MT) Smad4 3′ UTR with a deletion of the miR-431 site. (D) Lysates (300 µg) from young or old muscle tissues were incubated with biotinylated (Bi)-miR431-5p or Bi-cel-miR-67 (Ctrl miR) at 4°C with rotation. Four hours later, the RNA was isolated from pull-down material using streptavidin beads and was analyzed by RT-qPCR for Smad4 mRNA enrichment; normalization was carried out using Gapdh mRNA for young or old muscle tissue. The data represent the means ± SEM of three independent experiments. (*) P < 0.05. (E) Lysates from young myoblasts were incubated with antisense oligomers (ASOs) complementary to Smad4 mRNA or a negative control RNA (Gm14635 lincRNA). The RNA isolated after pull-down using streptavidin beads and Smad4 mRNA (normalized to Gapdh mRNA) (left) or for miR431-5p (normalized to RNU6B) (right) were measured by RT-qPCR analysis. (F) Levels of SMAD4 in I-miR-431 transfected young myoblasts (left) and in M-miR-431 transfected old myoblasts (right), each relative to control cells.