R eight and 14 days (as for Figure 1) was assessed by qPCR. A important improve in miR-125b expression was observed in sorted aMHC-GFP+ hESCs at day eight and sustained through day 14 of differentiation. ZEN-3862 Technical Information Information shown are mean6s.e.m. (N = 3). , p,0.001. B) Relative expression of miR-125b in undifferentiated hESC cultures transfected with 30 nM anti-miR-125b inhibitor (anti-125b) or pre-miR-125b (pre-125b), as analyzed by qPCR, showed appropriate down- or up-regulation of miR-125b expression, respectively, in comparison with untransfected control (Ctl) hESCs. C) Relative binding and activity of miR-125b in undifferentiated hESC cultures transfected with anti-125b and pre-125b, as assessed by luciferase reporter activity, Rose Bengal web demonstrated proper up- and down-regulation of luciferase activity inside a dose-dependent manner, respectively, in comparison to hESCs transfected with luciferase reporter alone (Ctl). Information shown are mean6s.e.m. (N = three). , p,0.01; , p,0.001. doi:10.1371/journal.pone.0036121.gmiR-125b targets the Lin28/let-7 axis in the course of hESC differentiationTo figure out regardless of whether Lin28 expression inversely parallels miR125b expression through CM differentiation, we analyzed Lin28 transcription by qPCR in undifferentiated hESCs when compared with aMHC-GFP+ myocardial precursors and CMs sorted from cultures grown in differentiation medium for 8 and 14 days, respectively (Figure 4C). We observed a substantial decrease in Lin28 mRNA over time (Day eight: 0.2760.02 vs. 1.0060.1, p,0.001; Day 14: 0.1360.06 vs. 1.0060.1, p,0.001), as will be predicted by damaging post-transcriptional regulation of Lin28 by miR-125b. To determine whether or not the alter in Lin28 expression with differentiation was mediated by miR-125b, we knocked down miR-125b in differentiating hESCs (Figure 5A), and assayed Lin28 protein expression (Figure 5B). In undiffer-entiated cells, expression of anti-miR-125b result in an increase in Lin28 expression. As Lin28 expression decreased with hESC differentiation, transfection with anti-miR-125b had a related impact compared to untransfected cells, despite the fact that to a lesser extent. This demonstrated that the effects of miR-125b on early hESC differentiation probably take place through inhibition of Lin28. Because Lin28 has been shown to exert its effects on pluripotency via binding to and inactivation of let-7, we examined the effect of miR-125b knockdown on let-7d and let-7d expression in differentiating hESCs. We focused on let-7d and let-7d due to the fact let-7d demonstrated regulated expression through our initial expression profiling screen (Table 1). MiR-125b knockdown resulted in parallel downregulation of let-7d expression (Figure 6A). Interestingly, a related effect on let-7d was not observed (data not shown), suggesting that its expression during hESC differentiation is regulated independent of miR-125b. SincePLoS A single | plosone.orgmiR-125b and Mesoderm Fate DeterminationFigure 3. miR-125b promotes the expression of cardiac-specific genes throughout hESC differentiation. hESCs had been transfected with premiR-125b (pre-125b) or anti-miR-125b inhibitor (anti-125b), cultured in differentiation medium for 2 or 8 days, and analyzed for expression of cardiacspecific mRNAs by qPCR. A) Overexpression of pre-125b induced premature expression in the early cardiac transcription issue, GATA4, in undifferentiated hESCs (Undiff), and promoted the early expression of each GATA4 and Nkx2-5 in hESCs cultured in differentiation medium for only 2 days, just before the commonly observed expression of these.
