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. 2017 Oct 9;45(22):12766–12779. doi: 10.1093/nar/gkx896

Figure 7.

Figure 7.

H19 regulates p53 peptide levels and activity and is required for activation of the AKT/mTOR pathway. (A) Expression of H19 lncRNA and of miRNAs 675-3p and 675-5p in H19ΔEx1/H19+ myoblasts. RNAs were assayed as described in Methods and are reported relative to their expression in wild type cells. The >200-fold change in H19lncRNA expression in LOI cells dwarfs the change in miR-675–3p and -5p expression. N = 3. (B) Maternal inheritance of H19ΔEx1 results in reduced hypertrophy that can be rescued by treatment with cAMP. Cells were cultured for 72 h in differentiation medium with 0 or with 50 μM cAMP and costained for DAPI (blue) and Myh3 (Green). Upon cAMP treatment, the H19Δex1/+ cells display hypertrophy similar to wild type cells. (C) Expression of let-7 miRNAs and of let7 and of p53 mRNA biomarkers in H19-deficient myotubes. Cells were cultured 48 h in Differentiation Medium (DM) and analyzed as described in A. N = 3. (D) Immunoblot analyses of wild type (W.T.) and of H19ΔEx1/H19+ myoblasts (DM–) and myotubes (DM+). AKT/p-AKT, p70/p-p70, and rpS6/p-rpS6 were analyzed to monitor activation of AKT/mTOR pathways. β-tubulin is included as a loading control. p53 expression stays high in H19ΔEx1/+ cells in DM, while Myogenin is downregulated. p-AKT, p-p70 and p-rpS6 are also reduced, indicating reduced activation of AKT/mTOR pathways. (E) cAMP treatment causes rapid activation of pAKT/mTOR pathways in both wild type and H19ΔEx1/H19+ myotubes. 50 μM cAMP was added for the times indicated when cells were moved to differentiation medium. (F) Cartoon summary of H19 lncRNA function in myotube hypertrophy in wild type and H19-deficient cells. p53 controls InsR/Igf1R expression, which in turn activates AKT/mTOR signaling and induces muscle hypertrophy. H19 lncRNA adds another layer of control in wild type cells, negatively regulating p53 bioavailability. In H19ΔEx1/+ cells, the lack of H19 expression results in increased p53 and lower InsR/Igf1R levels, causing reduced AKT/mTOR signaling and less hypertrophy. The addition of cAMP to these cells bypasses the H19-p53-InsR/Igf1R control, directly activating AKT/mTOR signaling and inducing hypertrophy. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.