Supplementary MaterialsSupplementary Shape. mechanisms of delaying Bmpr2 effect of MEF2A on VEC cell senescence was SIRT1-expression activation through the PI3K/p-Akt pathway. Moreover, the plasma MEF2A levels may be a potential biomarker for CAD risk prediction. 0.05; **, 0.01; ***, 0.001. MEF2A up-regulated the PI3K/p-Akt/SIRT1 signaling pathway The PI3K/p-Akt/SIRT1 signaling pathway plays an important role in regulating cell proliferation, cell survival, development, cell senescence, and other biological processes. To understand the molecular mechanism of the effect of MEF2A on cellular senescence, real-time fluorescent quantitative PCR and immune-blotting were used to detect the effect of inhibition or MEF2A overexpression on the expression of several key genes in the PI3K/p-Akt/SIRT1 signaling pathway. When MEF2A was knocked down in HUVECs, the mRNA levels of PIK3CA, PIK3CG, and SIRT1 decreased significantly compared with the negative control group (Figure 2A). Western blotting showed that PIK3CA, PIK3CG, SIRT1, and p-AKT levels were down-regulated with the inhibition of MEF2A, and the P53 level was up-regulated (Figure 2B). Conversely, PIK3CA, PIK3CG, and SIRT1 mRNA levels were significantly up-regulated in the MEF2A-overexpression HUVECs (Figure 2C), and the protein levels of PIK3CA, PIK3CG, SIRT1, and p-Akt notably increased but P53 decreased (Figure 2D). When the specific inhibitors of PIK3CA and PIK3CG were added while overexpressing MEF2A, the activities of p-Akt and P53 did not change with increased MEF2A, and SIRT1 expression did not increase with increased MEF2A (Figure 2C and ?and2D).2D). This result suggested that MEF2A up-regulated the expression of the downstream gene SIRT1 by positively regulating the expression of PIK3CA and PIK3CG genes. Compared with the empty vector group, the transfection of MEF2A expression plasmid decreased the proportion of the SA–gal staining-positive cells, but when PI3K inhibitor was added, it significantly enhanced SA–gal staining and eliminated the effect of MEF2A overexpression on HUVECs (Figure 2E). This result implicated that MEF2A delayed HUVEC aging by activating AN3365 PI3K/p-Akt/SIRT1 pathway. Open in a separate window Figure 2 The influence of changes in expression of MEF2A on downstream gene expression. (A) Changes in downstream gene mRNA levels after transfection of si-MEF2A in HUVEC; (B) Changes in downstream gene protein levels after transfection of si-MEF2A in HUVEC; (C) Effect of transfection of MEF2A overexpression plasmid or transfection of MEF2A overexpression plasmid plus PI3K inhibitor on the mRNA level of downstream gene in HUVEC; (D) Influence of transfection of MEF2A overexpression plasmid or transfection of MEF2A overexpression plasmid plus PI3K inhibitors on downstream gene protein levels in HUVEC. (E) Impact of transfection of MEF2A overexpression plasmid or transfection of MEF2A overexpression plasmid plus PI3K inhibitor on HUVEC phenotype. The mRNA level, protein level and senescence rate were expressed as the mean fold changes relative to the control group, and the error bars represent the standard error of the AN3365 fold changes in 3 independent experiments. *, 0.05; **, 0.01; ***, 0.001; ns, no significance. Down-regulation of MEF2A may be one of the mechanisms underlying oxidative-stress-induced senescence in HUVECs Oxidative stress is AN3365 one of the main factors inducing cell senescence. Cellular senescence is accelerated when cells are exposed to oxidative stress elements such as for example hydrogen peroxide in vitro. In this scholarly study, we noticed that the treating HUVECs with H2O2 demonstrated a reduction in cell viability in focus- and time-dependent manners (Body 3A). In the next experiments, the cells had been treated by us with 100 M H2O2 for 1 h. Results demonstrated that the treating HUVECs with 100 M H2O2 for 1 h considerably accelerated cell senescence, reduced cell viability (Body 3B), considerably down-regulated the appearance of MEF2A as well as the downstream genes: PI3K, p-AKT and SIRT1, and elevated the appearance of P53 (Body 3C). When MEF2A was overexpressed in.