3.5 miR-26a suppresses ECM deposition by directly targeting CTGF.
CTGF is essential for the synthesis of collagen (Liang et al. , 2014). We hypothesized that miR-26a attenuates ECM accumulation by regulating CTGF. In vivo data shown in Figure 5a support this hypothesis: CTGF protein level was significantly downregulated in SHR thoracic aortas with rAAV-miR-26a overexpression. The effect of miR-26a was further confirmed in vitro: miR-26a mimic suppressed CTGF expression in AngII-induced VSMCs and miR-26a inhibitor increased CTGF protein and mRNA levels in vitro (Fig. 5b, c). Bioinformatics analysis by using Targetscan (www.targetscan.org) predicted CTGF as a potential target gene of miR-26a (Fig. 5d). By using a reporter construct with the putative 3′-UTR miR-26a binding site of CTGF downstream of the luciferase gene, we found that miR-26a reduced luciferase activity, which provides experimental validation of CTGF as a target for miR-26a. Accordingly, after mutation of the binding site, the activity of luciferase was not altered (Fig. 5e), which confirms that the binding of miR-26a to the 3′-UTR site was necessary for silencing CTGF