3.7 AngII regulates miR-26a expression via Smad3 activation.
PROMO analysis revealed three potential Smad3 binding sites in the miR-26a promoter (Fig. 7a). ChIP assay with VSMC lysates and anti‐Smad3 antibody validated that Smad3 directly binds to the promoter of miR‐26a to suppress its expression. Additionally, Smad3 exhibited stronger binding to the miR-26a promoter in AngII-induced VSMCs as compared with controls (Fig. 7b, c). Thus, AngII promoted Smad3 binding to the miR-26a promoter, thereby inhibiting miR-26a transcription. To further elaborate whether miR-26a downregulation was mediated by Smad3 in VSMCs, we used loss-of-function studies. We first constructed three siRNAs fragments of Smad3 to transfect VSMCs. Both qRT-PCR and western blot analysis confirmed that siRNA-3 silenced Smad3 the best (Supplemental Fig. S2a, b). Next, we used siRNA or SB431542, which inhibited Smad3 expression or activation, respectively, and observed increased miR-26a expression in AngII-induced VSMCs (Fig. 7d-f), which indicates that Smad3 negatively regulates miR-26a expression. These results indicate that AngII regulates miR-26a expression via Smad3 activation.