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.