3.6 EZH2/p21 pathway mediates miR-26a dysregulation-induced VSMC proliferation.
To explore the mechanisms of miR-26a-inhibited proliferation of VSMCs, we investigated the regulation of miR-26a of proliferation-related genes. Considering that rAAV-miR-26a overexpression decreased EZH2 protein level in the thoracic aorta of SHRs (Fig. 6a), we assumed that miR-26a inhibiting VSMC proliferation is mediated by EZH2. Consistent with in vivo findings, in AngII-induced VSMCs, the expression of EZH2 at both the protein and mRNA levels was significantly decreased after transfection with miR-26a mimic (Fig. 6b, e). Furthermore, Targetscan revealed that the miR-26a binding site within the 3’UTR of EZH2 mRNA is highly conserved (Fig. 6f). We further performed luciferase reporter assay and found reduced luciferase activity with miR-26a mimic transfection. However, miR-26a did not affect activity of the binding site mutation, which confirms that EZH2 is a target of miR-26a (Fig. 6g).
p21 belongs to a cell-cycle regulator that contributes to cell growth inhibition, and the expression of p21 is indirectly inhibited by EZH2 (Lu et al. , 2011; Li et al. , 2019). In our study, p21 expression was negatively related to the expression of EZH2 both in vitro and in vivo (Fig. 6a, c), which suggests that miR-26a upregulated p21 level by directly suppressing EZH2 expression. In other words, miR-26a inhibited VSMC proliferation by regulating the EZH2/p21 pathway.
miR-26a can also regulate cell proliferation by directly targeting cyclin D2 (Zhou et al. , 2016). To explore whether miR-26a regulated cyclin D2 expression in VSMCs, we transfected the miR-26a mimic and inhibitor in AngII-induced VSMCs. The expression of cyclin D2 was markedly upregulated in AngII-induced VSMCs on transfection with miR-26a inhibitor and was reversed by transfection with the miR-26a mimic (Fig. 6d). Thus, miR-26a mitigates excessive VSMC proliferation by targeting the EZH2/p21 pathway and cyclin D2.