MiR‐9 mediates Nox4‐dependent regulation of myocardin and serum response factor in human smooth muscle cells (866.6)

Abstract

Myocardin is a transcriptional coactivator of serum response factor (SRF) responsible for vascular smooth muscle cell (SMC) differentiation. Changes in Nox4 NADPH oxidase expression are associated with changes in myocardin and SRF levels and have been implicated in the phenotypic switching of SMCs. However, the mechanisms underlying this molecular switch in response to vascular injury are poorly understood. We examined the role of microRNAs (miR) in the expression of Nox4, SRF and myocardin in human SMCs. Analysis of the 3’UTR of Nox4 revealed a single binding site for miR‐25, and two putative miR‐9 binding sites in the 5’ region of the 3’UTR. Treatment of human SMCs with a miR‐25 or a miR‐9 mimic suppressed mRNA expression of Nox4. We created a Nox4 3’UTR luciferase construct and found that the miR‐25 mimic decreased relative light units (RLU) by 40% which was prevented with mutation of the single miR‐25 site. The miR‐9 mimic decreased Nox4 3’UTR RLU by 60% and mutation of the two miR‐9 sites had a synergistic effect in restoring RLU to wild type levels. In addition, both miR‐9 and miR‐25 mimics decreased mRNA expression of myocardin and SRF. Finally, the miR‐25 mimic increased levels of miR‐9, but the miR‐9 mimic had no effect on miR‐25 levels. We conclude that miR‐25‐dependent regulation of Nox4 regulates SMC differentiation in part via miR‐9 dependent regulation of SRF and myocardin.