MiR-665 regulates VSMCs proliferation via targeting FGF9 and MEF2D and modulating activities of Wnt/β-catenin signaling.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to the development of cardiovascular diseases. Studies have showed the great impact of microRNAs (miRNAs) on the cell proliferation in VSMCs. This study examined the in vitro functional roles of miR-665 in the VSMCs and explored the underlying molecular mechanisms. The mRNA and protein expression levels were determined by qRT-PCR and western blot assays, respectively. CCK-8, transwell invasion and wound healing assays were performed to measure VSMCs proliferation, invasion and migration, respectively. The miR-665 targeted-3'UTR of fibroblast growth factor 9 (FGF9) and myocyte enhancer factor 2D (MEF2D) was confirmed by luciferase reporter assay. Platelet-derived growth factor-bb (PDGF-bb) and 20% serum promoted cell proliferation and suppressed the expression of miR-665 in VSMCs. In vitro functional assays demonstrated that miR-665 inhibited VSMCs proliferation, invasion and migration. Bioinformatics analysis showed that FGF9 and MEF2D were found to be downstream targets of miR-665. Luciferase report assay confirmed that FGF9 and MEF2D 3'UTRs are direct targets of miR-665, and miR-665 overexpression suppressed both the mRNA and protein expression levels of FGF9 and MEF2D. Furthermore, rescue experiments showed that enforced expression of FGF9 or MEF2D attenuated the inhibitory effects of miR-665 on VSMCs proliferation. More importantly, overexpression of miR-665 also suppressed the mRNA and protein expression levels of β-catenin, c-myc and cyclin D1. In summary, miR-665 suppressed the VSMCs proliferation, invasion and migration via targeting FGF9 and MEF2D, and the in vitro effects of miR-665 on VSMCs may be associated with modulation of Wnt/β-catenin signaling activities.