Suppression of endothelial nitric oxide synthase expression and endothelial cell proliferation by an intronic 27-ntmiRNA and it's a novel link to AP-1.

This study aims to investigate the role of activator protein 1 (AP-1) in the effects of 27nt-miRNA on expression of endothelial nitric oxide synthase (eNOS) gene and proliferation of endothelial cells. Cell proliferation was analyzed by cell number counting, colony formation assay and MTT assay. Cell migration and invasion was detected by transwell assay and invasion assay. Expression of eNOS and AP-1 was measured by real-time RT-PCR (mRNA level) and Western blotting (protein level). Luciferase reporter assay was performed to detect the binding of 27nt-miRNA to AP-1. Overexpression of 27nt-miRNA significantly inhibited endothelial cells proliferation, invasion and migration in vitro. And, eNOS and AP-1 expression at mRNA and protein levels were down-regulated by overexpression of 27nt-miRNA. Interestingly, overexpression of AP-1 protein partially restored eNOS expression and endothelial cell proliferation. Furthermore, the luciferase reporter assay demonstrated that AP-1 was a direct target of 27nt-miRNA. These data demonstrate that overexpression of 27nt-miRNA inhibits endothelial cell proliferation, invasion, migration, eNOS expression and AP-1 expression. Moreover, AP-1, a direct target of 27nt-miRNA, reverses the inhibitory effects of 27nt-miRNA. Thus, the effects of 27nt-miRNA might be acted through targeting AP-1.