LPS-induced downregulation of microRNA-204/211 upregulates and stabilizes Angiopoietin-1 mRNA in EA.hy926 endothelial cells

Abstract

Angiopoietin-1 (ANG-1), a ligand of the endothelial cell-specific TIE2 surface receptor, acts in a complementary and coordinated manner with vascular endothelial growth factor during the process of angiogenesis. ANG-1 can be used as a clinically informative biomarker of disease severity and outcome in severe sepsis. The epithelium-specific Ets transcription factor 1 can activate ANG-1 transcription in the setting of inflammation; however, relatively little is known about the regulation of ANG-1 by microRNAs (miRs). It was observed that lipopolysaccharide (LPS) significantly increased ANG-1 mRNA and protein expression in EA.hy926 cells. ANG-1 was identified as a potential target gene of MIR-204 and MIR-211. Overexpression of MIR-204/211 partially reversed the LPS-induced ANG-1 expression in EA.hy926 cells. Furthermore, overexpression of MIR-204/211 significantly reduced the activity of a luciferase reporter gene containing the wild-type ANG-13'-untranslated region (UTR), but did not influence the activity of a luciferase reporter gene containing the ANG-13'-UTR with a mutated MIR-204/211 binding site, confirming that MIR-204/211 can bind to the ANG-13'-UTR and post-transcriptionally regulate ANG-1. Additionally, LPS enhanced the stability of ANG-1 mRNA by reducing the abundance of MIR-204/211. Overexpression of MIR-204/211 reduced the migration of EA.hy926 cells in vitro. The present study demonstrated that ANG-1 is a novel direct target gene of MIR-204 and MIR-211; in addition, LPS was able to inhibit this effect by reducing the expression of MIR-204 and MIR-211.