title: Thrombospondin-1-induced vascular smooth muscle cell migration and proliferation are functionally dependent on microRNA-21.
abstract: OBJECTIVE
  Thrombospondin-1 (TSP-1) is a matricellular glycoprotein released from platelets at sites of arterial injury and is important in neointima development after balloon angioplasty. MicroRNAs are small noncoding RNAs that function by binding target gene mRNA and inhibiting protein translation. MicroRNA-21 (miR-21) is up-regulated after angioplasty, and inhibition of miR-21 leads to decreased intimal hyperplasia. In this study, we examined the effects of miR-21 inhibition on vascular smooth muscle cell (VSMC) processes.
  
  
  METHODS
  VSMCs were exposed to TSP-1 and miR-21 inhibitor for 20 minutes. TSP-1-induced migration was assessed with a modified Boyden microchemotaxis chamber and proliferation with calcein-AM fluorescence. Phosphorylated extracellular signaling kinase (ERK) 1/2 expression was determined by Western Blot and densitometry. Quantitative real-time polymerase chain reaction for TSP-1, hyaluronic acid synthase 2 (HAS2), and transforming growth factor beta 2 (TGFβ2) was performed. Statistical analysis was performed with analysis of variance (P < .05).
  
  
  RESULTS
  Inhibition of miR-21 blocked TSP-1-induced VSMC migration, proliferation, and ERK 1/2 phosphorylation (P < .05) and had no effect on TSP-1-stimulated expression of genes for TSP-1, HAS2, or TGFβ2 (P > .05).
  
  
  CONCLUSIONS
  Acute inhibition of miR-21 led to a decrease in VSMC migration and proliferation caused by TSP-1. The decrease in TSP-1's activation of ERK 1/2 after acute miR-21 inhibition indicates an active role for miR-21 in TSP-1's cell signaling cascade. No effect on TSP-1-induced expression of the pro-stenotic genes thbs1, tgfb2, or has2, occurred after acute miR-21 inhibition. These data indicate that miR-21 directly modulates cell function and signaling pathways in ways other than inhibition of protein translation.
authors: Department of Surgery, SUNY Upstate Medical University, Syracuse, NY; Department of Veterans Affairs Healthcare Network Upstate New York at Syracuse, Syracuse, NY.
authors: Jeffrey J
authors: JJ
authors: Stein
authors: Department of Surgery, SUNY Upstate Medical University, Syracuse, NY; Department of Veterans Affairs Healthcare Network Upstate New York at Syracuse, Syracuse, NY.
authors: Chinenye
authors: C
authors: Iwuchukwu
authors: Department of Surgery, SUNY Upstate Medical University, Syracuse, NY; Department of Veterans Affairs Healthcare Network Upstate New York at Syracuse, Syracuse, NY.
authors: Kristopher G
authors: KG
authors: Maier
authors: Department of Surgery, SUNY Upstate Medical University, Syracuse, NY; Department of Veterans Affairs Healthcare Network Upstate New York at Syracuse, Syracuse, NY. Electronic address: gahtanv@upstate.edu.
authors: Vivian
authors: V
authors: Gahtan
mesh_terms: D002465:Cell Movement / Q000502:physiology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006083:Graft Occlusion, Vascular / Q000209:etiology / Q000378:metabolism; D006801:Humans; D000076002:Hyaluronan Synthases; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D010766:Phosphorylation; D019700:Thrombospondin 1 / Q000502:physiology*; D053781:Transforming Growth Factor beta2 / Q000378:metabolism*
chemical_list: C524940:MIRN21 microRNA, human; D035683:MicroRNAs; C509041:TGFB2 protein, human; D019700:Thrombospondin 1; D053781:Transforming Growth Factor beta2; D014453:Glucuronosyltransferase; C101754:HAS2 protein, human; D000076002:Hyaluronan Synthases; D048049:Extracellular Signal-Regulated MAP Kinases