SMemb-specific siRNA expression vector inhibited bound thrombin-induced upregulation of SMemb gene in cultured rabbit vascular smooth muscle cells

Abstract

Post-clotting thrombin (bound thrombin) could induce phenotypic modulation of vascular smooth muscle (VSM) cells from contractile to synthetic type through the upregulation of embryonic myosin heavy chain isoform (SMemb) and plasminogen activator inhibitor type1 (PAI-1) mRNAs. To examine whether the bound thrombin-induced phenotypic modulation was prevented through knockdown of SMemb gene, we constructed siRNA expression plasmid (pSilencer^TM) vectors targeting different sites of SMemb mRNA. Two kinds of siRNA vectors were designed to target open reading frame (ORF-1 and ORF-2), and the other to target 3’ untranslated region (3’ UTR). To test which siRNA vectors inhibit SMemb mRNA expression, the 0.5 μg/ml of siRNA vectors were transfected into cultured VSM cells using the lipofection method. Northern blot analysis revealed that the ORF-2 siRNA vector strongly inhibited the SMemb mRNA expression. Immunofluorescent study revealed that SMemb protein expression was depressed in ORF-2 siRNA vector-treated cells. When VSM cells were transfected with ORF-2 siRNA vector at 48 hr after we stimulation using 10 unit/ml of bound thrombin, SMemb mRNA expression was significantly inhibited at 48 hr after transfection. SMemb mRNA expression was not downregulated and became increased in empty siRNA vector-treated cells (control). Thus, ORF-2 siRNA vector may prevent bound thrombin-induced phenotypic modulation of VSM cells. [Jpn J Physiol 55 Suppl:S236 (2005)]