SMemb-specific siRNAs inhibited SMemb mRNA expression in cultured rabbit vascular smooth muscle cells

Abstract

We have investigated a role of SMemb gene expression in the phenotype conversion of vascular smooth muscle (VSM) cells. In the present study, we employed SMemb specific short interfering RNAs (siRNAs) to inhibit SMemb mRNA expression. We designed three different siRNAs targeting SMemb mRNA. The two siRNAs were designed to target open reading frame (named as ORF-1 and ORF-2), and the one siRNA was designed to target 3' untranslated region (named as 3' UTR). The siRNAs at different concentrations (1, 10 and 100 nM) were transfected into rabbit cultured VSM cells by the lipofection method. After 6 hours transfection, 10% fetal bovine serum (FBS)-culture medium was added and incubated for another 42 hours. We extracted total RNA to determine the level of SMemb mRNA expression by RT-PCR. Consequently, the 3' UTR and ORF-2 siRNAs dose-dependently decreased the SMemb mRNA expression. The SMemb protein expression in the cells was evaluated by immunohistochemistry. The number of FITC-positive cells in siRNA transfected group was less than that in the control group. We also extracted crude myosin extract to determine the level of SMemb protein expression by dot blot analysis. All the SMemb protein expressions were inhibited to about half by 100 nM of the three siRNAs. Other myosin heavy chain (MHC) isoform mRNA expressions such as SM1 and SM2 were not changed by treatment with the three siRNAs. Thus, the three different siRNAs specific for SMemb could inhibit SMemb mRNA and protein expressions. [Jpn J Physiol 54 Suppl:S80 (2004)]