title: RNA interference targeting embryonic myosin heavy chain isoform inhibited mRNA expressions of phenotype markers in rabbit cultured vascular smooth muscle cells.
abstract: To investigate whether the knockdown of SMemb gene expression induces phenotypic modulation of vascular smooth muscle (VSM) cells toward a contractile type, we constructed a siRNA targeting the 3' untranslated region (UTR) of SMemb gene (SMemb-siRNA). The SMemb-siRNA was introduced into cultured rabbit VSM cells for 48 h at 37 degrees C by the lipofection method. The mRNA expressions were estimated by comparative reverse transcription-polymerase chain reaction (RT-PCR). SMemb-siRNA significantly decreased the ratio of SMemb to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in a dose-dependent manner (P < 0.01): 0 nM, 0.90 +/- 0.08; 100 nM, 0.43 +/- 0.07. Immunofluorescence and immunoblot analyses demonstrated that SMemb-siRNA markedly decreased SMemb protein expression to 56% +/- 7.8% (P < 0.01). Other MHC isoform (SM1 and SM2) mRNA expressions were not changed. The relative mRNA expressions of other phenotype markers (plasminogen activator inhibitor (PAI)-1 and beta-actin) were significantly decreased by SMemb-siRNA to 71% +/- 7.5% and 61% +/- 7.5%, respectively (P < 0.01). Expression of smooth muscle (SM) alpha-actin protein and cell proliferation was not changed by SMemb-siRNA. Thus, SMemb gene might be involved in the transcription of PAI-1 and beta-actin, but not involved in SM alpha-actin and cell proliferation in cultured VSM.
authors.affiliation: First Department of Physiology, Unit of Physiological Science, School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa, 903-0215, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Shimada
authors.forename: Masanori
authors.initials: M
authors.lastname: Sunagawa
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Hanashiro
authors.forename: Mariko
authors.initials: M
authors.lastname: Nakamura
authors.forename: Tadayoshi
authors.initials: T
authors.lastname: Kosugi
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000378:metabolism*; D010641:Phenotype; D020033:Protein Isoforms; D034622:RNA Interference / Q000502:physiology*; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D020033:Protein Isoforms; D018995:Myosin Heavy Chains; D016650:Fluorescein-5-isothiocyanate