RNA interference targeting embryonic myosin heavy chain isoform inhibited mRNA expressions of phenotype markers in rabbit cultured vascular smooth muscle cells.
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.
Shimada
Seiji
S
First Department of Physiology, Unit of Physiological Science, School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa, 903-0215, Japan.
Sunagawa
Masanori
M
Hanashiro
Kazuhiko
K
Nakamura
Mariko
M
Kosugi
Tadayoshi
T
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
D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D020033:Protein Isoforms; D018995:Myosin Heavy Chains; D016650:Fluorescein-5-isothiocyanate