Inhibition of vascular smooth muscle cell proliferation by DNA-RNA chimeric hammerhead ribozyme targeting to rat platelet-derived growth factor A-chain mRNA

Background Spontaneously hypertensive rats (SHR)derived vascular smooth mus cle cells (VSMC) show exaggerated growth and increasingly express platelet- derived growth factor (PDGF) A-chain mRNA compared to VSMC from normotensiv e Wistar-Kyoto (WKY) rats, Objective To investigate the effects of designed DNA-RNA chimeric hammerhea d ribozyme to rat PDGF A-chain on exaggerated growth of VSMC from SHR, Design and methods We designed and synthesized a 38-base DNA-RNA chimeric h ammerhead ribozyme with two phosphorothioate linkages at the 3' terminal to cleave rat PDGF A-chain mRNA at the GUC sequence at nucleotide 921, We con firmed the cleavage activity of designed ribozyme by in vitro cleavage reac tion and by lipofectin-mediated transfection of ribozyme into VSMC, Results Doses of 0.1 and 1 mu mol/l DNA-RNA chimeric ribozyme dose-dependen tly inhibited basal DNA synthesis in VSMC from SHR. A dose of 1 mu mol/l DN A-RNA chimeric ribozyme time-dependently inhibited basal DNA synthesis in V SMC from SHR. However, the same doses of all-RNA ribozyme had no effects on DNA synthesis in VSMC from SHR, Fluorescein isothiocyanate-labeled DNA-RNA chimeric ribozyme was recognized in cytosol at 30 min, and in nucleus at 6 0 min after lipofectin transfection. A dose of 1 mu mol/l DNA-RNA chimeric ribozyme significantly inhibited expressions of both PDGF A-chain mRNA and PDGF-AA protein in VSMC from SHR, but not from WKY rats. Conclusion These results indicated that the designed DNA-RNA chimeric riboz yme to PDGF A-chain mRNA effectively and specifically inhibited the exagger ated growth of VSMC from SHR at low concentrations, which were mediated by the reduction of PDGF A-chain mRNA and PDGF-AA protein expressions, (C) 200 1 Lippincott Williams & Wilkins.