Abstract
The purpose of this study was to develop DNA-RNA chimeric hammerhead ribozyme against transforming growth factor-beta(1) (TGF-beta(1)) mRNA as a gene therapy agent for arterial proliferative diseases. A 38-base hammerhead ribozyme against rat TGF-beta(1) mRNA, to produce cleavage at the GUC sequence at nucleotide 825 according to the secondary structure of rat TGF-beta(1) mRNA was designed. To enhance its stability, we synthesized a DNA-RNA chimeric ribozyme with two phosphorothioate linkages at the 3'-terminal. We also synthesized a mismatch ribozyme with single base change in the catalytic loop region as a control. These ribozymes were delivered into rat vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats by lipofectin-mediated transfection, and their biological effects were investigated. According to in vitro cleavage studies, the synthetic ribozyme can cleave the synthetic substrate RNA into two RNA fragments. Chimeric ribozyme significantly inhibited DNA synthesis in VSMC from SHR but not in cells from WKY rats. Mismatch ribozyme showed only a little effect on growth of VSMC from SHR. Chimeric ribozyme significantly inhibited proliferation of VSMC from SHR; in contrast, the proliferation of VSMC from WKY rats was significantly increased by this chimeric ribozyme. Mismatch ribozyme did not affect proliferation of VSMC from either rat strain. Chimeric hammerhead ribozyme to rat TGF-beta(1) dose-dependently inhibited TGF-beta(1) mRNA expression detected by reverse transcription and polymerase chain reaction analysis in VSMC from both rat strains. Chimeric hammerhead ribozyme to rat TGF-beta(1) also dose-dependently inhibited TGF-beta(1) protein production detected by Western blot analysis. The present results demonstrated that our designed DNA-RNA chimeric hammerhead ribozyme to TGF-beta(1) mRNA might be a useful gene therapy agent for hypertensive vascular diseases.
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