Effect of hammerhead ribozyme that specifically cleaves c-myc mRNA on proliferation of vascular smooth muscle cells

摘要

Ojective: To investigate the effect of hammerhead ribozyme that specifically cleaves c-myc mRNA on the proliferation of vascular smooth muscle cells ( VSMCs ). Methods: Based on the computer analysis of the secondary structure of c-myc mRNA, nt 2029 in rat c-myc oncogene was selected as a cleaving site for hammerhead ribozyme and the ribozyme was designed. With automatic DNA synthesizer, the two complementary DNA strands of the ribozyme were synthesized. The ribozyme gene was cloned into pGEM3Zf ( + ) vector and subcloned into eukaryotic expression pcD-NA3 vector. The recombinant pcDNA-Rz was transfected into the cultured rat VSMCs by lipofectAMINE mediated DNA transfection protocol and individual cell clones were selected by G418. Results: The sequence of ribozyme gene inserted in pGEM3Zf ( + ) vector was proved to be perfectly correct. In VSMCs transfected with recombinant pcDNA-Rz, flow cytometry analysis showed that the S phase and G_2/M fractions were decreased significantly and cell proliferation stagnated in the G_0/G_1 phase. Conclusion: The results suggest that hammerhead ribozyme that specifically cleaves c-myc mRNA can significantly inhibit the proliferation of VSMCs.