[Inhibition of mutant p53 in hepatocellular carcinoma cells by hammerhead ribozyme]

Abstract

Objectives: To investigate the inhibition of mutant type p53 in hepatocellular carcinoma by hammerhead ribozyme in both cell-free system and MHCC97 cells.

Methods: Hammerhead ribozyme genes (RZ) and control ribozyme (asRZ) directed against mutant p53 (249 codons, AGG --> AGT) were designed by computer. The in vitro transcription plasmid and eukaryotic expression plasmid were constructed into the vector pBSKU6 and pEGFPC1. Human mutant and wild type p53 gene fragment were cloned into the pGEM-T vector under T7 promoter control. In vitro cleavage reaction was carried out by mixing the RZ and target mRNAs which were labeled with [alpha-32P] dUTP. RZ was introduced into MHCC97 cells by LipofectAMINEAM2000 and mtp53 expression was analyzed by RT-PCR.

Results: In cell-free systems, RZ showed a specific cleavage activity against mtp53 with cleavage efficiency of 42%, while the wild type p53 was not cleaved. The mRNA level of mtp53 in MHCC97 cells after transfection was reduced by RT-PCR analysis.

Conclusion: These findings suggest that the hammerhead ribozyme against the mtp53 is a new promosing gene therapeutic agent against hepatocellular carcinoma.