锤头状核酶对肝癌突变基因p53的抑制作用

Inhibition of mutant p53 in hepatocellular carcinoma cells by hammerhead ribozyme

目的 探讨p53核酶对肝癌细胞突变型抑癌基因p53的抑制作用。方法 应用计算机设计并合成针对突变型p53(249位密码子AGG→AGT)的锤头状核酶RZ,构建其体外转录和真核表达载体,检测核酶对突变型p53(mtp53)的体外切割作用,并在Lipofect AMINE^(TM)2000的介导下转染肝癌细胞MHCC97,应用逆转录聚合酶联反应(RT—PCR)检测核酶对肝癌细胞突变型p53的抑制作用。结果 测序证实核酶基因被正确克隆入体外转录载体pBSKU6和真核载体pEGFPC1中。体外切割效率为42％,而野生型p53(wtp53)没有被切割。在LipofectAMINETM2000的介导下成功转染肝癌细胞MHCC97,RT—PCR检测证实突变型p53的mRNA水平明显下降,细胞内的切割效率为69％。结论 p53核酶可成功抑制肝癌细胞中突变型p53的表达,为肝癌的基因治疗提供了一个新的选择。

Objective 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 pEGFPCl. 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 [ a -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.