摘要
A hammerhead ribozyme which site-specifically cleaved the GUC position in canon 880 of the mdr1 mRNA was designed. The target site was chosen between the two ATP binding sites, which may be important for the function of the P-Gp as an ATP-dependent pump. A DNA sequence encoding the ribozyme gene was then incorporated into a eukaryotic expression vector (pH Apr-1 neo) and transfected into the breast cancer cell line MCF-7/Adr, which is resistant to adriamycin and expresses the MDR phenotype. The ribozyme was stably expressed in the cell line by the RNA dot blotting assay. The result of Northern blot assay showed that the expressed ribozyme could decrease the level of mdrl mRNA expression by 83. 5 %; and the expressed ribozyme could inhibite the formation of p-glycoprotein detected by immuno- cy-tochemistry assay and could reduce the cell’s resistance to adrimycin; this means that the resistant cells were 1 000-fold more resistant than the parental cell line(MCF-7), whereas those cell clones that showed ribozyme expression were only 6-fold more resistant than the parental cell line. These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.
A hammerhead ribozyme which site-specifically cleaved the GUC position in canon 880 of the mdr1 mRNA was designed. The target site was chosen between the two ATP binding sites, which may be important for the function of the P-Gp as an ATP-dependent pump. A DNA sequence encoding the ribozyme gene was then incorporated into a eukaryotic expression vector (pH Apr-1 neo) and transfected into the breast cancer cell line MCF-7/Adr, which is resistant to adriamycin and expresses the MDR phenotype. The ribozyme was stably expressed in the cell line by the RNA dot blotting assay. The result of Northern blot assay showed that the expressed ribozyme could decrease the level of mdrl mRNA expression by 83. 5 %; and the expressed ribozyme could inhibite the formation of p-glycoprotein detected by immuno- cy-tochemistry assay and could reduce the cell's resistance to adrimycin; this means that the resistant cells were 1 000-fold more resistant than the parental cell line(MCF-7), whereas those cell clones that showed ribozyme expression were only 6-fold more resistant than the parental cell line. These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.
基金
This research was supported by the National Natural ScienceYouth Grant.
关键词
多药耐药
P-糖蛋白
乳癌
核糖酶
耐药机制
hammerhead ribozyme
multidrug resistance reversion
human breast cancer cell line MCF-7/Adr
