Reversal MDR in breast carcinoma cells by transfection of ribozyme designed according the secondary structure of mdr1 mRNA.
Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. The present study aims to investigate whether the ribozyme could reverse MDR in breast carcinoma cells. In this study, two GUC sites (GUC106 and GUC135) on the surface of mdr1 mRNA were selected according to the secondary structure of the 5'-region of mdrl mRNA. The ribozyme gene RZ106 and RZ135 complementary to two sides bases of the target GUC were synthesized and cloned into the plasmid pEGFP -C1 which has EGFP (Enhanced Green Fluorescence Protein) as report gene and Kan/Neo as selection gene. After transfection with the recombinant plasmid and selected by G418, the stable cell clones were produced and used for detection. The alteration of mdr1 mRNA and P-gp in the treated cells was detected by RT-PCR, flow cytometry and Rh123 retention. The reversal efficiency of the drug resistance for adriamycin was determined by MTT assay. The results showed that after transfection with RZ106 and RZ135, the amount of the mdr1 mRNA and P-gp decreased significantly and the efflux function of P-gp was inhibited accordingly. Nine-fold and 16-fold reduction of resistance for adriamycin was observed in the two groups of treated cells. These results suggested that both ribozymes can reverse the MDR phenotype by inhibiting the expression of mdr1 mRNA and P-gp, and the RZ135 showed the better cleavage efficiency. The ribozyme strategy designed according the secondary structure of the target RNA could be a useful therapy for reversal of MDR.
Gao
Peng
P
Department of Pathology, School of Medicine, Shandong University, Jinan 250012, Shandong, PR China.
Zhou
Geng-Yin
GY
Zhang
Qing-Hui
QH
Li
Hong
H
Mu
Kun
K
Yuan
Yin-Ping
YP
Zhang
Jing
J
Wang
Bao-Heng
BH
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000008:administration & dosage; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004317:Doxorubicin / Q000008:administration & dosage*; D018432:Drug Resistance, Multiple / Q000235:genetics; D006801:Humans; D017433:Protein Structure, Secondary; D016337:RNA, Catalytic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection / Q000379:methods*; D016896:Treatment Outcome
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D016337:RNA, Catalytic; D012333:RNA, Messenger; D004317:Doxorubicin