[Study on RNA interference reversing the multidrug resistance of leukemia cell].
OBJECTIVE
To downregulate the expression of mdr1 gene in K562/A02 cell line by RNA interference.


METHODS
The eukaryotic expression vectors of shRNA aiming at two mdr1 mRNA target sequences were constructed and used to transfect the drug resistance cell line K562/A02 with liposome-induced gene transfection. The mRNA of mdr1 gene was identified by Q-PCR. The P-gp expression of was detected by Western blot. The function of P-gp was measured by daunorubicin (DNR) efflux experiment and the sensitivity of cell lines to doxorubicin (ADM) was detected by MTT test.


RESULTS
Two shRNA plasmids targeting mdr1 mRNA were constructed and cloned. Two mdrl-targeted shRNA could down-regulate mdr1 mRNA expressions with 89.74% and 87.18% respectively, almost completely down-regulate the P-gp expression. The intracellular DNR increased after RNAi treating. The daunorubicin efflux ratio at 60 min were 13.16% and 22.02%, compared with control 40.44% , 45.31%, P<0.05. MTT test demonstrated the relative reversing efficiencies to doxorubicin were 84.36% and 76.69% respectively. CONCLUSION RNA interference can effectively reverse multidrug resistance caused by mdr1.
Gu
Ling
L
Department of Hematology, West China Hospital, Sichuan University, Chengdu 610041, China.
Liu
Ting
T
Gong
Yu-ping
YP
Xi
Ya-ming
YM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D003630:Daunorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007938:Leukemia / Q000235:genetics / Q000473:pathology*; D008969:Molecular Sequence Data; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D012333:RNA, Messenger; D003630:Daunorubicin