[Reversal of multidrug resistance by MDR1 shRNA expression vector in human leukemia K562/A02 cells].
OBJECTIVE
To construct a short hairpin RNA (shRNA) eukaryotic expression vector specific to MDR1 gene in multidrug resistance (MDR) human leukemia cell line K562/A02 to observe its silencing effect on MDR1 and P-glycprotein (P-gp) expression.


METHODS
The shRNA expression vector was constructed by gene recombination, then transfected into the cultured K562/A02 cells. The transcription of MDR1 gene was detected by semi-quantitative RT-PCR and the expression level of P-gp was determined by Western blot. 50% inhibition concentration (IC50) of ADM in K562/A02 cells was determined by MTT method. The intracellular doxorubicin (ADM) concentration was determined by HPLC.


RESULTS
The introduction of pEGFP-C1/U6/MDR1-A or pEGFP-C1/U6/MDR1-B expression vector was shown to efficiently and specifically inhibit the expression of P-gp according to results of Western blot, with an inhibitory rate of 50.67%. Semi-quantitative RT-PCR showed that mRNA transcription of MDR1 gene was reduced by (48.2 +/- 2.5)%. On the contrast, the control plasmid did not exhibit inhibitory effect on the protein expression and mRNA transcription of MDR1. The relative efficiency of K562/A02 to ADM was 40.8% or 62.4%, respectively, and the intracellular accumulation of ADM increased after shRNA treatment.


CONCLUSIONS
The shRNA expression vector targeting MDR1 gene showed dramatic inhibition on RNA transcription and protein expression. It could effectively restore the sensitivity of K562/A02 cells to conventional chemotherapeutic agents.
Xiao
Xi-Bin
XB
Department of Hematology, Xiangya Hospital, Central South University, Changsha 410008, China. xzxchangsha@tom.com
Xie
Zhao-Xia
ZX
Qin
Qun
Q
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism; D000903:Antibiotics, Antineoplastic / Q000378:metabolism / Q000494:pharmacology; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000378:metabolism / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D004317:Doxorubicin