[Functional study of mdr1 and GSTpi expression reversed by hairpin siRNA in K562/A02 cell line].
OBJECTIVE
To investigate the effect of hairpin small interference RNA (shRNA) on mdr1 and GSTpi protein expression in multidrug resistance human leukemia cell line K562/A02.


METHODS
The shRNAs were synthesized targeting the coding region sequences of mdr1 (79 - 99 nt) and GSTpi (308 - 327 nt) respectively, and cloned to plasmid pSilencer2.1-U6 neo. The cloned products pSilence mdr1 and pSilence GSTpi were transfected into K562/A02 cells. Western blot and immunofluorescence analysis were used to detect the effectiveness and the specificity of the gene silence. 50% inhibition concentration (IC(50)) of doxorubicin (ADM) on K562/A02 cells was determined by MTT method.


RESULTS
pSilence mdr1 and pSilence GSTpi reduced the expression of P-gp and GSTpi protein from 0.75 +/- 0.02 and 0.54 +/- 0.02 to 0.48 +/- 0.05 and 0.39 +/- 0.02 (P < 0.01) respectively, with no effect on alpha-tubulin expression in comparison with the mock treatment. Transfection of pSilence lamin A/C into K562/A02 decreased lamin A/C expression but had no effect on the expression of P-gp and GSTpi. Immunofluorescence assay also showed that shRNAs significantly reduced the P-gp and GSTpi positive cells from (71.25 +/- 9.65)% and (81.25 +/- 6.49)% to (35.25 +/- 5.97)% and (41.25 +/- 4.43)% (P < 0.01), respectively, compared with the mock treatment. The resistance indexes after transfection were decreased to 8 (pSilence mdr1) and 10 (pSilence GSTpi) respectively from 23 (mock transfection) (P < 0.01).


CONCLUSIONS
The shRNA could effectively and specifically reverse the multidrug resistance on K562/A02 cell line.
Gu
Jing-wen
JW
Department of Hematology, Central Laboratory, Huashan Hospital, Fudan University, Shanghai 200040, China.
Zhang
Tao
T
Chen
Bo-bin
BB
Lu
Yuan
Y
Lin
Guo-wei
GW
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D051549:Glutathione S-Transferase pi / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051549:Glutathione S-Transferase pi