[发夹状小干扰RNA逆转K562/A02细胞系中mdr1和GSTpi表达的功能研究]

[Functional study of mdr1 and GSTpi expression reversed by hairpin siRNA in K562/A02 cell line].

作者信息

Gu Jing-wen, Zhang Tao, Chen Bo-bin, Lu Yuan, Lin Guo-wei

机构信息

Department of Hematology, Central Laboratory, Huashan Hospital, Fudan University, Shanghai 200040, China.

出版信息

Zhonghua Xue Ye Xue Za Zhi. 2006 Jan;27(1):17-20.

PMID:16732933

Abstract

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.

METHOD

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.

RESULT

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).

CONCLUSION

The shRNA could effectively and specifically reverse the multidrug resistance on K562/A02 cell line.

摘要

目的

研究发夹状小干扰RNA（shRNA）对多药耐药人白血病细胞系K562/A02中mdr1和GSTpi蛋白表达的影响。

方法

分别合成靶向mdr1编码区序列（79 - 99 nt）和GSTpi编码区序列（308 - 327 nt）的shRNAs，并克隆至质粒pSilencer2.1-U6 neo。将克隆产物pSilence mdr1和pSilence GSTpi转染至K562/A02细胞。采用蛋白质免疫印迹法和免疫荧光分析法检测基因沉默的有效性和特异性。采用MTT法测定阿霉素（ADM）对K562/A02细胞的50%抑制浓度（IC(50)）。

结果

与空载体处理相比，pSilence mdr1和pSilence GSTpi分别将P-gp和GSTpi蛋白表达从0.75±0.02和0.54±0.02降至0.48±0.05和0.39±0.02（P<0.01），对α-微管蛋白表达无影响。将pSilence lamin A/C转染至K562/A02可降低lamin A/C表达，但对P-gp和GSTpi表达无影响。免疫荧光分析还显示，与空载体处理相比，shRNAs使P-gp和GSTpi阳性细胞分别从（71.25±9.65）%和（81.25±6.49）%显著降至（35.25±5.97）%和（41.25±4.43）%（P<0.01）。转染后的耐药指数分别从23（空载体转染）降至8（pSilence mdr1）和10（pSilence GSTpi）（P<0.01）。