[通过mdr1和GSTpi基因沉默逆转K562/A02细胞系的多药耐药性]

[Reversal of multidrug resistance of K562/A02 cell line by mdr1 and GSTpi gene silence].

作者信息

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

机构信息

Department of Hematology, Central Laboratory, Hua-shan Hospital, Fudan University, Shanghai 200040, China.

出版信息

Zhonghua Xue Ye Xue Za Zhi. 2005 Dec;26(12):719-22.

PMID:16620573

Abstract

OBJECTIVE

To investigate the effect of short hairpin RNA (shRNA) on mdr1 and GSTpi expression of human multidrug resistant leukemia cell line K562/A02.

METHODS

shRNAs were synthesized according to the sequence targeting mdr1 and GSTpi coding region of 79-99nt and 308 approximately 327nt, and cloned into pSilencer 2.1-U6 neo vector. The cloned products, pSilence-mdr1 and pSilence-GSTpi, were transfected into K562/A02 cell line. Expression of mdr1 and GSTpi mRNA was assayed by real time PCR. 50% inhibition concentration (IC(50)) of doxorubicin (ADM) for K562/A02 cell line was determined by MTT method.

RESULTS

After transfected with pSilence mdr1, the expression of mdr1 mRNA in K562/A02 cells was reduced by 71.5%, from (2.80 +/- 1.65) x 10(8) copy/microg RNA to (3.90 +/- 2.37) x 10(7) copy/microg RNA(P < 0.01). While the expression of GSTpi mRNA in pSilence-GSTpi transfected K562/A02 cells reduced by 39.8%, from (2.30 +/- 1.14) x 10(5) copy/microg RNA to (5.40 +/- 2.45) x 10(4) copy/microg RNA (P < 0.01). The resistance indexes after transfection were decreased to 8 and 10 respectively as compared to 23 of the mock transfection (P < 0.01).

CONCLUSION

The shRNA could effectively reverse the multidrug resistance of K562/A02 cell line.

摘要

目的

研究短发夹RNA（shRNA）对人多药耐药白血病细胞系K562/A02中mdr1和GSTpi表达的影响。

方法

根据靶向mdr1编码区79 - 99nt以及GSTpi编码区约308至327nt的序列合成shRNAs，并克隆至pSilencer 2.1 - U6 neo载体。将克隆产物pSilence - mdr1和pSilence - GSTpi转染至K562/A02细胞系。通过实时PCR检测mdr1和GSTpi mRNA的表达。采用MTT法测定阿霉素（ADM）对K562/A02细胞系的50%抑制浓度（IC(50)）。

结果

转染pSilence mdr1后，K562/A02细胞中mdr1 mRNA的表达降低了71.5%，从(2.80 ± 1.65)×10(8)拷贝/μg RNA降至(3.90 ± 2.37)×10(7)拷贝/μg RNA（P < 0.01）。而转染pSilence - GSTpi的K562/A02细胞中GSTpi mRNA的表达降低了39.8%，从(2.30 ± 1.14)×10(5)拷贝/μg RNA降至(5.40 ± 2.45)×10(4)拷贝/μg RNA（P < 0.01）。与空载体转染的23相比，转染后的耐药指数分别降至8和10（P < 0.01）。