[小干扰RNA沉默mdr1基因表达并逆转K562/ADM细胞系的凋亡抗性]

[siRNA silences mdr1 gene expression and reverses apoptosis resistance of K562/ADM cells line].

作者信息

Wei Hu-lai, Gao Li-ping, Jing Tao, Zhao Huai-shun, Yi Juan, Sun Jin, Han Jian

机构信息

Laboratory Center for Medical Science, Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Lanzhou University, Lanzhou 730000, China.

出版信息

Zhonghua Xue Ye Xue Za Zhi. 2007 Jun;28(6):388-90.

DOI:

PMID:17939404

Abstract

OBJECTIVE

To explore the effect of small interfering RNA(siRNA) on silence of mdr1 gene and reversal of apoptosis resistance in multidrug-resistant (MDR) human leukemia K562/ADM cell.

METHODS

Human MDR leukemia cell line K562/ADM was used as the target cells. Two siRNAs (mdr1 siRNA-1 and mdr1 siRNA-2) targeted mdr1 gene were chemically synthesized and transfected into K562/ADM cells with liposome. Expression of mdr1 mRNA was determined by real-time PCR, P-glycoprotein (P-gp) expression and caspase-3 activity were measured with flow cytometry (FCM), and the cell apoptosis was observed by optical and electronic microscopy for morphology and Annexin V/PI staining.

RESULTS

The mdr1 siRNA-1 and mdr1 siRNA-2 could markedly down-regulate the expression of mdr1 gene in K562/ADM cells, the expression of mdr1 mRNA decreased by 91.2% and 82.0% , and the P-gp by 74.1% and 84.4%, respectively. The caspase-3 activity was markedly enhanced, and the active caspase-3 in K562/ADM cells increased by about 40% compared to liposome alone and non-silencing controls. the sensitivity of K562/ADM cells to adriamycin-induced apoptosis was significantly augmented, the apoptotic rate of the cells treated with siRNA plus adriamycin increased by about 60% compared to adriamycin alone.

CONCLUSION

siRNAs silence the expression of mdr1/P-gp to overcome the P-gp-mediated apoptosis resistance in drug-resistant K562/ADM cells.

摘要

目的

探讨小干扰RNA（siRNA）对多药耐药（MDR）人白血病K562/ADM细胞中mdr1基因沉默及凋亡抗性逆转的影响。

方法

以人MDR白血病细胞系K562/ADM为靶细胞。化学合成两种靶向mdr1基因的siRNA（mdr1 siRNA-1和mdr1 siRNA-2），并用脂质体将其转染到K562/ADM细胞中。通过实时PCR测定mdr1 mRNA的表达，用流式细胞术（FCM）检测P-糖蛋白（P-gp）的表达和半胱天冬酶-3的活性，通过光学和电子显微镜观察细胞凋亡的形态及用Annexin V/PI染色。

结果

mdr1 siRNA-1和mdr1 siRNA-2可显著下调K562/ADM细胞中mdr1基因的表达，mdr1 mRNA的表达分别下降了91.2%和82.0%，P-gp分别下降了74.1%和84.4%。半胱天冬酶-3的活性显著增强，与单独使用脂质体和非沉默对照相比，K562/ADM细胞中活性半胱天冬酶-3增加了约40%。K562/ADM细胞对阿霉素诱导凋亡的敏感性显著增强，与单独使用阿霉素相比，用siRNA加阿霉素处理的细胞凋亡率增加了约60%。