靶向siRNA沉默mdr1基因表达及对白血病耐药细胞系K562/ADM的耐药逆转作用

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

目的研究小干扰 RNA(siRNA)对白血病多药耐药细胞系 K562/ADM 细胞 mdr1基因表达的沉默作用和凋亡抑制的逆转效应。方法 K562/ADM 为靶细胞,设汁、筛选和合成2对针对mdr1基因 mRNA 的 siRNA(mdr1 siRNA-1和 mdr1 siRNA-2),用脂质体介导转染 K562/ADM 细胞;实时荧光定量 PCR(real-tMe PCR)法检测 mdr1 mRNA 的表达;流式细胞术测定 P-糖蛋白(P-gp)水平和caspase-3活性;细胞形态学和 FITC 标记的膜联蛋白 V/碘化丙锭(Annexin V-FITC/PI)双染色法检测细胞的凋亡。结果筛选出的 mdr1 siRNA-1和 mdr1 siRNA-2显著抑制 K562/ADM 细胞 mdr1的表达,mdr1 mRNA 的表达分别降低91.2%和82.0%,P-gp 水平下降74.1%和84.4%;增强 caspase-3活性,活化 caspase-3增加约40%;K562/ADM 耐药细胞对阿霉素诱导凋亡的敏感性增强,Annexin V-FITC/PI染色检测细胞凋亡率提高约60%。结论 siRNA 通过沉默 mdr1/P-gp 表达而逆转 K562/ADM 多药耐药细胞的凋亡抑制现象。

Objective To explore the effect of small interfering RNA（siRNA） on silence of mdr1 gene and reversal of apoptosis resistance in muhidrug-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 I（562/ 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 obserued 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.