WAVE1基因在K562/A02白血病细胞多药耐药中的作用

Role of WAVE1 in drug resistance of K562/A02 leukemia cells

目的探讨 WASP 家族 Verprolin 同源蛋白1(WAVE1)基因在 K562/A02白血病细胞多药耐药机制中的作用。方法将 pEFBOS-WAVE1真核表达质粒转染 K562细胞构建 WAVE1高表达的 K562细胞,将 WAVE1基因的特异性小片段干扰 RNA(WAVE1 siRNA)转染 K562/A02细胞构建WAVE1低表达的 K562/A02细胞。Western blot 和 RT-PCR 法检测基因转染前后 K562/A02细胞及K562细胞 WAVE1基因及蛋白的表达;可溶性噻唑盐 WST-8染色法检测阿霉素对转染前后细胞的半数抑制浓度(IC_(50));Hoechest33258染色法检测细胞形态学改变并计算凋亡细胞百分率;RT-PCR 检测多药耐药基因 mdrl mRNA 表达;Western blot 检测 Bcl-2表达。结果①与 K562细胞相比,K562/A02细胞 WAVE1 mRNA 表达水平增加70%,蛋白表达水平增加63%。②转染 WAVE1基因的 K562细胞WAVE1过表达,并降低了对阿霉素的药物敏感性,使 IC_(50)从转染前的(0.35±0.00)μg/ml,增加到(2.99±0.12)μg/ml,在阿霉素浓度为1、5μg/ml时,凋亡细胞分别下降30%、35%。③转染 WAVE1siRNA 的 K562/A02细胞 WAVE1蛋白表达水平与未转染组相比明显降低,并可增强对阿霉素的药物敏感性,使 IC_(50)从转染前的(4.29±0.15)μg/ml下降到(1.85±0.07)μg/ml,阿霉素浓度为1、5μg/ml时,凋亡细胞分别增加24%、21%。④转染 WAVE1的 K562细胞 mdr1基因和 Bcl-2蛋白表达水平均增高,而转染 WAVE1 siRNA 的 K562/A02细胞 mdr1基因和 Bcl-2蛋白表达水平比转染前明显降低。结论 WAVE1参与了 K562/A02细胞多药耐药的形成,其机制可能与调控 mdr1和 Bcl-2水平有关。

Objective To investigate if WAVE1 is involved in muh drug-resistance （MDR） of human leukemia cell line K562/A02. Methods The level of WAVE1 in K562 and K562/A02 cells was assayed by Western blot and RT-PCR; K562 cells and K562/A02 cells were transient transfected with pEFBOSWAVE1 reconstructed plasmid or specifically siRNA to WAVE1. 50 % inhibition concentration （IC50） of doxorubicin on K562/A02 was determined by WST-8 assay. Hoechst33258 staining was used to examine cell morphological changes and to calculate percentage of apoptotic nuclei. The mRNA level of mdr1 was assayed by RT-PCR. The Bcl-2 protein was assayed by Western blot. Results （1） The WAVE1 expression at mRNA and protein level in K562/A02 cells was increased by about 70% and 63% respectively as compared with that in K562 cells. （2）Overexpression of WAVE1 in K562 cells by transient transfection significantly increased the resistance to doxorubicin, and increased IC50 from （0.05 ±0.00） μg/ml to （2.99 ±0.12） μg/ml, and at 1 μg/ml or 5 μg/ml of doxorubicin treatment, cell apoptotic nuclei rate was decreased by 30% or 35% respectively. （3）Suppression of WAVE1 in K562/A02 cells by siRNA resulted in a reversal of MDR to doxorubicin, and decreased IC50 from （4.29 ± 0.15 ） μg/ml to （ 1.85 ± 0.07 ） μg/ml, and at 1 μg/ml or 5 μg/ml of doxorubicin treatment, cell apoptotic nuclei rate was increased by 24% or 21% respectively. （4）Overexpression of WAVE1 in K562 cells significantly increased the mdr1 mRNA and the Bcl-2 protein, and suppression of WAVE1 in K562/A02 cells by siRNA decreased the mRNA and the protein. Conclusion WAVE1 involves in the MDR mechanisms in K562/A02 leukemia cells through regulation the level of mdr1 and Bcl-2.