发夹状siRNA逆转白血病K562/A02细胞株mdr1和GSTπ功能的研究

Functional study of mdrl and GSTπ expression reversed by hairpin siRNA in K562/A02 cell line

目的研究发夹状小分子干扰RNA(small RNA interference,siRNA)对白血病多药耐药细胞株K562/A02多药耐药基因(mdr1)和谷胱甘肽S-转移酶(GSTπ)基因功能的影响。方法以 mdr1 mRNA第79-99和GSTπ mRNA第308-327核苷酸为作用靶点,合成针对靶区域序列的发夹状 siRNA,克隆入pSilencer2．1-U6 neo,克隆产物为pSilence mdr1和pSilence GSTπ,转染K562/A02细胞株。用Western blot和荧光免疫组化法观察P糖蛋白(P-gp)和GSTπ蛋白的表达;MTT法检测阿霉素对K562/A02细胞半数抑制浓度(IC50)。结果 Western blot检测显示转染pSilence mdr1和pSilence GSTπ的K562/A02细胞株与K562/A02细胞对照组相比,P-gp和GSTπ的表达明显下降,分别从0．75 ±0．02和0．54±0．02下降到0．48±0．05和0．39±0．02(P值均<0．01),α-微管蛋白的表达没有变化;转染pSilence核纤层蛋白A/C的K562/A02细胞株与对照组相比,核纤层蛋白A/C表达明显下降,但P-gp和GSTπ的表达不变;荧光免疫组化显示P-gp和GSTπ阳性细胞比例从转染前的(71．25± 9．65)％和(81．25±6．49)％下降到转染后的(35．25±5:97)％和(41．25±4．43)％(P值均<0．01); 转染空载体后K562/A02细胞对阿霉素的耐药指数为23,转染pSilence mdr1和pSilence GSTπ后分别下降为8和10,差异有统计学意义(P<0．01),结论 siRNA可有效、特异地逆转K562/A02细胞株 mdr1和GSTπ的多药耐药性。

Objective To investigate the effect of hairpin small interference RNA （shRNA） on mdr1 and GSTπ 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 GSTπ （308 ～327 nt） respectively, and cloned to plasmid pSilencer2, 1-U6 neo. The cloned products pSilence mdr1 and pSilence GSTπ 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 （IC50） of doxorubicin（ADM） on K562/A02 cells was determined by MTF method. Result pSilence mdr1 and pSilence GSTπ reduced the expression of P-gp and GSTπ 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 α-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 GSTπ. Immunofluorescence assay also showed that shRNAs significantly reduced the P-gp and GSTπ 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 transfeetion were decreased to 8 （pSilence mdr1 ） and 10 （pSilence GSTπ） respectively from 23 （ mock transfection） （P 〈 0.01 ）. Conclusion The shRNA could effectively and specifically reverse the muhidrug resistance on K562/A02 cell line.