mdr1和GSTπ基因缄默逆转K562/A02细胞多药耐药性的研究

Reversal of multidrug resistance of K562/A02 cell line by mdr1 and GSTπ gene silence

目的研究短发夹状小分子干扰RNA(short hairpin RNA,shRNA)对白血病多药耐药细胞株K562/A02 mdr1和谷胱甘肽S-转移酶(GST)π基因的表达和功能的影响.方法根据mdr1mRNA第79～99位和GSTπ mRNA第308～327位核苷酸为作用靶点,合成针对靶区域序列的shRNA,克隆入pSilencer2.1-U6 neo,克隆产物为pSilence-mdrl和pSilence-GSTπ,转染K562/A02细胞株.用实时荧光定量PCR检测K562/A02细胞mdr1和GSTπ mRNA的表达;MTT法检测阿霉素对K562/A02细胞半数抑制浓度(IC50).结果经pSilence-mdr1转染后的K562/A02细胞mdr1 mRNA表达量下降了71.5%,从(2.80±1.65)×108拷贝/μgRNA下降至(3.90±2.37)×107拷贝/μg RNA(P＜0.01);同时pSilence-GSTπ作用后,K562/A02细胞GSTπ mRNA表达量较对照组下降了39.8%,从(2.30±1.14)×105拷贝/μg RNA下降至(5.40±2.45)×104拷贝/μg RNA(P＜O.01);空载体转染后阿霉素的耐药指数为23,pSilence-mdr1转染后为8,pSilence-GSTπ转染后为10,差异有统计学意义(P＜0.01).结论shRNA可有效逆转K562/A02细胞mdr1和GSTπ的多药耐药性.

Objective To investigate the effect of short hairpin RNA（shRNA） on mdrl and GSTπ expression of human muhidrug resistant leukemia cell line K562/A02. Methods shRNAs were synthesized according to the sequence targeting mdrl and GSTπ coding region of 79 - 99nt and 308 - 327nt, and cloned into pSilencer 2. 1-U6 neo vector. The cloned products, pSilence-mdrl and pSilence-GSTπ, were transfected into K562/A02 cell line. Expression of mdrl and GSTπ mRNA was assayed by real time PCR. 50% inhibition concentration （IC50）of doxorubicin （ADM） for K562/A02 cell line was determined by MTF method. Results After transfected with pSilence mdrl, the expression of mdrl mRNA in K562/A02 cells was reduced by 71.5% , from （2.80 ± 1.65） × 10^8 copy/μg RNA to （3.90 ± 2.37） × 10^7 copy/μg RNA （P 〈 0.01）. While the expression of GSTπ mRNA in pSilence-GSTπ transfected K562/A02 cells reduced by 39.8% , from （2.30±1.14） × 10^5copy/μg RNA to （5.40 ± 2.45） × l0^4 copy/μg 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.