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目的探讨碱基错配对短发夹RNA(shRNA)逆转白血病细胞耐药作用的影响。方法针对mdr1基因mRNA合成一条序列正确的shRNA及正义链少1个碱基的对应错配shRNA,构建一对抗mdr1 shRNA真核表达载体,脂质体介导转染白血病耐药细胞株K562／A02。采用实时荧光定量RT- PCR检测mdr1 mRNA表达;柔红霉素泵出实验检测P-gp外排泵功能;MTT法检测细胞对阿霉素的敏感性。结果序列正确和对应错配shRNA真核表达载体均明显下调mdr1 mRNA的表达,降低细胞膜P-gp外泵功能,60 min柔红霉素泵出率分别为6％和10％。显著低于对照组的45％;细胞内柔红霉素平均荧光强度分别为9．51和7．09,明显高于对照组的2．80;对阿霉素药物敏感性的相对逆转率为90％和87％。结论正义链碱基错配shRNA对RNA干扰功能无明显影响,与序列正确的shRNA一样可有效逆转mdr1所致的耐药。
Objective To investigate the effect of base mismatch on short-hairpin RNA (shRNA) -mediated reversal of drug resistance in leukemia cells. Methods The mdr1 gene mRNA was synthesized with a sequence-correct shRNA and a mismatch shRNA of 1 base less than that of sense strand. A pair of anti-mdr1 shRNA eukaryotic expression vectors were constructed. Liposome-mediated transfection of leukemia cell line K562 / A02 . The expression of mdr1 mRNA was detected by real-time fluorescent quantitative RT-PCR. The function of P-gp efflux pump was detected by pump-in of daunorubicin and the sensitivity of cells to doxorubicin was detected by MTT assay. The correct sequence and the corresponding mismatch shRNA eukaryotic expression vector significantly down-regulated the expression of mdr1 mRNA and decreased the P-gp external pump function. The pump-out rates of daunorubicin at 6 and 10 min were 6% and 10%, respectively. Significantly lower than 45% of the control group; intracellular daunorubicin average fluorescence intensity was 9.51 and 7.09, significantly higher than the control group 2.80; the relative reversal rate of doxorubicin drug sensitivity was 90% and 87%. Conclusion Mismatched sense strand base mismatch shRNA had no significant effect on RNA interference function, as the correct shRNA sequence could effectively reverse the resistance induced by mdr1.