小分子干扰RNA联合反义脱氧寡核苷酸靶向逆转人乳腺癌细胞MCF-7/ADR多药耐药的研究

Reversal of multidrug resistance of human breast cancer MCF-7/ADR cells by small interfering RNAs combined with antisense oligodeoxyribonucleotides for inhibition of MDR1 gene expression

目的探讨针对多药耐药基因(MDR-1)的小分子干扰RNA(siRNAs)和反义脱氧寡核苷酸(asODNs)联合应用逆转人乳腺癌细胞MCF-7/ADR的作用效果。方法设计并合成针对MDR-1基因同一序列的siRNAs和asODNs及阴性对照siRNAs,采用转染试剂lipofectamineTM2000分别转染人乳腺癌耐药细胞MCF-7/ADR;利用RT-PCR检测MDR-1mRNA和Western blot检测MDR-1蛋白质的表达;采用罗丹明123外排实验检测P-gp的转运功能,MTT法检测MCF-7/ADR细胞对阿霉素的耐药逆转效果。结果siRNAs、asODNs、asODNs和siRNAs联合应用均能降低MDR-1mRNA及其蛋白质表达,提高P-gp的转运功能,使细胞对阿霉素的敏感性明显恢复;asODNs和siRNAs联合应用效果明显提高;低浓度siRNAs(200nmol/L)比高浓度asODNs(5μmol/L)的效果强。结论siRNAs、asODNs能有效逆转人乳腺癌细胞MCF-7/ADR的多药耐药,asODNs和siRNAs联合应用效果明显加强。

Objective To study the effect of small interfering RNAs（siRNAs） combined with antisense oligodeoxyribonucleotides （asODNs） , which targeted MDR1 gene, on reversing multidrug resistance of human breast cancer cell line MCF-7/ADR. Methods The siRNAs and asODNs which targeted the same sequences of MDR1 gene were designed and synthesized. Human breast cancer MCF-7/ADR cells were cultured and tranfected with asODNs, siRNAs ＋ asODNs, siRNAs, and negative siRNAs using lipofectamineTM 2000, respectively. MDR-1mRNA was assayed by RT-PCR and the protein expression was detected by Western blotting. The function of P-glycoprotein （P-gp） was detected by rhodamine 123 retention and the resistant efficiency of MCF-7/ADR to ADM was determined by MTT method. Results The expressions of MDR-lmRNA and protein decreased significantly after transfection of asODNs, siRNAs ＋ asODNs, and siRNAs respectively. The transporting function of P-gp increased and the resistance of MCF-7/ADR to ADM reversed significantly. The reversing effect increased significantly by siRNAs combined with asODNs. The inhibition effect of the siRNAs with lower concentration （200 nmol/L）was greater than that of asODNs with higher concentration （5 umol/L）. Conclusion siRNAs and asODNs could reverse multidmg resistance of human breast cancer cell line MCF-7/ADR. The inhibition effect is increased significantly by siRNAs combined with asODNs.