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乳腺癌耐药蛋白(BCRP)在肿瘤细胞中的过表达可能导致肿瘤耐药性。RNA干扰是一种选择性抑制目的基因表达的实用技术。本研究旨在应用RNA干扰技术调控BCRP的表达和功能。首先在过表达BCRP的耐药细胞MCF-7/MX100中,评价了三种靶向于BCRP的si RNAs(si-BCRP1,si-BCRP2和si-BCRP3)。研究发现si-BCRP2和si-BCRP3对BCRP的m RNA和蛋白的抑制率分别超过90%和70%,进而导致MCF-7/MX100细胞中米托蒽醌的积聚显著上升。进一步的研究将生成si-BCRP2和si-BCRP3的shR NA序列克隆至慢病毒表达载体pT RIPZ中,并采用慢病毒介导的转基因体系建立了稳定表达si RNA的MCF-7/MX100细胞。在该细胞系中,si-BCRP2和si-BCRP3对BCRP的mR NA的抑制率分别达到72%和56%,对其蛋白的抑制率分别为70%和53%。在进一步的研究中,该细胞系被用于中药活性成分的筛选,以评价BCRP的底物和抑制剂。结果显示,BCRP低表达的MCF-7/MX100细胞可能作为良好的细胞模型被用于临床前研究。
Overexpression of breast cancer resistance protein (BCRP) in tumor cells may result in tumor resistance. RNA interference is a potent technique that selectively inhibits the expression of a gene of interest. This study aimed to use RNA interference technology to regulate the expression and function of BCRP. Three siRNAs targeted to BCRP (si-BCRP1, si-BCRP2 and si-BCRP3) were first evaluated in BCRP-overexpressing drug-resistant cells MCF-7 / MX100. The inhibition of m RNA and protein of BCRP by si-BCRP2 and si-BCRP3 was found to be over 90% and 70%, respectively, leading to a significant increase in mitoxantrone accumulation in MCF-7 / MX100 cells. Further studies cloned the shR NA sequence of si-BCRP2 and si-BCRP3 into the lentiviral expression vector pT RIPZ. MCF-7 / MX100 cells stably expressing si RNA were constructed by using lentivirus-mediated transgenic system. In this cell line, the inhibitory rates of si-BCRP2 and si-BCRP3 on BCRP mR NA were 72% and 56%, respectively, and their protein inhibition rates were 70% and 53%, respectively. In a further study, the cell line was used for the screening of the active ingredients of traditional Chinese medicine to evaluate the substrates and inhibitors of BCRP. The results show that MCF-7 / MX100 cells with low BCRP expression may be used as preclinical studies as a good cell model.