Silencing the expression and function of breast cancer resistance protein in MCF-7/MX100 cells by shRNA expressing lentivirus

应用shRNA慢病毒技术抑制MCF7/MX100细胞中乳腺癌耐药蛋白的表达和功能(英文)

Overexpression of breast cancer resistance protein （ABCG2/BCRP） in cancer cells may cause tumor resistance to chemotherapeutic drugs. RNA interference （RNAi） can selectively silence the expression of a target gene of interest. In the present study, we aimed to modulate the BCRP expression and examine the functional consequence using RNAi approach. Three siRNAs （si-BCRP1, si-BCRP2 and si-BCRP3） targeting BCRP were evaluated in drug-resistant MCF-7/MX100 cells overexpressing BCRP. The BCRP expression at the mRNA and protein levels was inhibited by si-BCRP2 and si-BCRP3 over 90% and 70%, respectively. As a result, the intracellular mitoxantrone accumulation was sharply increased in MCF-7/ MX100 cells after the transfection. Furthermore, shRNA sequences bearing si-BCRP2 and siBCRP3 were cloned into lentiviral expression plasmid （pTRIPZ） to package lentivirus, and MCF-7/MX100 cells stably expressing siRNA targeted to human ABCG2/BCRP were established by lentivector-mediated gene transfer system. The stable cells exhibited an increased miotxantrone accumulation, among which the BCRP expression at the mRNA level was reduced by Lenti-BCRP2 and Lenti-BCRP3 around 72% and 56%, respectively. Moreover, the BCRP expression at the protein level was reduced by 70% and 53%, respectively. Furthermore, the cell lines were used to screen active ingredients in traditional herbal medicines in order to evaluate BCRP substrates or inhibitors. Our data suggested that the BCRP knockdown cell lines could serve as good cell models for preclinical studies.

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。