Breast cancer resistance protein BCRP/ABCG2 regulatory microRNAs (hsa-miR-328, -519c and -520h) and their differential expression in stem-like ABCG2+ cancer cells.
Breast cancer resistance protein BCRP/ABCG2 regulatory microRNAs (hsa-miR-328, -519c and -520h) and their differential expression in stem-like ABCG2+ cancer cells.
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DOI：
10.1016/j.bcp.2010.12.018
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发表时间：
2011-03-15
影响因子：
5.8
通讯作者：
Yu AM
中科院分区：
文献类型：
--
作者：
Li X;Pan YZ;Seigel GM;Hu ZH;Huang M;Yu AM
最近的研究表明，许多microRNAs(miRNA或miR)可能调节人乳腺癌耐药蛋白(BCRP/ABCG2)，BCRP/ABCG2是负责细胞药物处置的重要外排转运体，但它们对ABCG2蛋白表达的影响尚未进行比较。在本研究中，我们首次通过计算分析在ABCG2 3‘-非翻译区(3’UTR)中发现了hsa-miR-519c的一个新的近端miRNA反应元件(MRE)。用双荧光素酶报告基因分析和定点突变证实了miR-519c的MRE位点。免疫印迹分析显示，hsa-miR-328-或-519c表达载体转染MCF-7/MX100细胞后，ABCG2蛋白表达显著下调，而miR-328或-519c反义寡核苷酸转染MCF-7细胞后，ABCG2蛋白表达显著上调。而在MCF-7/MX100细胞中，hsa-miR-520h表达载体的ABCG2蛋白表达没有变化，这与未检测到miR-520h的表达有关。此外，在转染miR-519c表达载体的细胞中，ABCG2mRNA的降解速度明显加快，提示这可能参与了mRNA的降解机制。通过流式细胞仪分析，干预miR-328或-519c信号导致细胞内米托蒽醌积聚的显著变化。此外，我们通过免疫磁选将RB143人视网膜母细胞瘤细胞分为干性细胞(ABCG2+)和非干性细胞(ABCG2−)，发现在ABCG2+细胞中miR-328、-519c和-520h的表达水平分别降低了9、15和3倍。我们的数据表明，miR-519c和-328比miR-520h对MCF-7人乳腺癌细胞ABCG2表达的影响更大，并且近端miR-519c的存在解释了miR-519c对ABCG2 3‘UTR缩短的作用。
Recent studies have shown that a number of microRNAs (miRNA or miR) may regulate human breast cancer resistance protein (BCRP/ABCG2), an important efflux transporter responsible for cellular drug disposition, whereas their effects on ABCG2 protein expression are not compared. In this study, we first identified a new proximal miRNA response element (MRE) for hsa-miR-519c within ABCG2 3′-untranslated region (3′UTR) through computational analyses. This miR-519c MRE site was confirmed using dual luciferase reporter assay and site-directed mutagenesis. Immunoblot analyses indicated that ABCG2 protein expression was significantly down-regulated in MCF-7/MX100 cells after transfection with hsa-miR-328- or -519c expression plasmids, and was markedly up-regulated in MCF-7 cells after transfection with miR-328 or -519c antagomir. However, ABCG2 protein expression was unchanged in MCF-7/MX100 cells after transfection with hsa-miR-520h expression plasmids, which was associated with undetectable miR-520h expression. Furthermore, ABCG2 mRNA degradation was accelerated dramatically in cells transfected with miR-519c expression plasmid, suggesting the involvement of mRNA degradation mechanism. Intervention of miR-328 or -519c signaling led to significant change in intracellular mitoxantrone accumulation, as determined by flow cytometry analyses. In addition, we separated RB143 human retinoblastoma cells into stem-like (ABCG2+) and non-stem-like (ABCG2−) populations through immunomagnetic selection, and found that miR-328, -519c and -520h levels were 9-, 15- and 3-fold lower in the ABCG2+ cells, respectively. Our data suggest that miR-519c and -328 have greater impact on ABCG2 expression than miR-520h in MCF-7 human breast cancer cells, and the presence of proximal miR-519c MRE explains the action of miR-519c on shortened ABCG2 3′UTR.
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影响因子：
14.9
作者：
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