title: Breast cancer resistance protein BCRP/ABCG2 regulatory microRNAs (hsa-miR-328, -519c and -520h) and their differential expression in stem-like ABCG2+ cancer cells.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14260-1200, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: Yu-Zhuo
authors.initials: YZ
authors.lastname: Pan
authors.forename: Gail M
authors.initials: GM
authors.lastname: Seigel
authors.forename: Zi-Hua
authors.initials: ZH
authors.lastname: Hu
authors.forename: Min
authors.initials: M
authors.lastname: Huang
authors.forename: Ai-Ming
authors.initials: AM
authors.lastname: Yu
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis*; D000818:Animals; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D006801:Humans; D049410:Luciferases, Renilla / Q000502:physiology; D035683:MicroRNAs / Q000096:biosynthesis* / Q000502:physiology; D009363:Neoplasm Proteins / Q000096:biosynthesis*; D014411:Neoplastic Stem Cells / Q000378:metabolism*
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; C548170:MIRN328 microRNA, human; C546194:MIRN519 microRNA, human; D035683:MicroRNAs; D009363:Neoplasm Proteins; D049410:Luciferases, Renilla