Elsevier

Biochemical Pharmacology

Volume 81, Issue 6, 15 March 2011, Pages 783-792
Biochemical Pharmacology

Breast cancer resistance protein BCRP/ABCG2 regulatory microRNAs (hsa-miR-328, -519c and -520h) and their differential expression in stem-like ABCG2+ cancer cells

https://doi.org/10.1016/j.bcp.2010.12.018Get rights and content

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.

Graphical abstract

Hsa-miR-519c and -328 showed a more profound impact on BCRP/ABCG2 expression than miR-520h in human breast cancer cells.

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Introduction

MicroRNAs (miRNA or miR) are a large group of short, noncoding RNAs that act on their mRNA targets by complementary Watson–Crick base pairings, and control posttranscriptional regulation of target genes through translation inhibition or mRNA cleavage [1], [2]. More than 1000 miRNAs have been identified within human genome, which may regulate thousands of protein-coding genes involved in essentially all critical biological processes [3]. There is also accumulating evidence supporting the hypothesis that miRNAs are involved in the regulation of drug metabolism and disposition [4], [5], [6], [7], [8]. Some miRNAs directly target the 3′-untranslated region (3′UTR) of genes encoding drug-metabolizing enzymes and/or drug transporters [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]. Other miRNAs act on the 3′UTR of transcriptional regulators (e.g., xenobiotic receptors) of drug-metabolizing enzymes and drug transporters [10], [20], [21], [22]. As a result, these miRNAs may determine the final protein expression levels of enzymes or transporters, modulate the capacity of drug metabolism and disposition, and affect the response of cells to xenobiotic drugs.

Breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette membrane transporter that is expressed ubiquitously in human tissues. ABCG2 plays an important role in cellular disposition of a variety of xenobiotic drugs (e.g., mitoxantrone, doxorubicin and topotecan) and metabolites (e.g., estrone-3-sulfate and estradiol-17-β-d-glucuronide) [23], [24]. Variations of ABCG2 gene expression and protein function may result in significant change in pharmacokinetics and drug response. Overexpression of ABCG2 may confer multidrug resistance in cancer cells, which could be caused by gene amplification [25], [26], [27] and malfunction of regulatory factors [11]. In addition, ABCG2 is revealed as a critical element of tumorigenic stem cells [28], [29], [30], and has been used as one selective biomarker for cancer stem cells.

Recent studies have shown that a number of miRNAs may regulate the expression of ABCG2 through their actions on ABCG2 3′UTR. Using a luciferase reporter assay, Liao et al. [31] found that hsa-miR-520h could act on the 3′UTR (30–52 nt from stop codon) of ABCG2 mRNA, and Wang et al. [18] showed that ABCG2 protein expression was indeed down-regulated by miR-520h in pancreatic cancer cells. Meanwhile, To et al. [14], [15] reported a distal miRNA response element (MRE; 1526–1548 nt) for hsa-miR-519c that was close to the poly(A) tail of ABCG2 3′UTR (Fig. 1A), and showed that a miR-519c inhibitor or mimic was able to alter ABCG2 protein expression in A549 human lung adenocarcinoma epithelial cells. Interestingly, the 3′UTR including that of ABCG2 was revealed to be commonly shortened in cancer cells, especially in embryonic stem cells, proliferating cells and drug-resistant cancer cells [14], [32], [33], [34]. The absence of this distal miR-519c MRE site in shortened ABCG2 3′UTR might limit the role of miR-519c in ABCG2 regulation. In addition, we found that ABCG2 3′UTR (599–626 nt) could be readily targeted by hsa-miR-328 when considering target-site accessibility or binding energy, and modulation of miR-328 signaling had significant effects on ABCG2 mRNA and protein expression in drug-sensitive MCF-7 and drug-resistant MCF-7/MX100 human breast cancer cells [11]. Nevertheless, it remains unknown which miRNA is more important in posttranscriptional regulation of ABCG2. Therefore, this study aimed to compare the contribution of miR-328, -519c and -520h to the regulation of ABCG2, and delineate the potential action of miR-519c on the shortened 3′UTR of ABCG2 lacking the distal miR-519c MRE site (Fig. 1A).

Section snippets

Materials

Mitoxantrone was bought from Sigma–Aldrich (St. Louis, MO). Fumitremorgin C (FTC) was purchased from Alexis (San Diego, CA). Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, trypsin, phosphate-buffered saline (PBS) and antibiotics were bought from Mediatech (Manassas, VA). Fetal bovine serum and calf serum were bought from Invitrogen (Carlsbad, AL). Selective miRNA and control antagomirs were purchased from Dharmacon (Lafayette, CO).

Identification of MRE sites

Putative MRE sites within the 3′UTR of human ABCG2

A new, conserved proximal MRE site (30–52 nt from stop codon) is identified for miR-519c through computational analysis

Bioinformatic analyses have shown that ABCG2 3′UTR may be targeted by a number of miRNAs [11], [15], [31], including the distal MRE site (1526–1548 nt; Fig. 1A) for miR-519c. Herein we show that the putative miR-520 MRE site from 30 to 52 nt within ABCG2 3′UTR [31] may be targeted by miR-519c as well (Fig. 1A). Complementary base pairings of miR-519c-3p to this site were supported by computational analyses with PITA (folding energy ΔG, −19.8 kcal/mol; difference between miRNA–mRNA binding energy

Discussion

There is emerging evidence supporting the role for noncoding miRNAs in epigenetic regulation of drug-metabolizing enzymes and membrane transporters [4], [5], [6], [7], [8], which are molecular determinants of drug absorption, distribution, metabolism and excretion processes. The full length 3′UTR of ABCG2 transcript consists of 1968 bases that may be targeted by regulatory miRNAs [11], [15], [18]. However, in contrast to the existence of both long (∼2000 nt) and short (100–400 nt) ABCG2 3′UTR

Acknowledgements

A.-M. Yu is supported by grant R01DA021172 from National Institute on Drug Abuse, National Institutes of Health (NIH), and by the Interdisciplinary Research Development Fund from University at Buffalo, The State University of New York. G.M. Seigel is supported by R21CA127061, Research to Prevent Blindness and U54CA143876 from the National Cancer Institute, NIH. Xin Li was supported by International Program of Project 985 at Sun Yat-Sen University and National Major Projects for science and

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