Abstract
Breast cancer resistance protein (BCRP/ABCG2) is a molecular determinant of pharmacokinetic properties of many drugs in humans. To understand post-transcriptional regulation of ABCG2 and the role of microRNAs (miRNAs) in drug disposition, we found that microRNA-328 (miR-328) might readily target the 3′-untranslated region (3′-UTR) of ABCG2 when considering target-site accessibility. We then noted 1) an inverse relation between the levels of miR-328 and ABCG2 in MCF-7 and MCF-7/MX100 breast cancer cells and 2) that miR-328 levels could be rescued in MCF-7/MX100 cells by transfection with miR-328 plasmid. Luciferase reporter assays showed that ABCG2 3′-UTR-luciferase activity was decreased more than 50% in MCF-7/MX100 cells after transfection with miR-328 plasmid, the activity was increased over 100% in MCF-7 cells transfected with a miR-328 antagomir, and disruption of miR-328 response element within ABCG2 3′-UTR led to a 3-fold increase in luciferase activity. Furthermore, the level of ABCG2 protein was down-regulated when miR-328 was over-expressed, and the level was up-regulated when miR-328 was inhibited by selective antagomir. Altered ABCG2 protein expression was associated with significantly declined or elevated levels of ABCG2 3′-UTR and coding sequence mRNAs, suggesting possible involvement of the mechanism of mRNA cleavage. Finally, miR-328-directed down-regulation of ABCG2 expression in MCF-7/MX100 cells resulted in an increased mitoxantrone sensitivity, as manifested by a significantly lower IC50 value (2.46 ± 1.64 μM) compared with the control (151 ± 32 μM). Together, these findings suggest that miR-328 targets ABCG2 3′-UTR and, consequently, controls ABCG2 protein expression and influences drug disposition in human breast cancer cells.
Footnotes
-
This project was supported by the Interdisciplinary Research Development Fund, the University at Buffalo, The State University of New York; and in part by the National Institutes of Health National Institute on Drug Abuse [Grant R01-DA021172] (to A.-M.Y.).
-
ABBREVIATIONS: 3′-UTR, 3′-untranslated region; miRNA, microRNA; miR-328, microRNA-328; ABCG2, ATP-binding cassette, subfamily G (white), member 2; BCRP, breast cancer resistance protein; FTC, fumitremorgin C; PBS, fetal bovine serum; bp, base pair(s); MRE, microRNA response element; qPCR, quantitative real-time polymerase chain reaction; PCR, polymerase chain reaction; CDS, coding sequence; hsa-miR-328, human miR-328.
- Received December 12, 2008.
- Accepted March 6, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|