MicroRNA-328 Negatively Regulates the Expression of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Cancer Cells

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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.