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MiR-28 regulates Nrf2 expression through a Keap1-independent mechanism

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Abstract

NF-E2-related factor 2 (Nrf2) is an important transcription factor involved in antioxidant response. Nrf2 binds antioxidant response elements (ARE) within promoters of genes encoding detoxification enzymes (e.g., NAD (P) H-quinone oxidoreductase 1 (NQO1)) leading to their transcriptional activation. Nrf2 function is regulated post-translationally by its negative regulator Kelch-like ECH-associated protein 1 (Keap1) that binds Nrf2 and induces cytoplasmic Nrf2 degradation. Our present studies provide new evidence that Nrf2 expression can be regulated by a Keap1-independent mechanism. Here, we utilized breast epithelial cells to explore the impact of microRNA (miRNA) on Nrf2 expression. We found that Nrf2 mRNA levels are reversibly correlated with miR-28 expression and that ectopic expression of miR-28 alone reduces Nrf2 mRNA and protein levels. We further investigated the molecular mechanisms by which miR-28 inhibits Nrf2 mRNA expression. Initially, the ability of miR-28 to regulate the 3′ untranslated region (3′UTR) of Nrf2 mRNA was evaluated via luciferase reporter assay. We observed that miR-28 reduces wild-type Nrf2 3′UTR luciferase reporter activity and this repression is eliminated upon mutation of the miR-28 targeting seed sequence within the Nrf2 3′UTR. Moreover, over-expression of miR-28 decreased endogenous Nrf2 mRNA and protein expression. We also explored the impact of miR-28 on Keap1-Nrf2 interactions and found that miR-28 over-expression does not alter Keap1 protein levels and has no effect on the interaction of Keap1 and Nrf2. Our findings, that miR-28 targets the 3′UTR of Nrf2 mRNA and decreases Nrf2 expression, suggest that this miRNA is involved in the regulation of Nrf2 expression in breast epithelial cells.

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Acknowledgments

This study was supported by grants from Flight Attendants Medical Research Institute (FAMRI YCSA072084 to QZ), and Maryland Stem Cell Research Fund (2010-MSCRFE-0179-00 to QZ).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

    Muhua Yang, Yuan Yao, Gabriel Eades, Yongshu Zhang & Qun Zhou

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  1. Muhua Yang
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  2. Yuan Yao
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  3. Gabriel Eades
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  4. Yongshu Zhang
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  5. Qun Zhou
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Correspondence to Qun Zhou.

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Yang, M., Yao, Y., Eades, G. et al. MiR-28 regulates Nrf2 expression through a Keap1-independent mechanism. Breast Cancer Res Treat 129, 983–991 (2011). https://doi.org/10.1007/s10549-011-1604-1

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  • DOI: https://doi.org/10.1007/s10549-011-1604-1

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