title: MiR-28 regulates Nrf2 expression through a Keap1-independent mechanism.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
authors.forename: Muhua
authors.initials: M
authors.lastname: Yang
authors.forename: Yuan
authors.initials: Y
authors.lastname: Yao
authors.forename: Gabriel
authors.initials: G
authors.lastname: Eades
authors.forename: Yongshu
authors.initials: Y
authors.lastname: Zhang
authors.forename: Qun
authors.initials: Q
authors.lastname: Zhou
mesh_terms: D020413:3' Untranslated Regions; D001483:Base Sequence; D001943:Breast Neoplasms / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D000072019:Kelch-Like ECH-Associated Protein 1; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; C570628:MIRN28 microRNA, human; D035683:MicroRNAs; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D012333:RNA, Messenger; D008156:Luciferases