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Potential role for EZH2 in promotion of asthma through suppression of miR-34b transcription by inhibition of FOXO3

Abstract

Highly expressed enhancer of zeste homolog 2 (EZH2) has been associated with many kinds of cancers and other diseases, while its functional role in asthma is largely unknown. In our study, we investigated the molecular mechanism of EZH2 in the development of asthma. An ovalbumin-induced mouse asthma model was established, followed by injection of short hairpin RNA (sh)-EZH2, overexpression-B-cell translocation gene 2 (oe-BTG2), microRNA (miR)-34b agomir as well as their corresponding controls. Next, primary bronchial epithelial cells were isolated and cultured, followed by treatment of oe-FOXO3, miR-34b inhibitor, sh-EZH2, oe-BTG2, and corresponding controls. The effects of EZH2 on inflammation were evaluated by determining levels of inflammatory factors interleukin (IL)-4, IL-5, IL-13, IL-17, and protein levels of transforming growth factor β, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinases-1. The interactions between EZH2 and forkhead box O3 (FOXO3), between FOXO3 and miR-34b promoter, and between miR-34b and BTG2 were analyzed by conducting dual-luciferase reporter and chromatin immunoprecipitation assays. Notably, EZH2 and BTG2 were significantly overexpressed, while FOXO3 and miR-34b were obviously underexpressed in asthma. EZH2 silencing led to inhibited inflammation though upregulation of FOXO3, which could bind to the miR-34b promoter and facilitate its expression. In turn, miR-34b reduced BTG2 expression by targeting its 3’untranslated region. Our study provides evidence that EZH2 promotes asthma progression by regulating the FOXO3-miR-34b-BTG2 axis.

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Fig. 1: EZH2 suppressed FOXO3 expression in asthma in an epigenetic manner.
Fig. 2: Silencing of EZH2 inhibited inflammation of epithelial cells by upregulation of FOXO3.
Fig. 3: FOXO3 directly binds to the miR-34b promoter and enhances its transcription.
Fig. 4: Overexpression of FOXO3 suppressed inflammation and expression of TGF-β, MMP9, and TIMP-1 by facilitating miR-34b expression.
Fig. 5: miR-34b suppressed inflammation in BECs by directly targeting BTG2.
Fig. 6: EZH2 silencing inhibited inflammatory cytokines expression by FOXO3-miR-34b-BTG2 axis.
Fig. 7: Depletion of EZH2 inhibited asthma progression in vivo by FOXO3-miR-34b-BTG2 axis.

Data availability

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We acknowledge and appreciate our colleagues for their valuable suggestions and technical assistance for this study.

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Contributions

HS designed the study. HL and CZ collated the data, carried out data analyses, and produced the initial draft of the manuscript. JW and BL contributed to drafting the manuscript. All authors have read and approved the final submitted manuscript.

Corresponding author

Correspondence to Changjuan Zhao.

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The authors declare no competing interests.

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All experimental procedures involving animals were approved by the Ethics Committee of Institutional Animal Care and Use Committee of Linyi People’s Hospital. Extensive efforts were made to numbers and discomfort the suffering of the included animals.

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Liu, B., Sun, H., Wang, J. et al. Potential role for EZH2 in promotion of asthma through suppression of miR-34b transcription by inhibition of FOXO3. Lab Invest (2021). https://doi.org/10.1038/s41374-021-00585-7

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