A novel fine tuning scheme of miR-200c in modulating lung cell redox homeostasis

Abstract

Oxidative stress induces miR-200c, the predominant microRNA (miRNA) in lung tissues; however, the antioxidant role and biochemistry of such induction have not been clearly defined. Therefore, a lung adenocarcinoma cell line (A549) and a normal lung fibroblast (MRC-5) were used as models to determine the effects of miR-200c expression on lung antioxidant response. Hydrogen peroxide (H₂O₂) upregulated miR-200c, whose overexpression exacerbated the decrease in cell proliferation, retarded the progression of cells in the G2/M-phase, and increased oxidative stress upon H₂O₂ stimulation. The expression of three antioxidant proteins, superoxide dismutase (SOD)-2, haem oxygenase (HO)-1, and sirtuin (SIRT) 1, was reduced upon H₂O₂ stimulation in miR-200c-overexpressed A549 cells. This phenomenon of increased oxidative stress and antioxidant protein downregulation also occurs simultaneously in miR-200c overexpressed MRC-5 cells. Molecular analysis revealed that miR-200c inhibited the gene expression of HO-1 by directly targeting its 3′-untranslated region. The downregulation of SOD2 and SIRT1 by miR-200c was mediated through zinc finger E-box-binding homeobox 2 (ZEB2) and extracellular signal-regulated kinase 5 (ERK5) pathways, respectively, where knockdown of ZEB2 or ERK5 decreased the expression of SOD2 or SIRT1 in A549 cells. LNA anti-miR-200c transfection in A549 cells inhibited the endogenous miR-200c expression, resulting in increased expressions of antioxidant proteins, reduced oxidative stress and recovered cell proliferation upon H₂O₂ stimulation. These findings indicate that miR-200c fine-tuned the antioxidant response of the lung cells to oxidative stress through several pathways, and thus this study provides novel information concerning the role of miR-200c in modulating redox homeostasis of lung.

Keywords:

• H₂O₂
• HO-1
• ERK5
• SIRT1
• SOD2
• antioxidant response

Acknowledgements

Y.-H.W., H.-R.L., Y.-H.L., P.-H.H. and H.-C.W. were responsible for performing experiments. Y.-H.W., H.-R.L. and D.T.-Y.C. were responsible for planning experiments, analysing data and writing the manuscript. A.S. was responsible for writing the article and English editing.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The supports for the current study by grants from Chang Gung University (BMRP098, CMRPD1F0461, CMRPD1C0773, CMRPD3D0161, and CMRPD3D0162), the Ministry of Science and Technology of Taiwan (MOST103-2320-B-182-026-MY2 and MOST105-2320-B-182-031-MY2) and the Ministry of Education of Taiwan (EMRPD1G0181) are greatly appreciated.