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miR-200a controls hepatic stellate cell activation and fibrosis via SIRT1/Notch1 signal pathway

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Abstract

Objectives

miR-200a has been established as a key regulator of HSC activation processes in liver fibrosis. Epigenetic silencing of miR-200a contributing to SIRT1 over-expression has been discussed in breast cancer; however, whether miR-200a controls SIRT1 gene expression in hepatic fibrosis is still unknown.

Methods and materials

We analyzed miR-200a regulation of SIRT1 expression in CCl4-induced liver fibrosis and TGF-β1-mediated activation of HSC. miR-200a, SIRT1, α-SMA, Col1A1, Notch1 and NICD expression were estimated by Western blotting, qRT-PCR and Immunohistochemistry. HSCs were transfected with miR-200a mimic, miR-200a inhibitor and SIRT1-RNAi. Luciferase reporter assays further confirmed the interaction between miR-200a and the SIRT1 mRNA 3'-UTR. Cell proliferation ability was assessed by MTT and cell cycle.

Results

We found that treatment activated HSC with miR-200a mimics, restored miR-200a expression and reduced SIRT1 levels. Conversely, treatment activated HSC with miR-200a inhibitors, decreased miR-200a expression and up-regulated SIRT1 levels. Restoration of miR-200a or the knockdown of SIRT1 prevented HSC activation and proliferation. We have established the SIRT1 transcript as subject to regulation by miR-200a, through miR-200a targeting of SIRT1 3’-UTR. Finally, HSC transfected with SIRT1-siRNA increased the levels of Notch1 protein and mRNA expression.

Conclusions

Our study demonstrated that miR-200a regulates SIRT1/Notch1 expression during HSC activation and fibrosis.

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Abbreviations

HSC:

Hepatic stellate cell

ECM:

Extracellular matrix

α-SMA:

α-Smooth muscle actin

TGF-β1:

Transforming growth factor-β1

SIRT1:

Silent information regulator 1

TIMP-1:

Tissue inhibitor of metalloproteinase-1

miR:

MicroRNA

3′-UTR:

3′-Untranslated region

NICD:

Notch1-intercellular domain

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Acknowledgements

This study was funded by Anhui Provincial Natural Science Foundation (1608085MH200) and National Natural Science Foundation of China (81600477) and Anhui Medical University Foundation (2015xjk014).

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Authors and Affiliations

  1. Department of Pharmacology, The Second Hospital of Anhui Medical University, Hefei, 230601, China

    Jing-Jing Yang, Li-Ping Liu & Wei Hu

  2. Department of Cardiothoracic Surgery, The Second Hospital of Anhui Medical University, Hefei, 230601, China

    Hui Tao

  3. Department of Scientific, The Second Hospital of Anhui Medical University, Hefei, 230601, China

    Zi-Yu Deng

  4. School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui, 230032, China

    Jun Li

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  1. Jing-Jing Yang
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  2. Hui Tao
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  4. Wei Hu
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Correspondence to Jun Li.

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All applicable international, national, and institutional guidelines for the care and use of animals were followed.

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Responsible Editor: Liwu Li.

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Yang, JJ., Tao, H., Liu, LP. et al. miR-200a controls hepatic stellate cell activation and fibrosis via SIRT1/Notch1 signal pathway. Inflamm. Res. 66, 341–352 (2017). https://doi.org/10.1007/s00011-016-1020-4

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  • DOI: https://doi.org/10.1007/s00011-016-1020-4

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