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MicroRNA-99b-3p promotes angiotensin II-induced cardiac fibrosis in mice by targeting GSK-3β

Abstract

Cardiac fibrosis is a typical pathological change in various cardiovascular diseases. Although it has been recognized as a crucial risk factor responsible for heart failure, there is still a lack of effective treatment. Recent evidence shows that microRNAs (miRNAs) play an important role in the development of cardiac fibrosis and represent novel therapeutic targets. In this study we tried to identify the cardiac fibrosis-associated miRNA and elucidate its regulatory mechanisms in mice. Cardiac fibrosis was induced by infusion of angiotensin II (Ang II, 2 mg·kg−1·d−1) for 2 weeks via osmotic pumps. We showed that Ang II infusion induced cardiac disfunction and fibrosis accompanied by markedly increased expression level of miR-99b-3p in heart tissues. Upregulation of miR-99b-3p and fibrotic responses were also observed in cultured rat cardiac fibroblasts (CFs) treated with Ang II (100 nM) in vitro. Transfection with miR-99b-3p mimic resulted in the overproduction of fibronectin, collagen I, vimentin and α-SMA, and facilitated the proliferation and migration of CFs. On the contrary, transfection with specific miR-99b-3p inhibitor attenuated Ang II-induced fibrotic responses. Similarly, intravenous injection of specific miR-99b-3p antagomir could prevent Ang II-infused mice from cardiac dysfunction and fibrosis. We identified glycogen synthase kinase-3 beta (GSK-3β) as a direct target of miR-99b-3p. In CFs, miR-99b-3p mimic significantly reduced the expression of GSK-3β, leading to activation of its downstream profibrotic effector Smad3, whereas miR-99b-3p inhibitor caused anti-fibrotic effects. GSK-3β knockdown ameliorated the anti-fibrotic role of miR-99b-3p inhibitor. These results suggest that miR-99b-3p contributes to Ang II-induced cardiac fibrosis at least partially through GSK-3β. The modulation of miR-99b-3p may provide a new approach for tackling fibrosis-related cardiomyopathy.

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Fig. 1: Ang II induces cardiac fibrosis accompanied by elevated miR-99b-3p expression.
Fig. 2: MiR-99b-3p mimic induces fibrotic responses in CFs.
Fig. 3: Inhibition of miR-99b-3p suppresses Ang II-induced fibrotic responses in CFs.
Fig. 4: GSK-3β is a direct target gene of miR-99b-3p.
Fig. 5: GSK-3β knockdown attenuates the antifibrotic effects of the miR-99b-3p inhibitor.
Fig. 6: MiR-99b-3p promotes fibrotic responses in CFs by modulating the GSK-3β/Smad3 cascade.
Fig. 7: Inhibition of miR-99b-3p alleviates Ang II-induced cardiac fibrosis in mice.
Fig. 8: MiR-99b-3p antagomir restores GSK-3β expression and inhibits Smad3 phosphorylation in the hearts of Ang II-treated mice.

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Acknowledgements

This work was supported by grants from the National Major Special Projects for the Creation and Manufacture of New Drugs (2019ZX09301104), the National Engineering and Technology Research Center for New Drug Druggability Evaluation (Seed Program of Guangdong Province, 2017B090903004), the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01Y093), the Guangdong Basic and Applied Basic Research Foundation (2020A1515011512), and the Young Teacher Training Program of Sun Yat-sen University (18ykpy26).

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YHY and JTY conceived and designed the experiments. YHY, YHZ, XYB, JY, HZ, and LLZ performed the experiments. YQD and PXW analyzed the data. YHY and JTY wrote the manuscript.

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Correspondence to Jian-tao Ye.

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The authors declare no competing interests.

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Yu, Yh., Zhang, Yh., Ding, Yq. et al. MicroRNA-99b-3p promotes angiotensin II-induced cardiac fibrosis in mice by targeting GSK-3β. Acta Pharmacol Sin 42, 715–725 (2021). https://doi.org/10.1038/s41401-020-0498-z

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Keywords

  • cardiac fibrosis
  • angiotensin II
  • miR-99b-3p
  • GSK-3β
  • Smad3

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