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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Frangogiannis NG. Cardiac fibrosis: cell biological mechanisms, molecular pathways and therapeutic opportunities. Mol Asp Med. 2019;65:70–99.
Travers JG, Kamal FA, Robbins J, Yutzey KE, Blaxall BC. Cardiac fibrosis the fibroblast awakens. Circ Res. 2016;118:1021–40.
Kong P, Christia P, Frangogiannis NG. The pathogenesis of cardiac fibrosis. Cell Mol Life Sci. 2014;71:549–74.
Gourdie RG, Dimmeler S, Kohl P. Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease. Nat Rev Drug Disco. 2016;15:620–38.
Hinderer S, Schenke-Layland K. Cardiac fibrosis—a short review of causes and therapeutic strategies. Adv Drug Deliv Rev. 2019;146:77–82.
Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–97.
Hammond SM. An overview of microRNAs. Adv Drug Deliv Rev. 2015;87:3–14.
Zhou SS, Jin JP, Wang JQ, Zhang ZG, Freedman JH, Zheng Y, et al. miRNAS in cardiovascular diseases: potential biomarkers, therapeutic targets and challenges. Acta Pharmacol Sin. 2018;39:1073–84.
Boen JRA, Gevaert AB, De Keulenaer GW, Van Craenenbroeck EM, Segers VFM. The role of endothelial miRNAs in myocardial biology and disease. J Mol Cell Cardiol. 2020;138:75–87.
Ali Syeda Z, Langden SSS, Munkhzul C, Lee M, Song SJ. Regulatory mechanism of MicroRNA expression in cancer. Int J Mol Sci. 2020;21:1723.
Creemers EE, van Rooij E. Function and therapeutic potential of noncoding RNAs in cardiac fibrosis. Circ Res. 2016;118:108–18.
Thum T, Gross C, Fiedler J, Fischer T, Kissler S, Bussen M, et al. MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts. Nature. 2008;456:980–4.
Nagpal V, Rai R, Place AT, Murphy SB, Verma SK, Ghosh AK, et al. MiR-125b is critical for fibroblast-to-myofibroblast transition and cardiac fibrosis. Circulation. 2016;133:291–301.
Zhang Y, Huang XR, Wei LH, Chung ACK, Yu CM, Lan HY. miR-29b as a therapeutic agent for angiotensin II-induced cardiac fibrosis by targeting TGF-beta/Smad3 signaling. Mol Ther. 2014;22:974–85.
Chang SE, Gao ZC, Yang Y, He K, Wang XF, Wang LM, et al. miR-99b-3p is induced by vitamin D3 and contributes to its antiproliferative effects in gastric cancer cells by targeting HoxD3. Biol Chem. 2019;400:1079–86.
Yao XB, Zhang HG, Liu YJ, Liu XM, Wang XH, Sun XF, et al. miR-99b-3p promotes hepatocellular carcinoma metastasis and proliferation by targeting protocadherin 19. Gene. 2019;698:141–9.
Hong HQ, Lu J, Fang XL, Zhang YH, Cai Y, Yuan J, et al. G3BP2 is involved in isoproterenol-induced cardiac hypertrophy through activating the NF-kappa B signaling pathway. Acta Pharmacol Sin. 2018;39:184–94.
Olson ER, Shamhart PE, Naugle JE, Meszaros JG. Angiotensin II-induced extracellular signal-regulated kinase 1/2 activation is mediated by protein kinase C delta and intracellular calcium in adult rat cardiac fibroblasts. Hypertension. 2008;51:704–11.
Lal H, Ahmad F, Woodgett J, Force T. The GSK-3 family as therapeutic target for myocardial diseases. Circ Res. 2015;116:138–49.
Huang XR, Chung ACK, Yang FY, Yue WS, Deng CX, Lau CP, et al. Smad3 mediates cardiac inflammation and fibrosis in angiotensin II-induced hypertensive cardiac remodeling. Hypertension. 2010;55:1165–71.
Bujak M, Ren G, Kweon HJ, Dobaczewski M, Reddy A, Taffet G, et al. Essential role of smad3 in infarct healing and in the pathogenesis of cardiac remodeling. Circulation. 2007;116:2127–38.
Guo X, Ramirez A, Waddell DS, Li ZZ, Liu XD, Wang XF. Axin and GSK3-beta control Smad3 protein stability and modulate TGF-beta signaling. Genes Dev. 2008;22:106–20.
Millet C, Yamashita M, Heller M, Yu LR, Veenstra TD, Zhang YE. A negative feedback control of transforming growth factor-beta signaling by glycogen synthase kinase 3-mediated Smad3 linker phosphorylation at Ser-204. J Biol Chem. 2009;284:19808–16.
Jinnin M, Ihn H, Tamaki K. Characterization of SIS3, a novel specific inhibitor of Smad3, and its effect on transforming growth factor-beta 1-induced extracellular matrix expression. Mol Pharmacol. 2006;69:597–607.
Pham TP, Kremer V, Boon RA. RNA-based therapeutics in cardiovascular disease. Curr Opin Cardiol. 2020;35:191–8.
Chandy M. A tangled tale of microRNA and cardiac fibrosis. Clin Sci. 2019;133:2217–20.
Pan ZW, Lu YJ, Yang BF. MicroRNAs: a novel class of potential therapeutic targets for cardiovascular diseases. Acta Pharmacol Sin. 2010;31:1–9.
Lu DC, Thum T. RNA-based diagnostic and therapeutic strategies for cardiovascular disease. Nat Rev Cardiol. 2019;16:661–74.
Poller W, Dimmeler S, Heymans S, Zeller T, Haas J, Karakas M, et al. Non-coding RNAs in cardiovascular diseases: diagnostic and therapeutic perspectives. Eur Heart J. 2018;39:2704–16.
Rockey DC, Bell PD, Hill JA. Fibrosis—a common pathway to organ injury and failure reply. N Engl J Med. 2015;373:96.
Leask A. Getting to the heart of the matter new insights into cardiac fibrosis. Circ Res. 2015;116:1269–76.
Weber KT, Sun Y, Bhattacharya SK, Ahokas RA, Gerling IC. Myofibroblast-mediated mechanisms of pathological remodelling of the heart. Nat Rev Cardiol. 2013;10:15–26.
Small EM, Olson EN. Pervasive roles of microRNAs in cardiovascular biology. Nature. 2011;469:336–42.
Tay YMS, Tam WL, Ang YS, Gaughwin PM, Yang H, Wang WJ, et al. MicroRNA-134 modulates the differentiation of mouse embryonic stem cells, where it causes post-transcriptional attenuation of Nanog and LRH1. Stem Cells. 2008;26:17–29.
Zhang SB, Lin SY, Liu M, Liu CC, Ding HH, Sun Y, et al. CircAnks1a in the spinal cord regulates hypersensitivity in a rodent model of neuropathic pain. Nat Commun. 2019;10:4119.
Cohen P, Frame S. The renaissance of GSK3. Nat Rev Mol Cell Biol. 2001;2:769–76.
Beurel E, Grieco SF, Jope RS. Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases. Pharmacol Ther. 2015;148:114–31.
Guo YJ, Gupte M, Umbarkar P, Singh AP, Sui JY, Force T, et al. Entanglement of GSK-3 beta, beta-catenin and TGF-beta 1 signaling network to regulate myocardial fibrosis. J Mol Cell Cardiol. 2017;110:109–20.
Lal H, Ahmad F, Zhou JB, Yu JE, Vagnozzi RJ, Guo YJ, et al. Cardiac fibroblast glycogen synthase kinase-3 beta regulates ventricular remodeling and dysfunction in ischemic heart. Circulation. 2014;130:419–30.
Matsuda T, Zhai P, Maejima Y, Hong C, Gao SM, Tian B, et al. Distinct roles of GSK-3 alpha and GSK-3 beta phosphorylation in the heart under pressure overload. Proc Natl Acad Sci U S A. 2008;105:20900–5.
Zeng ZF, Wang QY, Yang XM, Ren YL, Jiao SH, Zhu QQ, et al. Qishen granule attenuates cardiac fibrosis by regulating TGF-beta /Smad3 and GSK-3 beta pathway. Phytomedicine 2019;62:152949.
Meng XM, Nikolic-Paterson DJ, Lan HY. TGF-beta: the master regulator of fibrosis. Nat Rev Nephrol. 2016;12:325–38.
Shi YG, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell. 2003;113:685–700.
Goumans MJ, ten Dijke P. TGF-beta signaling in control of cardiovascular function. Cold Spring Harb Perspect Biol. 2018;10:39.
Shi J, Liu H, Wang H, Kong XQ. MicroRNA expression signature in degenerative aortic stenosis. BioMed Res Int. 2016;2016:4682172.
Ramaraj R, Sorrell VL. Degenerative aortic stenosis. BMJ. 2008;336:550–5.
Krayenbuehl HP, Hess OM, Monrad ES, Schneider J, Mall G, Turina M. Left ventricular myocardial structure in aortic valve disease before, intermediate, and late after aortic valve replacement. Circulation. 1989;79:744–55.
Dweck MR, Joshi S, Murigu T, Alpendurada F, Jabbour A, Melina G, et al. Midwall fibrosis is an independent predictor of mortality in patients with aortic stenosis. J Am Coll Cardiol. 2011;58:1271–9.
Thum T. Noncoding RNAs and myocardial fibrosis. Nat Rev Cardiol. 2014;11:655–63.
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).
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
- cardiac fibrosis
- angiotensin II