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. 2019 Nov 3:2019:1512326.
doi: 10.1155/2019/1512326. eCollection 2019.

miR-200a Attenuated Doxorubicin-Induced Cardiotoxicity through Upregulation of Nrf2 in Mice

Affiliations

miR-200a Attenuated Doxorubicin-Induced Cardiotoxicity through Upregulation of Nrf2 in Mice

Xiaoping Hu et al. Oxid Med Cell Longev. .

Abstract

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was closely involved in doxorubicin- (DOX-) induced cardiotoxicity. MicroRNA-200a (miR-200a) could target Keap1 mRNA and promote degradation of Keap1 mRNA, resulting in Nrf2 activation. However, the role of miR-200a in DOX-related cardiotoxicity remained unclear. Our study is aimed at investigating the effect of miR-200a on DOX-induced cardiotoxicity in mice. For cardiotropic expression, male mice received an injection of an adeno-associated virus 9 (AAV9) system carrying miR-200a or miR-scramble. Four weeks later, mice received a single intraperitoneal injection of DOX at 15 mg/kg. In our study, we found that miR-200a mRNA was the only microRNA that was significantly decreased in DOX-treated mice and H9c2 cells. miR-200a supplementation blocked whole-body wasting and heart atrophy caused by acute DOX injection, decreased the levels of cardiac troponin I and the N-terminal probrain natriuretic peptide, and improved cardiac and adult cardiomyocyte contractile function. Moreover, miR-200a reduced oxidative stress and cardiac apoptosis without affecting matrix metalloproteinase and inflammatory factors in mice with acute DOX injection. miR-200a also attenuated DOX-induced oxidative injury and cell loss in vitro. As expected, we found that miR-200a activated Nrf2 and Nrf2 deficiency abolished the protection provided by miR-200a supplementation in mice. miR-200a also provided cardiac benefits in a chronic model of DOX-induced cardiotoxicity. In conclusion, miR-200a protected against DOX-induced cardiotoxicity via activation of the Nrf2 signaling pathway. Our data suggest that miR-200a may represent a new cardioprotective strategy against DOX-induced cardiotoxicity.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
miR-200a was decreased in DOX-induced cardiac injury: (a) the levels of miRNAs in the hearts (n = 6); (b) the levels of miRNAs in the H9c2 cells (n = 6); (c, d) the levels of miR-200a in the H9c2 cells (n = 6). P < 0.05 compared with the group with saline or PBS.
Figure 2
Figure 2
miR-200a overexpression improved cardiac function in mice: (a) schedule of the acute DOX regime experiment; (b) the levels of miR-200a in the hearts (n = 6); (c) the alteration in body weight (n = 12); (d) alterations in the ratio of heart weight to tibial length (n = 12); (e, f) the level of cTnI and NT-proBNP (n = 6), (g, h) the alteration in +dP/dt and EF in mice (n = 10); (i) cardiac output in the mice (n = 10). P < 0.05 compared with the group with saline. #P < 0.05 compared with mice after DOX injection.
Figure 3
Figure 3
miR-200a improved adult cardiomyocyte contractile properties: (a) the levels of miR-200a; (b) resting cell length; (c) peak shortening; (d, e) maximal velocity of shortening (+dL/dt) and maximal velocity of relengthening (-dL/dt). P < 0.05 compared with the group with saline. #P < 0.05 compared with mice after DOX injection. n = 50 cells from 4 mice per group.
Figure 4
Figure 4
miR-200a reduced oxidative stress in DOX-treated mice: (a) the level of MMP2 and MMP9 in the hearts (n = 6); (b) the level of TNF-α and IL-6 in the hearts (n = 6); (c, d) the levels of GSH and cardiac MDA in the hearts (n = 6); (e) the level of 4-HNE in the hearts (n = 6); (f) the level of SOD2 mRNA in the hearts (n = 6); (g) protein expression of Nrf2 and HO-1 (n = 6). P < 0.05 compared with the group with saline. #P < 0.05 compared with mice after DOX injection.
Figure 5
Figure 5
miR-200a suppressed cardiac apoptosis in DOX-treated mice. (a) TUNEL staining (n = 6). (b) Western analysis indicated the expression of Bax and Bad in the hearts (n = 6). P < 0.05 compared with the group with saline. #P < 0.05 compared with mice after DOX injection.
Figure 6
Figure 6
miR-200a provided cardioprotection via activating the Nrf2 signaling pathway: (a, b) the level of Keap1 and Nrf2 in the H9c2 cells (n = 6); (c) the protein expression of Nrf2 (n = 6); (d) DCF-DA staining; (e, f) the levels of 4-HNE and GSH in the cells (n = 6); (g) the level of Nrf2 in the cells (n = 6); (h) cell viability (n = 6); (i) caspase 3 activity (n = 6). P < 0.05.
Figure 7
Figure 7
miR-200a could not provide cardiac protection against DOX-induced acute cardiotoxicity in Nrf2-deficient mice: (a) schedule of Nrf2 deficiency in mice with the acute DOX treatment; (b) the level of Nrf2 in the hearts (n = 6); (c) EF (n = 8); (d, e) the levels of NT-proBNP and MDA in the hearts (n = 6); (f) caspase 3 activity (n = 6). P < 0.05.
Figure 8
Figure 8
miR-200a protected the hearts from DOX-induced chronic cardiotoxicity: (a) schedule of the acute DOX regime experiment; (b) the level of miR-200a in the hearts (n = 6); (c) survival rate in the groups (n = 15); (d) EF in the four groups (n = 10); (e) the level of 4-HNE in the hearts (n = 6); (f) caspase 3 activity (n = 6). P < 0.05 compared with the group with saline. #P < 0.05 compared with mice after DOX injection.

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