Microrna-146b Targets Hif-1α and Attenuates Cardiomyocyte Apoptosis and Fibrosis in Doxorubicin-Induced Heart Failure

Fig. 1. Expression of microRNA-146b is declined significantly in human end-stage heart failure. A, HE staining and (B) Masson’s staining for myocardial tissues. C, Collagen volume Fraction of LV in end-stage HF patients (n = 12) and controls (n = 6). D, RT-PCR for the microRNA expression analysis in myocardial tissue specimens of 12 end-stage heart failure patients and 6 controls. NS: P>0.05, *P < 0.05, **P < 0.01, ***P < 0.001. HE, hematoxylin eosin; HF, heart failure; LV, left ventricle.

Fig. 2. MiR-146b expression is downregulated by doxorubicin-induced myocardial injury in vitro and in vivo. A, RT-PCR for miR-146b expression in primary neonatal mouse cardiomyocytes. (n = 3). B, CCK-8 assay for the viability of primary neonatal mouse cardiomyocytes. (n = 3). C, TUNEL staining for apoptosis analysis in mice myocardial tissues. D, Apoptosis rate in mice myocardial tissues. (n = 3). E, RT-PCR for miR-146b expression in mice myocardial tissues. (n = 3). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Dox, doxorubicin; WT, wild type.

Fig. 3. Knockout of miR-146b increases the doxorubicin-induced cardiac damage in mice. A, Experimental strategy in a mouse model of doxorubicin-induced cardiac damage. The adult male C57BL/6 wild type mice and male miR-146b knockout mice were intraperitoneal injected doxorubicin (2.5 mg/kg) three times a week for 2 weeks and maintained for an additional 4 weeks before echocardiography and collection of heart tissue. B, Echocardiography performance of mice. C, RT-PCR for miR-146b relative expression in mice myocardial tissues of each group. (n = 6). D, LV Ejection Fraction, LV Fraction Shorting, LV Mass and LV Volume of mice at week 6. (n = 6). Data are expressed as mean ± SD. NS: P>0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Dox, doxorubicin; LV, left ventricle; echo: echocardiography; KO: knockout; miR-146b −/−: microRNA-146b knockout mice; WT, wild type.

Fig. 4. Knockout of miR-146b exacerbates doxorubicin-induced apoptosis and fibrosis of cardiomyocytes in mice. A, HE staining and Masson’s staining in mice myocardial tissues. B, Collagen volume fraction in mice myocardial tissues. (n = 6). C, TUNEL staining for apoptosis analysis in mice myocardial tissues. D, Cardiomyocyte apoptosis percent in mice myocardial tissues. (n = 6). Data are expressed as mean ± SD. NS: P>0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Dox, doxorubicin; KO, knockout; WT, wild type.

Fig. 5. MiR-146b inhibits doxorubicin-associated cardiotoxicity by targeting HIF-1α. A, The binding site between miR-146b and HIF-1α. B, Dual luciferase reporter assay verified the regulatory effect of miR-146b on the target gene HIF-1α. (n = 3). C and D, IHC analysis for HIF-1α protein in left ventricular myocardial tissues of end-stage HF patients (n = 12) and controls (n = 6). E and F, IHC analysis for HIF-1α protein in left ventricular myocardial tissues of mice (n = 6). G, Western blotting analysis for HIF-1α in primary neonatal mouse cardiomyocytes after assigned miR-146b inhibitor transfection with or without doxorubicin treatment. H, RT-PCR analysis for HIF-1α in primary neonatal mouse cardiomyocytes after assigned miR-146b mimic transfection with or without doxorubicin treatment. (n = 3). I, Western blotting analysis for HIF-1α in primary neonatal mouse cardiomyocytes after assigned miR-146b mimic transfection with or without doxorubicin treatment. Data were presented as mean ± SEM. NS: P>0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Dox, doxorubicin; KO, knockout; MUT, mutation; NC, negative control; WT, wild type.