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miR-499-5p Attenuates Mitochondrial Fission and Cell Apoptosis via p21 in Doxorubicin Cardiotoxicity

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miR-499-5p Attenuates Mitochondrial Fission and Cell Apoptosis via p21 in Doxorubicin Cardiotoxicity

Qinggong Wan et al. Front Genet.

Abstract

Doxorubicin (DOX) is a broad-spectrum anti-tumor drug, but its cardiotoxicity limits its clinical application. A better understanding of the molecular mechanisms underlying DOX cardiotoxicity will benefit clinical practice and remedy heart failure. Our present study observed that DOX caused cardiomyocyte (H9c2) apoptosis via the induction of abnormal mitochondrial fission. Notably, the expression levels of p21 increased in DOX-treated cardiomyocytes, and the silencing of p21 using siRNA greatly attenuated mitochondrial fission and apoptosis in cardiomyocytes. We also found that miR-499-5p could directly target p21 and attenuated DOX-induced mitochondrial fission and apoptosis. The role of the miR-499-5p-p21 axis in the prevention of DOX cardiotoxicity was also validated in the mice model. DOX treatment induced an upregulation of p21, which induced subsequent abnormal mitochondrial fission and myocardial apoptosis in mouse heart. Adenovirus-harboring miR-499-5p-overexpressing mice exhibited significantly reduced p21 expression, mitochondrial fission and myocardial apoptosis in hearts following DOX administration. The miR-499-5p-overexpressing mice also exhibited improved cardiomyocyte hypertrophy and cardiac function after DOX treatment. However, miR-499-5p was not involved in the DOX-induced apoptosis of cancer cells. Taken together, these findings reveal an emerging role of p21 in the regulation of mitochondrial fission program. miR-499-5p attenuated mitochondrial fission and DOX cardiotoxicity via the targeting of p21. These results provide new evidence for the miR-499-5p-p21 axis in the attenuation of DOX cardiotoxicity. The development of new therapeutic strategies based on the miR-499-5p-p21 axis is a promising path to overcome DOX cardiotoxicity as a chemotherapy for cancer treatment.

Keywords: apoptosis; cardiotoxicity; doxorubicin; miR-499-5p; mitochondrial; p21.

Figures

FIGURE 1
FIGURE 1
miR-499-5p attenuates mitochondrial fission and apoptosis in cardiomyocytes treated with DOX. (A) Measurement of miR-499-5p mRNA levels in H9c2 cells treated with 2 μM DOX at the indicated times. (B) Transfection with a miR-499-5p mimic forced the expression of miR-499-5p in cardiomyocytes. Cells were transfected with miR-499-5p mimic (miR-mimic) or negative control (NC) for 24 h. The expression levels of miR-499-5p were detected using real-time PCR. (C) miR-499-5p antagomiR knocked down endogenous miR-499-5p. Cells were transfected with a miR-499-5p antagomiR (Anti-miR-499) or antagomiR control for 24 h. The expression levels of miR-499-5p were detected using real-time PCR. (D) Overexpression of miR-499-5p inhibited 2 μM DOX-induced apoptosis in cardiomyocytes. Cardiomyocytes were transfected with miR-499-5p mimic or negative control (NC) for 24 h and treated with 2 μM DOX for 24 h. Cell death was detected using the Tunel assay. Representative images show Tunel staining results (blue, DAPI; green, Tunel). Scale bar: 100 μm. (E) Statistical analysis of Tunel-positive cells in each group. Data are expressed as the means ± SD, n = 3 experiment P < 0.05. (F) Knockdown of miR-499-5p sensitized cardiomyocytes to undergo apoptosis induced by DOX (0.2 μM). Cell death was detected using the Tunel assay. Data are expressed as the means ± SD; n = 3 experiment. P < 0.05. (G) Overexpression of miR-499-5p inhibited DOX-induced mitochondrial fission in cardiomyocytes. Cells were transfected with a miR-499-5p mimic or negative control for 24 h. Representative images were captured using confocal microcopy. Scale bar: 2 μM. (H) Knockdown of miR-499-5p sensitized cardiomyocytes to undergo 0.2 μM DOX-induced cell death. Cell death was detected using the trypan blue assay. Data are expressed as the means ± SD, n = 3 experiment. P < 0.05. (I) Overexpression of miR-499-5p inhibited 2 μM DOX-induced cell death in cardiomyocytes. Cell death was measured using trypan blue assays. Data are expressed as the means ± SD, n = 3 experiment. P < 0.05. (J) Statistical analysis of the percentage of cells undergoing mitochondrial fission. Data are expressed as the means ± SD, n = 3 experiment. P < 0.05. Cardiomyocytes were transfected with a miR-499-5p mimic or negative control for 24 h and treated with 2 μM DOX for 24 h. (K) Statistical analysis of the percentage of cells undergoing mitochondrial fission. Data are expressed as the means ± SD, n = 3 experiment. P < 0.05. H9c2 cells were transfected with a miR-499-5p antagomiR or antagomiR control for 24 h and treated with 0.2 μM DOX for 24 h. The percentage of cells undergoing mitochondrial fission was determined.
FIGURE 2
FIGURE 2
miR-499-5p attenuates mitochondrial fission and apoptosis in vivo. (A) miR-499-5p mRNA levels in adenovirus-harboring, miR-499-5p-overexpressing mice (Ad-miR-499) and the control mice administered DOX. n = 3 mice per group. Data are presented as the means ± SD, P < 0.05. (B) Serum miR-499-5p expression was detected in the mice administered DOX by real-time PCR. n = 3 mice per group. Data are presented as the means ± SD, P < 0.05. (C,D) Mitochondrial fission was analyzed using scanning electron microscope (SEM) in heart tissues from miR-499-5p-overexpressing mice and the control mice administered DOX. Scale bar, 2 μm. Arrows indicate mitochondria that underwent fission. (C) Statistical analyses of the percentage of mitochondrial fission. Mitochondrial fission was calculated as described in the Section “Materials and Methods.” Data are presented as the means ± SD, P < 0.05; n = 5 mice per group. (D,E) Representative images revealed Tunel-positive cells in heart tissues. Green, Tunel-positive nuclei; blue, DAPI (4,6-diamidino-2-phenylindole)-stained nuclei; red, cardiomyocytes labeled with α-actinin antibody; scale bar, 20 μm. n = 5 mice per group. (F) Statistical analysis of Tunel-positive cells in each group. Data are presented as the means ± SD, P < 0.05. (G) Western blotting shows the expression of p21. Actin served as a loading control.
FIGURE 3
FIGURE 3
miR-499-5p attenuates DOX cardiotoxicity in mice. (A–E) miR-499-5p-overexpressing mice are resistant to left ventricular remodeling after DOX treatment. (A,B) The expression levels of ANP and β-myosin heavy chain (B-MHC) were detected using qRT-PCR. Data are expressed as the means ± SD, n = 3 experiment. P < 0.05. (C–E) miR-499-5p-overexpressing mice or control mice were exposed to DOX or saline as described in the panel, and echocardiography was used to test heart function. LVIDs, systolic left ventricular internal diameter (C); LVIDd, diastolic left ventricular internal diameter (D); FS, fractional shortening of the left ventricular diameter (E). Data are presented as the means ± SD, n = 8 mice per group. P < 0.05.
FIGURE 4
FIGURE 4
miR-499-5p participates in the regulation of p21 expression. (A) Analysis of the p21 3′UTR potential binding site of miR-499-5p using RNA hybrid. Potential complementary residues are shown in red. (B) Cardiomyocytes were transfected with a miR-499-5p mimic or negative control for 24 h. The expression levels of p21 were determined using Western blot. (C) Cardiomyocytes were treated with DOX (2 μM) at the indicated time points. The expression levels of p21 were detected using Western blots. (D–F) miR-499-5p directly targets the p21 3′UTR. (D) Schematic diagram of the reporter containing the putative miR-499-5p binding in the p21 3′UTR. (E) Luciferase activity detected in HEK-293 cells transfected with synthesized miR-499-5p mimic or negative control along with luciferase reporter constructs, as indicated. (F) Luciferase activity of the luciferase construct p21 3′UTR-Wt decreased with DOX treatment in cardiomyocytes. Data are expressed as the means ± SD, n = 3 except in panel; P < 0.05.
FIGURE 5
FIGURE 5
p21 attenuates mitochondrial fission and cell death in H9c2 cells treated with DOX. (A) The expression levels of p21 were detected using Western blot in cardiomyocytes transfected with p21 siRNA. (B) The expression levels of p21 were detected using Western blot in cardiomyocytes transfected with a p21-overexpressing vector. Actin served as the loading control. (C,D) Knockdown of p21 inhibited 2 μM DOX-induced mitochondrial fission and cell death. Cardiomyocytes were transfected with p21 siRNA or p21 scramble for 24 h and treated with DOX (2 μM) for 24 h. (E,F) Overexpression of p21 increased mitochondrial fission and cell death induced by low-dose DOX (0.2 μM). Cardiomyocytes were transfected with p21 or empty vector for 24 h and treated with 0.2 μM DOX for 24 h. P < 0.05; Data are expressed as the means ± SD, n = 3 experiment. P < 0.05.
FIGURE 6
FIGURE 6
miR-499-5p attenuates mitochondrial fission and apoptosis via targeting p21. (A,B) miR-499-5p attenuated the sensitivity of cardiomyocytes to DOX treatment, which was abolished by p21 overexpression. Statistical analysis of the mitochondrial fission (A) and cell death (B) in cardiomyocytes following DOX (2 μM) treatment. (C,D) Knockdown of miR-499-5p increased the sensitivity of cardiomyocytes to DOX (0.2 μM) treatment, which was abolished by p21 knockdown. Statistical analysis of the mitochondrial fission (C) and cell death (D) in cardiomyocytes following DOX (0.2 μM) treatment. (E) Schematic diagram of this study. Data are presented as the means ± SD, n = 3 experiment. P < 0.05.
FIGURE 7
FIGURE 7
miR-499-5p is not involved in DOX-induced apoptosis in cancer cells. (A) Real-time PCR results show the miR-499-5p expression levels in H9c2, SGC-7901, A-549, SW-480, and HepG-2 cells. P < 0.05 versus cardiomyocytes. (B,C) Real-time PCR results show miR-499-5p levels in SG-7901 (B) and A-549 (C) cells treated with DOX (2 μM) for the indicated times. (D,E) Statistical analysis of the cell death in SG-7901 (D) and A-549 (E) cells transfected with miR-499-5p mimic following treatment with DOX (2 μM). Data are expressed as the means ± SD, n = 3 experiment.

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