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. 2020 Jun 5;17(10):1415-1427.
doi: 10.7150/ijms.41980. eCollection 2020.

Inhibition of miR-25 attenuates doxorubicin-induced apoptosis, reactive oxygen species production and DNA damage by targeting PTEN

Zhiqiang Li  1 , Hongqiang Li  1 , Baoxin Liu  1 , Jiachen Luo  1 , Xiaoming Qin  1 , Mengmeng Gong  1 , Beibei Shi  1 , Yidong Wei  1
Affiliations

Inhibition of miR-25 attenuates doxorubicin-induced apoptosis, reactive oxygen species production and DNA damage by targeting PTEN

Zhiqiang Li et al. Int J Med Sci. .

Abstract

Background: Doxorubicin (DOX) is one of the widely used anti-cancer drugs, whereas it can induce irreversible cardiac injury in a dose-dependent manner which limits its utility in clinic. Our study aimed to investigate the relationship between miR-25 and DOX-induced cardiac injury and its underlying mechanism. Methods: Mice and H9c2 cells were exposed to DOX. The overexpressed or knockdown of miR-25 in H9c2 cells was achieved by miR-25 mimic or inhibitor and the efficiency of transfection was identified by qRT-PCR or Western blotting. Cell viability, apoptotic cell rate, and levels of apoptosis-related proteins were determined by CCK-8, flow cytometry, and Western blotting, respectively. Furthermore, Western blotting and immunofluorescence staining (IF) were performed to assess the expression levels of reactive oxygen species and degree of DNA damage. Results: As a result, DOX significantly upregulated miR-25 expression in mice and H9c2 cells and reduced cell viability and increased cell apoptosis in vitro and in vivo. miR-25 overexpression expedited cell injury induced by DOX in H9c2 cells demonstrated by the increased cell apoptosis and reactive oxygen species (ROS) production, whereas miR-25 inhibition attenuated the cell injury. Furthermore, miR-25 negatively controlled the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Intervention the expression of PTEN using si-PTEN reversed the beneficial effects of miR-25 inhibition on DOX-injured H9c2 cells. Conclusion: In conclusion, this study demonstrated that miR-25 is involved in DOX-induced cell damage through the regulation of PTEN expression.

Keywords: H9c2 cells; PTEN; doxorubicin-induced cardiotoxicity; miR-25.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
DOX upregulates the level of miR-25 in H9c2 cells. (A) CCK8 assay shows the reduced proliferation of H9c2 cells treated with increased concentration of DOX for 6, 12, 24, and 48h. Effect of exposure to (B)5μM DOX for different time points and (C) different concentration of DOX for 24h on the expression of miR-25 determined by qRT-PCR in H9c2 cells. (*P < 0.05; **P < 0.01, compared with cells in 0h, n=4)
Figure 2
Figure 2
miR-25 expression is augmented after treatment of DOX in mice. (A) After stimulated with DOX or saline for 4 weeks, representative images of H&E staining from mice (upper panel, scale bar=50μm) and representative M-mode echocardiography of left ventricular chamber change (lower panel). Left ventricular performance was measured in mice and the variables (left ventricular ejection fraction (EF), fraction shortness (FS) left ventricular end-diastolic dimension (LVEDD) and end-systolic dimension (LVESD) between different treatment groups are shown in (B). (C) DOX decreases the protein level of Bcl-2, whereas increases the level of Bax in heart tissue. Antioxidant enzyme activities of SOD, CAT and GSH-Px are reduced by DOX (D-F). (G) DOX decreases the heart weight to tibial length ratio compared with control group in mice. (H)The expression of miR-25 in mice after treated with DOX. (*P < 0.05; **P < 0.01; ***P < 0.001, n=7)
Figure 3
Figure 3
The protective effect of miR-25 inhibition in DOX-induced apoptosis. The relative expression of miR-25 after transfection of miR-25 mimic(A) and miR-25 inhibitor(B) (**P < 0.01, n=4). (C) Representative dot plots of flow cytometric images in H9c2 cells after treated as indicated. (D)TUNEL assay results of different groups (original magnification ×400). Nuclei are stained in blue, and TUNEL staining is shown in green. Quantification of the results in C (E), and D (F). (*P < 0.05; **P < 0.01; ***P < 0.001 compared with control group; #p < 0.05; ##p < 0.01 compared with DOX+NC-treated group, n = 5)
Figure 4
Figure 4
miR-25 mediates ROS production and DNA damage process. (A) Intracellular reactive oxygen species (ROS) level in DOX-treated H9c2 cells with transfection of miR-25 inhibitor or miR-25 mimic. (B) The image shows the expression level of γ-H2AX in DOX-treated H9c2 cells with transfection of miR-25 inhibitor or miR-25 mimic. Nuclei are stained in blue, cytoskeleton in red and γ-H2AX in green. The bar graphs show the percentages of ROS levels (C) and γ-H2AX-positive cardiomyocytes of each group (D). (*P < 0.05; **P < 0.01; ***P < 0.001 compared with control group; #p < 0.05; ##p < 0.01 compared with DOX group, n = 5). (E) Parallel gels were run for γ-H2AX protein and GAPDH, under the gels are the quantification results. (*P < 0.05; **P < 0.01; ***P < 0.001 compared with control group; #p < 0.05; ##p < 0.01 compared with DOX group, n = 3)
Figure 5
Figure 5
miR-25 regulates the expression of PTEN. (A) Relative expression of PTEN measured from mice heart tissue lysates treated with DOX or CTRL. (B) After exposure to 5μM DOX for 6, 12, 24, 48h, PTEN levels were determined by Western blot analysis. (*P < 0.05; **P < 0.01 compared with cells in 0h; ##p < 0.01 compared with cells in 6h, n = 3) (C) After transfection of miR-25/NC mimic or miR-25/NC inhibitor, H9c2 cells were treated with 5μM DOX or control, and the PTEN expression was measured. GAPDH was served as the loading control. (*P < 0.05; **P < 0.01 compared with control group; #p < 0.05; ##p < 0.01 compared with DOX group, n = 3) (D) Sequence alignment of wild-type (WT) PTEN and mutated (MUT) PTEN mRNA 3′-UTR binding site of miR-25. (E) Luciferase activity levels upon co-transfection of a luciferase construct containing PTEN-3′UTR-WT or PTEN-3′UTR-MUT with miR-25 mimic or NC mimic in HEK293T cells. (**P < 0.01, n = 3)
Figure 6
Figure 6
Silencing of PTEN blunts the protective effect of miR-25 inhibition. H9c2 cells were co-transfected with miR-25 inhibitor or NC inhibitor and si-PTEN or si-NC (5μM) and then stimulated with DOX (5μm) for 24 h. Then, the TUNEL stainin (A), ROS (B) and γ-H2AX (C) levels in control and PTEN knockdown cells were measured. (D) Protein expression levels of PTEN, p-AKT, AKT, p-PI3K, PI3K, γ-H2AX, Bcl-2, and Bax in H9c2 cells were determined by Western blot analysis and quantification of the relative expression using Actin as an internal control (lower panel). (*p < 0.05, ** p < 0.01, *** p < 0.001, compared with the miR-25 inhibitor + si-NC group, #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the NC inhibitor + si-NC group. n = 3). Quantification of the results in A (E), B (F), and C(G). (*p < 0.05, ** p < 0.01, *** p < 0.001, compared with the miR-25 inhibitor + si-NC group, #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the NC inhibitor + si-NC group. n = 5)
Figure 7
Figure 7
miR-25 inhibition exerts no effect on DOX treatment in DLBCL cells. After NUDUL-1 and TMD8 cells were co-transfected with miR-25 inhibitor or NC inhibitor, DOX (5μm) was added into the cells for 24, 48, 72 and 96 h. CCK-8 results show the cell viability changes in NUDUL-1(A) and TMD8(B) cells.
Figure 8
Figure 8
A role of miR-25 in DOX-induced cardiotoxicity through PTEN/PI3K/AKT pathway.
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