MicroRNA therapy confers anti-senescent effects on doxorubicin-related cardiotoxicity by intracellular and paracrine signaling

Figures

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  Figure 1. Dox-treated cardiomyocytes exhibited increased cellular senescence. (A) Cell cycle analysis was performed with PI staining by flow cytometry. (B) Percentages of cells in the three phases of the cell cycle. (C) Cellular proliferation was measured using CCK-8 assay. (D) Representative images of SA-β-gal staining (senescent cells are stained green). Scale bars, 20 μm. (E) Percentage of senescent cells was calculated. (F) Representative p16INK4a staining (green). Scale bars, 20 μm. (G, H) Expression of miR-199a-3p in cardiomyocytes and cardiac tissue was quantified by qRT-PCR. *P < 0.05 versus control, n = 6 per group. (I) Representative photomicrographs of p16INK4a-positive cardiomyocytes in the Dox treated cardiac tissue. Scale bars, 100 μm.

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  Figure 2. A slowdown of Dox-induced aging via the overexpression of miR-199a-3p. (A) Expression of miR-199a-3p was quantified by qRT-PCR. *P < 0.05 versus the miR-199a-3p mimic in repeated-measures ANOVA, n = 6 per group. (B) Percentages of cells in the three phases of the cell cycle determined by flow cytometry. (C) Cellular proliferation was measured by the CCK-8 assay. (D) SASP factor protein levels quantified by Luminex of the medium. The medium was collected from such cells as follows: transfection with the miR-199a-3p mimic or miR-NC mimic, followed by treatment with Dox. The untreated cardiomyocytes were used as control. (E) The percentage of senescent cells was calculated. (F) Representative images of SA-β-gal staining (senescent cells are stained green). Scale bars, 20 μm. (G) Telomere length was detected by qRT-PCR. (H) Telomerase activity was determined using a telomerase repeat amplification protocol (TRAP). *P < 0.05 versus control; ▲P < 0.05 versus Dox + miR-199a-3p mimic in repeated-measures ANOVA, n = 6 per group.

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  Figure 3. MiR-199a-3p directly targeted GATA4. (A) Predicted miR-199a-3p binding site in the 3’-UTR of GATA4. The corresponding sequence in the mutated (MUT) version is also shown. (B) Luciferase activity was analyzed 48 h after transfection of the miR-199a-3p mimic or miR-NC mimic. *P < 0.05 versus the miR-199a-3p mimic (WT group) in repeated-measures ANOVA, n = 6 per group. (C, D) Western blot analysis of GATA4. The size of markers (in kDa) is indicated. *P < 0.05 versus control; ▲P < 0.05 versus Dox + miR-199a-3p mimic in repeated-measures ANOVA, n = 6 per group.

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  Figure 4. MiR-199a-3p repressed GATA4 contributing to the anti-senescent effect against Dox. (A–C) Cardiomyocytes were infected with Ad-GATA4, Ad-Ctrl, or Ad-GATA4+miR-199a-3p mimic. After 24 h, the cell lysates were harvested for qRT-PCR (A) and western blot analysis (B, C). *P < 0.05 versus Ad-GATA4 in repeated-measures ANOVA, n = 6 per group. (D) The percentages of cells in the three phases of the cell cycle determined by flow cytometry. (E) Cellular proliferation was measured using the CCK-8 assay. (F) Representative images of SA-β-gal staining (senescent cells are stained green). Scale bars, 20 μm. (G) The percentage of senescent cells was calculated. (H) SASP factor protein levels quantified by Luminex of the medium. The medium was collected from such cells as follows: transfection with the miR-199a-3p mimic or the miR-199a-3p mimic + Ad-GATA4, or the miR-199a-3p mimic + Ad-Ctrl, followed by exposure to Dox. The untreated cardiomyocytes were used as the control. (I) Telomere length was detected by qRT-PCR. (J) Telomerase activity was determined using telomerase repeat amplification protocol (TRAP). *P < 0.05 versus control; ▲P < 0.05 versus Dox; #P < 0.05 versus Dox + miR-199a-3p…

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  Figure 5. MiR-199a-3p eliminated the spread of senescence among cardiomyocytes. (A) Representative electron micrograph of isolated exosomes. Scale bar: 100 nm. (B) Size of exosomes measured using a Zetasizer Nano ZS instrument. (C) Protein levels of CD63 and CD81, two exosome markers. (D) DiI-labeled exosomes (red) were internalized into DAPI-labeled cardiomyocytes (blue). Scale bar, 20 μm. (E) Cellular proliferation was measured using the CCK-8 assay. (F) Representative images of SA-β-gal staining (senescent cells are stained green). Scale bars, 20 μm. (G) The percentage of senescent cells was calculated. (H) SASP factor protein levels quantified by Luminex of the medium. The medium was collected from cells as follows: treated with exosome, exosomeDox, exosomeDox+miR-199a-3p mimic, and exosomeDox+miR-NC mimic. The untreated cardiomyocytes were used as control. *P < 0.05 versus control; ▲P < 0.05 versus exosome; #P < 0.05 versus exosomeDox+ miR-199a-3p mimic in repeated-measures ANOVA, n = 6 per group.