miR-488-3p Protects Cardiomyocytes against Doxorubicin-Induced Cardiotoxicity by Inhibiting CyclinG1

Abstract

Objective: To investigate the protective effects and regulatory mechanism of miR-488-3p on doxorubicin-induced cardiotoxicity.

Methods: The C57BL/6 mice and primary cardiomyocytes were used to construct doxorubicin-induced cardiomyocyte injury models in vivo and in vitro. The levels of miR-488-3p and its downstream target genes were analyzed by quantitative real-time PCR. Mouse cardiac function, cell survival, cellular injury-related proteins, and the apoptosis level of cardiomyocytes were analyzed by echocardiography, MTT analysis, Western blotting, and DNA laddering separately.

Results: Cardiomyocyte injury caused by a variety of stimuli can lead to the reduction of miR-488-3p level, especially when stimulated with doxorubicin. Doxorubicin led to significant decrease in cardiac function, cell autophagic flux blockage, and apoptosis in vivo and in vitro. The expression of miR-488-3p's target gene, CyclinG1, increased remarkably in the doxorubicin-treated neonatal mouse cardiomyocytes. Overexpression of miR-488-3p inhibited CyclinG1 expression, increased cardiomyocyte viability, and attenuated doxorubicin-induced cardiomyocyte autophagic flux blockage and apoptosis.

Conclusions: miR-488-3p is one of the important protective miRNAs in doxorubicin-induced cardiotoxicity by inhibiting the expression of CyclinG1, which provides insight into the possible clinical application of miR-488-3p/CyclinG1 as therapeutic targets in doxorubicin-induced cardiovascular diseases.

Figure 1

Downregulation of miR-488-3p is correlated with myocardial injury. (a) miR-488-3p sequences in the mouse, rat, and human for the evolutionary conservation analysis. (b) The changes in miR-488-3p expression in different stimulus-induced myocardial injury models (n = 3). (c) Identification of miR-488-3p expression in cardiomyocytes treated with 0.5 μM and 1 μM doxorubicin (n = 3). ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Figure 2

Doxorubicin induces myocardial injury with decreased miR-488-3p expression in vivo. (a) Schematic representation of the doxorubicin (Dox) treatment protocol in male C57BL/6 mice. (b–e) Echocardiographic (Echo) measurement of the left ventricular anterior wall (LVAW), left ventricular posterior wall (LVPW), ejection fraction (EF%), and fractional shortening (FS%) after doxorubicin treatment for 6 days (n = 6). (f) Heart weight- (HW-) to-tibia length (TL) ratios of mice in the control (Con) or Dox group (n = 6). (g) Representative image of the hearts of Con group and Dox group mice. (h) H&E staining of sections of mouse hearts of the Con group and Dox group. (i) The number of vacuoles was measured in the cytoplasm (n = 5). (j, k) The number of apoptotic cells was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining in the mouse hearts of the Con group and Dox group. Blue staining shows TUNEL-positive cells (n = 5). (l) Relative expression of miR-488-3p in mouse left ventricles of the Con group and Dox group was determined by real-time qPCR (n = 6). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 3

miR-488-3p expression is negatively correlated with the seriousness of doxorubicin-induced cardiotoxicity. (a) The cell viability of left ventricle cardiomyocytes treated with different concentrations of Dox for 24 hours, as demonstrated by the MTT assay (n = 4). (b, c) Cardiomyocytes were treated with Dox for 24 hours, and MDC staining images showing autophagosomes (b) and autophagy in cardiomyocytes were quantified (c) (n = 6). (d, e) Representative photos and the average data of cardiomyocyte apoptosis were analyzed by TUNEL staining in different concentrations of Dox treatment for 24 hours (n = 3). (f) DNA laddering of myocardial cells treated with Dox (0, 0.1 0.5, 1, 3, 5, and 10 μM) for 24 hours. (g, h) Autophagy- and apoptosis-associated protein level in cardiomyocytes treated with different concentrations of Dox (0, 0.1, 0.5, and 1 μM) for 24 h (n = 3). (i) Relative expression of miR-488-3p in cardiomyocytes with different concentrations of Dox was analyzed by qPCR (n = 3). (j) The viability of myocardial cells treated with Dox or dexrazoxane (Dex) alone or in combination (n = 4). (k) Levels of miR-488-3p were measured in myocardial cells treated with Dox or Dex alone or in combination (n = 4). (l) The viability of cardiomyocytes in the Con and 0.5 μM Dox group after transfection with NC or miR-488-3p mimics (miR-488-3p m) (n = 4). (m, n) Fluorescence images showing NC- or miR-488-3p mimic-transduced cardiomyocytes that were treated with 0.5 μM Dox or left untreated (m) and quantification of the MDC-stained autophagosomes (n) (n = 6). (o) Cardiomyocytes were transduced with NCi or miR-488-3p inhibitor (miR-488-3p i) and were treated with 0.5 μM Dox or without Dox for 24 h. The formation of autophagosomes was examined using MDC staining after treatment with 0.5 μM Dox (n = 5). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 4

CyclinG1 is a downstream target gene of miR-488-3p. (a) The schematic of the miRNA-488-3p binding site to the target gene predicted and analyzed by using TargetScan. (b, c) Luciferase activity of pmirGLO-CyclinG1-3′-UTR (pmirGLO-C) in HEK 293A cells. The HEK 293A cells were cotransfected with pmirGLO, pmirGLO-CyclinG1-3′-UTR, and miR-488-3p mimic plasmid (n = 3). (d) Relative expression of miR-488-3p in cardiomyocytes after transfection with miR-488-3p mimics (n = 3). (e) The levels of CyclinG1 mRNA in cardiomyocytes after transfection with miR-488-3p mimics (n = 5). (f, g) Relative protein expression of CyclinG1 in cardiomyocytes after transfection with miR-488-3p mimics (n = 3). (h) The levels of miR-488-3p in cardiomyocytes after transfection with the miR-488-3p inhibitor (miR-488-3p i) (n = 3). (i) Relative mRNA expression of CyclinG1 in cardiomyocytes after transfection with the miR-488-3p inhibitor (n = 3). (j, k) The levels of CyclinG1 protein in cardiomyocytes after transfection with the miR-488-3p inhibitor (n = 3). ^∗∗P < 0.01, ^∗∗∗P < 0.001. ns indicates that no statistically significant difference was observed (P > 0.05).

Figure 5

CylcinG1 knockdown attenuates doxorubicin-induced cardiotoxicity in vitro. (a) Relative mRNA expression of CyclinG1 in cardiomyocytes treated with 1 μM Dox (n = 3). (b, c) Expression of CyclinG1 in control or 0.5 μM Dox-treated cardiomyocytes (n = 3). (d) MDC staining of the cardiomyocytes treated for 24 h with and without 0.5 μM Dox and after knockdown of CyclinG1 with siRNA or NC. (e) Graph showing the MDC fluorescence intensity quantified using the ImageJ program (n = 5). (f–k) The levels of CyclinG1 and autophagy- and apoptosis-associated protein expression were analyzed by Western blotting (n = 3). ^∗P < 0.05, ^∗∗P < 0.01. ns indicates that no statistically significant difference was observed (P > 0.05).