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Overexpression of microRNA-135b-5p attenuates acute myocardial infarction injury through its antioxidant and anti-apptotic properties
doi: 10.2478/fzm-2021-0011
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Abstract:
Objective: Myocardial infarction (MI) remains the leading cause of morbidity and mortality due partly to the limited regenerative capacity of cardiomyocytes to replace cardiomyocyte lost due to apoptosis. Inhibiting cardiomyocyte apoptosis is recognized as an effective therapeutic approach for MI. MicroRNAs (miRNAs, miRs), which regulate target genes at the post-transcriptional level, play a significant role in the regulation of cardiovascular diseases such as MI. MicroRNA-135b (miR-135b) has a protective effect on cardiomyocytes. However, the role of miR- 135b in cardiomyocyte apoptosis in infarct myocardium needs further clarification. Methods: We generated α-MHC-miR-135b transgenic mice to investigate the role of miR-135b in myocardial injury after MI. MiR- 135b mimic and negative control (NC) were transfected into H2O2-induced cardiomyocytes to evaluate the effect of overexpression of miR-135b on the levels of reactive oxygen species (ROS) and apoptosis. Results: Our results showed that overexpression of miR-135b had protective effect on cardiomyocyte injury both in vivo and in vitro. MiR-135b inhibited cardiomyocyte apoptosis and ROS generation, downregulated proapoptosis proteins (cleaved-caspase-3 and Bax), and increased antiapoptosis protein (Bcl-2). Moreover, miR-135b showed an inhibitory effect on apoptosis-related protein target transient receptor potential vanilloid-type 4 (TRPV4) cation channel. Conclusion: MiR-135b might be considered a new molecular target for potential replacement therapy as antiapoptotic cardioprotection in the setting of MI.
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Key words:
- myocardial infarction /
- apoptosis /
- microRNA-135b /
- TRPV4
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Figure 1. MI induced functional and morphological changes in mouse hearts
(A) M-mode echocardiograms. (B) Ejection fraction and (C) Fractional shortening. (D) TTC staining results. (E) Election microscope images. Scale bars = 500 nm. (F) TUNEL staining results. The mRNA expression of miR-135b (G) in vivo and (I) in vitro. (H) CCK8 assay result. n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 vs. Sham or Ctrl. The data are presented as mean ± SD.
Figure 2. Overexpression of miR-135b in transgenic (Tg) mice reverses MI-induced impairments of cardiac function
(A) The mRNA expression of miR-135b in vitro. (B) M-mode echocardiograms. (C) Ejection fraction and (D) Fractional shortening. (E) TTC staining results. (F) Election microscope images. Scale bars = 500 nm. (G) TUNEL staining results. n = 3; ***P < 0.001 vs. WT, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. WT+MI. &&&P < 0.001 vs. Tg+MI. The data are presented as mean ± SD.
Figure 3. Forced expression of miR-135b suppresses ROS generation and apoptosis in primary cultured neonatal mouse ventricular cells (NMVCs)
(A) The mRNA expression of miR-135b in vitro. (B) CCK8 assay result. (C) Flow cytometry results and (D) corresponding statistical result in vitro. (E) ROS generation in vitro. n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 vs. Ctrl or NC, ###P < 0.001 vs. H2O2. The data are presented as mean ± SD.
Figure 4. Overexpression of miR-135b alters the expression of apoptosis-related protein regulators in Tg mice
The mRNA expression of (A) Caspase-3 (B) Bax (C) Bcl-2 and (D) Bax/Bcl-2 mRNA ratio in vitro. The protein expression of (E) Caspase-3 (F) Bax (G) Bcl-2 and (H) Bax/Bcl-2 protein ratio in vitro. n = 3; **P < 0.01, ***P < 0.001 vs. WT, ##P < 0.01, ###P < 0.001 vs. WT+MI. The data are presented as mean ± SD.
Figure 5. Forced expression of miR-135b alters the expression of apoptosis-related protein regulators in cultured NMVCs
The mRNA expression of (A) Caspase-3 (B) Bax (C) Bcl-2 and (D) Bax/Bcl-2 mRNA ratio in vitro. The protein expression of (E) Caspase-3 (F) Bax (G) Bcl-2 and (H) Bax/Bcl-2 protein ratio in vitro. n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. WT+MI. The data are presented as mean ± SD.
Figure 6. miR-135b represses the expression of TRPV4 both in vivo and in vitro
(A) The predicted binding site for miR-135b in TRPV4 sequences of humans and mice. (B) The mRNA and (C) protein expression of TRPV4 in vivo. (D) The mRNA and (E) protein expression of TRPV4 in vitro. n = 3; **P < 0.01 vs. WT or Ctrl, #P < 0.05, ##P < 0.01 vs. WT+MI or H2O2. The data are presented as mean ± SD.
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