MicroRNA-137 Negatively Regulates H₂O₂-Induced Cardiomyocyte Apoptosis Through CDC42 - PubMed (original) (raw)
MicroRNA-137 Negatively Regulates H₂O₂-Induced Cardiomyocyte Apoptosis Through CDC42
Junnan Wang et al. Med Sci Monit. 2015.
Abstract
Background: Oxidative stress, inducing cardiomyocyte apoptosis or myocardial ischemia, is the major denominator of many cardiac diseases. In this study, we intended to explore the regulatory function of microRNA-137 (miR-137) in oxidative stress-induced cardiomyocyte apoptosis.
Material and methods: Cardiomyocytes were extracted from newborn C57BL/6 mice and cultured in vitro. Apoptosis was induced by H2O2, and evaluated by TUNEL assay. The effect of cardiomyocyte apoptosis on gene expression of miR-137 was evaluated by qRT-PCR. Lentivirus was used to stably down-regulate miR-137, and the subsequent effects of miR-137 down-regulation on cardiomyocyte apoptosis, its targeted gene CDC42, and caspase pathway were evaluated by TUNEL assay, dual-luciferase reporter assay, and Western blot assay, respectively. Finally, CDC42 was down-regulated by siRNA and its effect on miR-137-mediated cardiomyocyte apoptosis protection was examined.
Results: H2O2 induced significant apoptosis and up-regulated miR-137 in cardiomyocytes, whereas lentivirus-mediated miR-137 down-regulation protected against apoptosis. CDC42 was the direct target gene of miR-137 and proteins of CDC42, caspase-3, and caspase-9 were all regulated by miR-137 down-regulation in cardiomyocyte apoptosis. SiRNA-mediated CDC42 down-regulation reversed the protection of miR-137 down-regulation against cardiomyocyte apoptosis.
Conclusions: Our work demonstrated miR-137 and CDC42 are critical regulators in cardiomyocyte apoptosis. It may help to identify the molecular targets to prevent myocardial injury in human patients.
Figures
Figure 1
The effect of H2O2 on cardiomyocyte apoptosis and gene expression of miR-137. Cardiomyocytes were extracted from P1–P3 mice and incubated with 100 μM H2O2 for 24 h. (A) Cardiomyocyte apoptosis was evaluated by a TUNEL assay (green). The nuclei were stained with DAPI (blue). (B) The percentages of non-apoptotic cardiomyocytes (DAPI-positive/TUNEL-negative) were compared between Control and H2O2 treatment (* P<0.05). (C) The gene expression levels of miR-137 were also compared between Control and H2O2 treatment (* P<0.05).
Figure 2
The effect of miR-137 down-regulation on H2O2 -induced apoptosis in cardiomyocytes. Cardiomyocytes were transfected with lentiviruses of mmu-miR-137 inhibitor (miR-137-In) or its negative control miRNA (miR-C). (A) Twenty-four hours after lentiviral transfection, qRT-PCR was performed to measure the efficacy of lentiviral infection (* P<0.05). (B) Cardiomyocytes were then treated with 100 μM H2O2 for another 24 h, followed by a TUNEL assay to evaluate the effect of miR-137 down-regulation on cardiomyocyte apoptosis. (C) The percentages of non-apoptotic cardiomyocytes (DAPI-positive/TUNEL-negative) were compared between the cardiomyocytes transfected with miR-C and the ones transfected with miR-137-In (* P<0.05).
Figure 3
MiR-137 regulated CDC42 and caspase proteins in H2O2 -induced cardiomyocyte apoptosis. (A) The putative binding sites of murine miR-137 on wild-type (WT) CDC42 3′-UTR was highlighted. A mutated (Mut) CDC42 3′-UTR sequence was generated for the application of luciferase assay. (B) In a dual-luciferase reporter assay, HEK293T cells were transfected with firefly luciferase reporter inserted with WT CDC42 3′-UTR (CDC42-WT), or the reporter inserted with Mut CDC42 3′-UTR (CDC42-WT). Also, cells were co-transfected with either miR-137 mimics or its negative control miRNA (miR-NC). Twenty-four hours after transfection, relative luciferase activities were evaluated and normalized to the values in cells transfected with CDC42-MU (* P<0.05). (C) Cardiomyocytes were transfected with either miR-C or miR-137-In lentiviruses for 24 h, followed by another 24 h treatment of 100 μM H2O2. Western blotting analysis was used to compare the protein expression levels of CDC42, caspase-3 and caspase-9.
Figure 4
CDC42 down-regulation reversed the effect of miR-137 on H2O2-induced cardiomyocyte apoptosis. (A) Cardiomyocytes were transfected with CDC42 specific siRNA (CDC42-siRNA) or its negative control siRNA (C-siRNA) for 24 h. QRT-PCR was used to evaluate the transfection efficiency (* P<0.05). (B) Cardiomyocytes were transfected with miR-137-In for 24 h, followed by another 24 h transfection of either CDC42-siRNA or C-siRNA. Then, cardiomyocytes were treated with 100 μM H2O2 for 24 h, followed by a TUNEL assay to evaluate the effect of CDC42 down-regulation on cardiomyocyte apoptosis. (C) Western blotting analysis was ALSO used to compare the protein expression levels of CDC42, caspase-3 and caspase-9.
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