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MicroRNA-378 Alleviates Cerebral Ischemic Injury by Negatively Regulating Apoptosis Executioner Caspase-3

View Article: PubMed Central - PubMed

ABSTRACT

miRNAs have been linked to many human diseases, including ischemic stroke, and are being pursued as clinical diagnostics and therapeutic targets. Among the aberrantly expressed miRNAs in our previous report using large-scale microarray screening, the downregulation of miR-378 in the peri-infarct region of middle cerebral artery occluded (MCAO) mice can be reversed by hypoxic preconditioning (HPC). In this study, the role of miR-378 in the ischemic injury was further explored. We found that miR-378 levels significantly decreased in N2A cells following oxygen-glucose deprivation (OGD) treatment. Overexpression of miR-378 significantly enhanced cell viability, decreased TUNEL-positive cells and the immunoreactivity of cleaved-caspase-3. Conversely, downregulation of miR-378 aggravated OGD-induced apoptosis and ischemic injury. By using bioinformatic algorithms, we discovered that miR-378 may directly bind to the predicted 3′-untranslated region (UTR) of Caspase-3 gene. The protein level of caspase-3 increased significantly upon OGD treatment, and can be downregulated by pri-miR-378 transfection. The luciferase reporter assay confirmed the binding of miR-378 to the 3′-UTR of Caspase-3 mRNA and repressed its translation. In addition, miR-378 agomir decreased cleaved-caspase-3 ratio, reduced infarct volume and neural cell death induced by MCAO. Furthermore, caspase-3 knockdown could reverse anti-miR-378 mediated neuronal injury. Taken together, our data demonstrated that miR-378 attenuated ischemic injury by negatively regulating the apoptosis executioner, caspase-3, providing a potential therapeutic target for ischemic stroke.

No MeSH data available.


Pri-miR-378 transfection significantly reduced 3 h OGD/24h reoxygenation- induced N2A cell apoptosis. (A,B) Representative TUNEL staining and statistical analysis showed that transfection of pri-miR-378 significantly attenuated, while anti-miR-378 increased the number of TUNEL-positive cells, * p < 0.05 vs. OGD non-trans, n = 6 per group; (C) Cleaved caspase-3 staining showed that overexpression of miR-378 could attenuate OGD-induced neuronal apoptosis, while anti-miR-378 aggravated N2A cell apoptosis induced by 3 h OGD/24 h reoxygenation. Scale bar = 50 µm.
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ijms-17-01427-f002: Pri-miR-378 transfection significantly reduced 3 h OGD/24h reoxygenation- induced N2A cell apoptosis. (A,B) Representative TUNEL staining and statistical analysis showed that transfection of pri-miR-378 significantly attenuated, while anti-miR-378 increased the number of TUNEL-positive cells, * p < 0.05 vs. OGD non-trans, n = 6 per group; (C) Cleaved caspase-3 staining showed that overexpression of miR-378 could attenuate OGD-induced neuronal apoptosis, while anti-miR-378 aggravated N2A cell apoptosis induced by 3 h OGD/24 h reoxygenation. Scale bar = 50 µm.

Mentions: To determine the role of miR-378 in OGD-induced cell injury, we used pri-miR-378 and anti-miR-378 to alter miR-378 levels in cultured N2A cells. As shown in Figure 1B, transfection of pri-miR-378 or anti-miR-378, but not their negative controls, significantly increased or decreased miR-378 levels in normoxia and 3 h OGD followed by 24 h reoxygenation (p < 0.05, n = 5 per group). Later, methylthiazolyldiphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays were employed to evaluate the effect of miR-378 on OGD-induced cell survival and cell death, respectively. 3 h OGD/24 h reoxygenation resulted in obvious N2A cell death. Overexpression of miR-378 substantially suppressed the cell death, whereas transfection of anti-miR-378 aggravated the N2A cell death induced by 3 h OGD/24 h reoxygenation (Figure 1C–F, p < 0.05, n = 6 per group). To confirm whether miR-378 could affect 3 h OGD/24 h reoxygenation-induced N2A cell apoptosis, a TUNEL assay was performed. TUNEL-positive cells were rarely seen in the normoxic group, whereas 3 h OGD/24 h reoxygenation increased the number of TUNEL-positive cells. Transfection of pri-miR-378 could significantly attenuate, while anti-miR-378 enhanced the number of TUNEL-positive cells. (Figure 2A,B, p < 0.05, n = 6 per group). As we know that TUNEL-positive cells also include some necroptotic cells, we further examined the activation of caspase-3 using a cleaved-caspase-3 specific antibody. The results confirmed that transfection of pri-miR-378 attenuated 3 h OGD/24 h reoxygenation induced cell apoptosis, while anti-miR-378 aggravated 3 h OGD/24 h reoxygenation induced cell apoptosis (Figure 2C).


MicroRNA-378 Alleviates Cerebral Ischemic Injury by Negatively Regulating Apoptosis Executioner Caspase-3
Pri-miR-378 transfection significantly reduced 3 h OGD/24h reoxygenation- induced N2A cell apoptosis. (A,B) Representative TUNEL staining and statistical analysis showed that transfection of pri-miR-378 significantly attenuated, while anti-miR-378 increased the number of TUNEL-positive cells, * p < 0.05 vs. OGD non-trans, n = 6 per group; (C) Cleaved caspase-3 staining showed that overexpression of miR-378 could attenuate OGD-induced neuronal apoptosis, while anti-miR-378 aggravated N2A cell apoptosis induced by 3 h OGD/24 h reoxygenation. Scale bar = 50 µm.
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ijms-17-01427-f002: Pri-miR-378 transfection significantly reduced 3 h OGD/24h reoxygenation- induced N2A cell apoptosis. (A,B) Representative TUNEL staining and statistical analysis showed that transfection of pri-miR-378 significantly attenuated, while anti-miR-378 increased the number of TUNEL-positive cells, * p < 0.05 vs. OGD non-trans, n = 6 per group; (C) Cleaved caspase-3 staining showed that overexpression of miR-378 could attenuate OGD-induced neuronal apoptosis, while anti-miR-378 aggravated N2A cell apoptosis induced by 3 h OGD/24 h reoxygenation. Scale bar = 50 µm.
Mentions: To determine the role of miR-378 in OGD-induced cell injury, we used pri-miR-378 and anti-miR-378 to alter miR-378 levels in cultured N2A cells. As shown in Figure 1B, transfection of pri-miR-378 or anti-miR-378, but not their negative controls, significantly increased or decreased miR-378 levels in normoxia and 3 h OGD followed by 24 h reoxygenation (p < 0.05, n = 5 per group). Later, methylthiazolyldiphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays were employed to evaluate the effect of miR-378 on OGD-induced cell survival and cell death, respectively. 3 h OGD/24 h reoxygenation resulted in obvious N2A cell death. Overexpression of miR-378 substantially suppressed the cell death, whereas transfection of anti-miR-378 aggravated the N2A cell death induced by 3 h OGD/24 h reoxygenation (Figure 1C–F, p < 0.05, n = 6 per group). To confirm whether miR-378 could affect 3 h OGD/24 h reoxygenation-induced N2A cell apoptosis, a TUNEL assay was performed. TUNEL-positive cells were rarely seen in the normoxic group, whereas 3 h OGD/24 h reoxygenation increased the number of TUNEL-positive cells. Transfection of pri-miR-378 could significantly attenuate, while anti-miR-378 enhanced the number of TUNEL-positive cells. (Figure 2A,B, p < 0.05, n = 6 per group). As we know that TUNEL-positive cells also include some necroptotic cells, we further examined the activation of caspase-3 using a cleaved-caspase-3 specific antibody. The results confirmed that transfection of pri-miR-378 attenuated 3 h OGD/24 h reoxygenation induced cell apoptosis, while anti-miR-378 aggravated 3 h OGD/24 h reoxygenation induced cell apoptosis (Figure 2C).

View Article: PubMed Central - PubMed

ABSTRACT

miRNAs have been linked to many human diseases, including ischemic stroke, and are being pursued as clinical diagnostics and therapeutic targets. Among the aberrantly expressed miRNAs in our previous report using large-scale microarray screening, the downregulation of miR-378 in the peri-infarct region of middle cerebral artery occluded (MCAO) mice can be reversed by hypoxic preconditioning (HPC). In this study, the role of miR-378 in the ischemic injury was further explored. We found that miR-378 levels significantly decreased in N2A cells following oxygen-glucose deprivation (OGD) treatment. Overexpression of miR-378 significantly enhanced cell viability, decreased TUNEL-positive cells and the immunoreactivity of cleaved-caspase-3. Conversely, downregulation of miR-378 aggravated OGD-induced apoptosis and ischemic injury. By using bioinformatic algorithms, we discovered that miR-378 may directly bind to the predicted 3&prime;-untranslated region (UTR) of Caspase-3 gene. The protein level of caspase-3 increased significantly upon OGD treatment, and can be downregulated by pri-miR-378 transfection. The luciferase reporter assay confirmed the binding of miR-378 to the 3&prime;-UTR of Caspase-3 mRNA and repressed its translation. In addition, miR-378 agomir decreased cleaved-caspase-3 ratio, reduced infarct volume and neural cell death induced by MCAO. Furthermore, caspase-3 knockdown could reverse anti-miR-378 mediated neuronal injury. Taken together, our data demonstrated that miR-378 attenuated ischemic injury by negatively regulating the apoptosis executioner, caspase-3, providing a potential therapeutic target for ischemic stroke.

No MeSH data available.