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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.


Effect of miR-378 on the mRNA and protein expression levels of its target gene Caspase-3. (A,B) Representative Western blot and quantitative analysis showed the caspase-3 protein levels increased significantly in N2A cells at 0–24 h of reoxygenation after 3 h of OGD, * p < 0.05, n = 5 per group; (C,D) Transfection of pri-miR-378 effectively decreased the expression of caspase-3, whereas anti-miR-378 increased the expression of caspase-3 in mouse N2A cells exposed to 3 h OGD/24 h reoxygenation, * p < 0.05 vs. OGD non-trans, n = 3 per group; (E) Upper panel, construction of luciferase reporter system; Lower panel, pri-miR-378 decreased luciferase activity of the reporter vector containing 3′-UTR of caspase-3, but had no effect on the mutated reporter vector, * p < 0.05, n = 6 per group; (F) Pri-miR-378 and anti-miR-378 had no effect on Caspase-3 mRNA levels, n = 5 per group.
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ijms-17-01427-f003: Effect of miR-378 on the mRNA and protein expression levels of its target gene Caspase-3. (A,B) Representative Western blot and quantitative analysis showed the caspase-3 protein levels increased significantly in N2A cells at 0–24 h of reoxygenation after 3 h of OGD, * p < 0.05, n = 5 per group; (C,D) Transfection of pri-miR-378 effectively decreased the expression of caspase-3, whereas anti-miR-378 increased the expression of caspase-3 in mouse N2A cells exposed to 3 h OGD/24 h reoxygenation, * p < 0.05 vs. OGD non-trans, n = 3 per group; (E) Upper panel, construction of luciferase reporter system; Lower panel, pri-miR-378 decreased luciferase activity of the reporter vector containing 3′-UTR of caspase-3, but had no effect on the mutated reporter vector, * p < 0.05, n = 6 per group; (F) Pri-miR-378 and anti-miR-378 had no effect on Caspase-3 mRNA levels, n = 5 per group.

Mentions: As described by the previous report, miR-378 could inhibit caspase-3 protein expression and attenuated ischemic injury in cardiomyocytes [16]. To examine whether caspase-3 is responsible for OGD-induced N2A cell apoptosis, we analyzed the possibility of caspase-3 being a putative target of miR-378 by bioinformatics algorithms. We found that there were potential binding sites between mmu-miR-378 and the 3′-UTR of Caspase-3 mRNA. In partial agreement with this prediction, we found that caspase-3 protein levels increased significantly in N2A cells at 0–24 h of reoxygenation after 3 h of OGD (Figure 3A,B, p < 0.05, n = 5 per group). In addition, pri-miR-378 effectively decreased the expression of caspase-3, whereas anti-miR-378 increased the expression of caspase-3 in mouse N2A cells exposed to 3 h OGD/24 h reoxygenation (Figure 3C,D, p < 0.05, n = 3 per group). However, pri-miR-378 and anti-miR-378 had no effect on Caspase-3 mRNA levels (Figure 3F, p < 0.05, n = 5 per group).


MicroRNA-378 Alleviates Cerebral Ischemic Injury by Negatively Regulating Apoptosis Executioner Caspase-3
Effect of miR-378 on the mRNA and protein expression levels of its target gene Caspase-3. (A,B) Representative Western blot and quantitative analysis showed the caspase-3 protein levels increased significantly in N2A cells at 0–24 h of reoxygenation after 3 h of OGD, * p < 0.05, n = 5 per group; (C,D) Transfection of pri-miR-378 effectively decreased the expression of caspase-3, whereas anti-miR-378 increased the expression of caspase-3 in mouse N2A cells exposed to 3 h OGD/24 h reoxygenation, * p < 0.05 vs. OGD non-trans, n = 3 per group; (E) Upper panel, construction of luciferase reporter system; Lower panel, pri-miR-378 decreased luciferase activity of the reporter vector containing 3′-UTR of caspase-3, but had no effect on the mutated reporter vector, * p < 0.05, n = 6 per group; (F) Pri-miR-378 and anti-miR-378 had no effect on Caspase-3 mRNA levels, n = 5 per group.
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ijms-17-01427-f003: Effect of miR-378 on the mRNA and protein expression levels of its target gene Caspase-3. (A,B) Representative Western blot and quantitative analysis showed the caspase-3 protein levels increased significantly in N2A cells at 0–24 h of reoxygenation after 3 h of OGD, * p < 0.05, n = 5 per group; (C,D) Transfection of pri-miR-378 effectively decreased the expression of caspase-3, whereas anti-miR-378 increased the expression of caspase-3 in mouse N2A cells exposed to 3 h OGD/24 h reoxygenation, * p < 0.05 vs. OGD non-trans, n = 3 per group; (E) Upper panel, construction of luciferase reporter system; Lower panel, pri-miR-378 decreased luciferase activity of the reporter vector containing 3′-UTR of caspase-3, but had no effect on the mutated reporter vector, * p < 0.05, n = 6 per group; (F) Pri-miR-378 and anti-miR-378 had no effect on Caspase-3 mRNA levels, n = 5 per group.
Mentions: As described by the previous report, miR-378 could inhibit caspase-3 protein expression and attenuated ischemic injury in cardiomyocytes [16]. To examine whether caspase-3 is responsible for OGD-induced N2A cell apoptosis, we analyzed the possibility of caspase-3 being a putative target of miR-378 by bioinformatics algorithms. We found that there were potential binding sites between mmu-miR-378 and the 3′-UTR of Caspase-3 mRNA. In partial agreement with this prediction, we found that caspase-3 protein levels increased significantly in N2A cells at 0–24 h of reoxygenation after 3 h of OGD (Figure 3A,B, p < 0.05, n = 5 per group). In addition, pri-miR-378 effectively decreased the expression of caspase-3, whereas anti-miR-378 increased the expression of caspase-3 in mouse N2A cells exposed to 3 h OGD/24 h reoxygenation (Figure 3C,D, p < 0.05, n = 3 per group). However, pri-miR-378 and anti-miR-378 had no effect on Caspase-3 mRNA levels (Figure 3F, p < 0.05, n = 5 per group).

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.