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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.

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Related in: MedlinePlus

Caspase-3 siRNAs blocked anti-miR-378-mediated neuronal injury in N2A cells. (A,B) Caspase-3 siRNA, but not its negative control effectively decreased caspase-3 protein expression in normoxia and OGD-treated groups, * p < 0.05 vs. non-OGD vehicle, # p < 0.05 vs. OGD vehicle, n = 3 per group; (C) MTT results showed that caspase-3 siRNAs had the ability to block anti-miR-378-mediated neuronal injury induced by OGD treatment, * p < 0.05 vs. OGD, # p < 0.05 vs. caspase-3 siRNA ctrl, n = 6 per group.
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ijms-17-01427-f005: Caspase-3 siRNAs blocked anti-miR-378-mediated neuronal injury in N2A cells. (A,B) Caspase-3 siRNA, but not its negative control effectively decreased caspase-3 protein expression in normoxia and OGD-treated groups, * p < 0.05 vs. non-OGD vehicle, # p < 0.05 vs. OGD vehicle, n = 3 per group; (C) MTT results showed that caspase-3 siRNAs had the ability to block anti-miR-378-mediated neuronal injury induced by OGD treatment, * p < 0.05 vs. OGD, # p < 0.05 vs. caspase-3 siRNA ctrl, n = 6 per group.

Mentions: To further analyze the contribution of miR-378 targeting caspase-3 to the biological function of miR-378 in OGD-induced N2A cell injury, we performed siRNA-mediated inhibition of caspase-3 protein expression. As shown in Figure 5A,B, caspase-3 protein levels decreased significantly following 48 h transfection of caspase-3 siRNAs in both normoxia and 3 h OGD/24 h reoxygenation-treated N2A cells (p < 0.05, n = 3 per group). MTT results showed that caspase-3 siRNA had the ability to block anti-miR-378-mediated neuronal injury induced by OGD treatment (Figure 5C, p < 0.05, n = 6 per group).


MicroRNA-378 Alleviates Cerebral Ischemic Injury by Negatively Regulating Apoptosis Executioner Caspase-3
Caspase-3 siRNAs blocked anti-miR-378-mediated neuronal injury in N2A cells. (A,B) Caspase-3 siRNA, but not its negative control effectively decreased caspase-3 protein expression in normoxia and OGD-treated groups, * p < 0.05 vs. non-OGD vehicle, # p < 0.05 vs. OGD vehicle, n = 3 per group; (C) MTT results showed that caspase-3 siRNAs had the ability to block anti-miR-378-mediated neuronal injury induced by OGD treatment, * p < 0.05 vs. OGD, # p < 0.05 vs. caspase-3 siRNA ctrl, n = 6 per group.
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ijms-17-01427-f005: Caspase-3 siRNAs blocked anti-miR-378-mediated neuronal injury in N2A cells. (A,B) Caspase-3 siRNA, but not its negative control effectively decreased caspase-3 protein expression in normoxia and OGD-treated groups, * p < 0.05 vs. non-OGD vehicle, # p < 0.05 vs. OGD vehicle, n = 3 per group; (C) MTT results showed that caspase-3 siRNAs had the ability to block anti-miR-378-mediated neuronal injury induced by OGD treatment, * p < 0.05 vs. OGD, # p < 0.05 vs. caspase-3 siRNA ctrl, n = 6 per group.
Mentions: To further analyze the contribution of miR-378 targeting caspase-3 to the biological function of miR-378 in OGD-induced N2A cell injury, we performed siRNA-mediated inhibition of caspase-3 protein expression. As shown in Figure 5A,B, caspase-3 protein levels decreased significantly following 48 h transfection of caspase-3 siRNAs in both normoxia and 3 h OGD/24 h reoxygenation-treated N2A cells (p < 0.05, n = 3 per group). MTT results showed that caspase-3 siRNA had the ability to block anti-miR-378-mediated neuronal injury induced by OGD treatment (Figure 5C, p < 0.05, n = 6 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.


Related in: MedlinePlus