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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 oxygen-glucose deprivation (OGD)-induced ischemic injury in N2A cells. (A) miR expression decreased in cultured N2A cells exposed to 3 h OGD following different reoxygenation times. * p < 0.05 vs. normoxia, n = 5 per group; (B) Transfection of pri-miR-378 or anti-miR-378 significantly increased or decreased the level of miR-378 in normoxia and 3 h OGD/24 h reoxygenation treatments, * p < 0.05 vs. normoxic non-trans, # p < 0.05 vs. OGD non-trans, n = 5 per group; (C,D) MTT assay showed that pri-miR-378 and anti-miR-378 transfection significantly increased or decreased cell survival, respectively, following OGD treatment; (E,F) LDH assay showed that overexpression of miR-378 substantially suppressed the cell death, while transfection of anti-miR-378 aggravated cell death induced by 3 h OGD/24 h reoxygenation, * p < 0.05 vs. normoxic non-trans, # p < 0.05 vs. OGD non-trans, n = 6 per group.
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ijms-17-01427-f001: Effect of miR-378 on oxygen-glucose deprivation (OGD)-induced ischemic injury in N2A cells. (A) miR expression decreased in cultured N2A cells exposed to 3 h OGD following different reoxygenation times. * p < 0.05 vs. normoxia, n = 5 per group; (B) Transfection of pri-miR-378 or anti-miR-378 significantly increased or decreased the level of miR-378 in normoxia and 3 h OGD/24 h reoxygenation treatments, * p < 0.05 vs. normoxic non-trans, # p < 0.05 vs. OGD non-trans, n = 5 per group; (C,D) MTT assay showed that pri-miR-378 and anti-miR-378 transfection significantly increased or decreased cell survival, respectively, following OGD treatment; (E,F) LDH assay showed that overexpression of miR-378 substantially suppressed the cell death, while transfection of anti-miR-378 aggravated cell death induced by 3 h OGD/24 h reoxygenation, * p < 0.05 vs. normoxic non-trans, # p < 0.05 vs. OGD non-trans, n = 6 per group.

Mentions: In accordance with the changes of miR-378 in the peri-infarct region of MCAO mice [15], we examined the miR-378 expression in N2A cells during reoxygenation after 3 h OGD exposure. The cells were collected and analyzed at 0, 6, 12, 24, and 48 h after 3 h OGD by quantitative real-time PCR. The results showed that miR-378 expression level decreased gradually and reached the platform at 24 h reoxygenation compared with the normoxic control (Figure 1A, 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 oxygen-glucose deprivation (OGD)-induced ischemic injury in N2A cells. (A) miR expression decreased in cultured N2A cells exposed to 3 h OGD following different reoxygenation times. * p < 0.05 vs. normoxia, n = 5 per group; (B) Transfection of pri-miR-378 or anti-miR-378 significantly increased or decreased the level of miR-378 in normoxia and 3 h OGD/24 h reoxygenation treatments, * p < 0.05 vs. normoxic non-trans, # p < 0.05 vs. OGD non-trans, n = 5 per group; (C,D) MTT assay showed that pri-miR-378 and anti-miR-378 transfection significantly increased or decreased cell survival, respectively, following OGD treatment; (E,F) LDH assay showed that overexpression of miR-378 substantially suppressed the cell death, while transfection of anti-miR-378 aggravated cell death induced by 3 h OGD/24 h reoxygenation, * p < 0.05 vs. normoxic non-trans, # p < 0.05 vs. OGD non-trans, n = 6 per group.
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ijms-17-01427-f001: Effect of miR-378 on oxygen-glucose deprivation (OGD)-induced ischemic injury in N2A cells. (A) miR expression decreased in cultured N2A cells exposed to 3 h OGD following different reoxygenation times. * p < 0.05 vs. normoxia, n = 5 per group; (B) Transfection of pri-miR-378 or anti-miR-378 significantly increased or decreased the level of miR-378 in normoxia and 3 h OGD/24 h reoxygenation treatments, * p < 0.05 vs. normoxic non-trans, # p < 0.05 vs. OGD non-trans, n = 5 per group; (C,D) MTT assay showed that pri-miR-378 and anti-miR-378 transfection significantly increased or decreased cell survival, respectively, following OGD treatment; (E,F) LDH assay showed that overexpression of miR-378 substantially suppressed the cell death, while transfection of anti-miR-378 aggravated cell death induced by 3 h OGD/24 h reoxygenation, * p < 0.05 vs. normoxic non-trans, # p < 0.05 vs. OGD non-trans, n = 6 per group.
Mentions: In accordance with the changes of miR-378 in the peri-infarct region of MCAO mice [15], we examined the miR-378 expression in N2A cells during reoxygenation after 3 h OGD exposure. The cells were collected and analyzed at 0, 6, 12, 24, and 48 h after 3 h OGD by quantitative real-time PCR. The results showed that miR-378 expression level decreased gradually and reached the platform at 24 h reoxygenation compared with the normoxic control (Figure 1A, 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.