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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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Effect of miR-378 overexpression on cerebral ischemia/reperfusion injury of mice following 1 h MCAO/24 h reperfusion. (A–C) Western blot analysis showed that intracerebroventricular administration of miR-378 agomir, but not its negative control, could downregulate caspase-3 protein level and the ratio of cleaved caspase-3 in cerebral ischemic cortex of mice at 24 h after transient MCAO, * p < 0.05, n = 4 per group; (D) Representative TTC staining slices showed that miR-378 agomir effectively attenuated infarction size of mice following transient focal cerebral ischemia; (E–G) Nissl staining showed that administration of miR-378 agomir decreased neural cell loss in ischemic brain of mice at 24 and 72 h of reperfusion following 1 h MCAO, * p < 0.05 vs. sham, # p < 0.05 vs. none, n = 4 per group. Scale bar = 50 µm.
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ijms-17-01427-f004: Effect of miR-378 overexpression on cerebral ischemia/reperfusion injury of mice following 1 h MCAO/24 h reperfusion. (A–C) Western blot analysis showed that intracerebroventricular administration of miR-378 agomir, but not its negative control, could downregulate caspase-3 protein level and the ratio of cleaved caspase-3 in cerebral ischemic cortex of mice at 24 h after transient MCAO, * p < 0.05, n = 4 per group; (D) Representative TTC staining slices showed that miR-378 agomir effectively attenuated infarction size of mice following transient focal cerebral ischemia; (E–G) Nissl staining showed that administration of miR-378 agomir decreased neural cell loss in ischemic brain of mice at 24 and 72 h of reperfusion following 1 h MCAO, * p < 0.05 vs. sham, # p < 0.05 vs. none, n = 4 per group. Scale bar = 50 µm.

Mentions: To further evaluate the biological role of miR-378 in cerebral ischemic injury in vivo, we employed a micro infusion pump to continuously deliver miR-378 agomir into the lateral ventricle to increase miR-378 expression. Western blot analysis showed that miR-378 agomir but not its negative control could downregulate caspase-3 protein level and cleaved-caspase-3 ratio in cerebral ischemic cortex of mice after 1 h MCAO/24 h reperfusion (Figure 4A–C p < 0.05, n = 4 per group).Then we examined the effect of miR-378 overexpression on MCAO-induced infarction and neural cell loss by TTC and Nissl staining, respectively. Consequently, miR-378 agomir effectively attenuated infarction size (Figure 4D) and neuronal cell loss (Figure 4E–G, p < 0.05, n = 4 per group) in ischemic brain of MCAO mice. The above results indicated that miR-378 can exert neuroprotective effect partially by reducing caspase-3 associated cell apoptosis.


MicroRNA-378 Alleviates Cerebral Ischemic Injury by Negatively Regulating Apoptosis Executioner Caspase-3
Effect of miR-378 overexpression on cerebral ischemia/reperfusion injury of mice following 1 h MCAO/24 h reperfusion. (A–C) Western blot analysis showed that intracerebroventricular administration of miR-378 agomir, but not its negative control, could downregulate caspase-3 protein level and the ratio of cleaved caspase-3 in cerebral ischemic cortex of mice at 24 h after transient MCAO, * p < 0.05, n = 4 per group; (D) Representative TTC staining slices showed that miR-378 agomir effectively attenuated infarction size of mice following transient focal cerebral ischemia; (E–G) Nissl staining showed that administration of miR-378 agomir decreased neural cell loss in ischemic brain of mice at 24 and 72 h of reperfusion following 1 h MCAO, * p < 0.05 vs. sham, # p < 0.05 vs. none, n = 4 per group. Scale bar = 50 µm.
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ijms-17-01427-f004: Effect of miR-378 overexpression on cerebral ischemia/reperfusion injury of mice following 1 h MCAO/24 h reperfusion. (A–C) Western blot analysis showed that intracerebroventricular administration of miR-378 agomir, but not its negative control, could downregulate caspase-3 protein level and the ratio of cleaved caspase-3 in cerebral ischemic cortex of mice at 24 h after transient MCAO, * p < 0.05, n = 4 per group; (D) Representative TTC staining slices showed that miR-378 agomir effectively attenuated infarction size of mice following transient focal cerebral ischemia; (E–G) Nissl staining showed that administration of miR-378 agomir decreased neural cell loss in ischemic brain of mice at 24 and 72 h of reperfusion following 1 h MCAO, * p < 0.05 vs. sham, # p < 0.05 vs. none, n = 4 per group. Scale bar = 50 µm.
Mentions: To further evaluate the biological role of miR-378 in cerebral ischemic injury in vivo, we employed a micro infusion pump to continuously deliver miR-378 agomir into the lateral ventricle to increase miR-378 expression. Western blot analysis showed that miR-378 agomir but not its negative control could downregulate caspase-3 protein level and cleaved-caspase-3 ratio in cerebral ischemic cortex of mice after 1 h MCAO/24 h reperfusion (Figure 4A–C p < 0.05, n = 4 per group).Then we examined the effect of miR-378 overexpression on MCAO-induced infarction and neural cell loss by TTC and Nissl staining, respectively. Consequently, miR-378 agomir effectively attenuated infarction size (Figure 4D) and neuronal cell loss (Figure 4E–G, p < 0.05, n = 4 per group) in ischemic brain of MCAO mice. The above results indicated that miR-378 can exert neuroprotective effect partially by reducing caspase-3 associated cell apoptosis.

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