MiR-485-5p promotes neuron survival through mediating Rac1/Notch2 signaling pathway after cerebral ischemia/reperfusion

Xuan Chen, Sumei Zhang, Peipei Shi, Yangli Su, Dong Zhang, Na Li
Current Neurovascular Research 2020 April 15

OBJECTIVE: Ischemia-reperfusion (I/R) injury is a pathological feature of ischemic stroke. This study investigated the regulatory role of miR-485-5p in I/R injury.

METHODS: SH-SY5Y cells were induced with oxygen and glucose deprivation/reoxygenation (OGD/R) to mimic I/R injury in vitro. Cells were transfected with designated constructs (miR485-5p mimics, miR-485-5p inhibitor, lentiviral vectors overexpressing Rac1 or their corresponding controls). Cell viability was evaluated using MTT assay. The concentrations of lactate dehydrogenase, malondialdehyde, and reactive oxygen species were detected to indicate the degree of oxidative stress. Flow cytometry and caspase-3 activity assay were used for apoptosis assessment. Dual-luciferase reporter assay was performed to confirm that Rac family small GTPase 1 (Rac1) was a downstream gene of miR-485-5p.

RESULTS: OGD/R resulted in decreased cell viability, elevated oxidative stress, increased apoptosis, and downregulated miR-485-5p expression in SH-SY5Y cells. MiR-485-5p upregulation alleviated I/R injury, evidenced by improved cell viability, decreased oxidative markers, and reduced apoptotic rate. OGD/R increased the levels of Rac1 and neurogenic locus notch homolog protein 2 (Notch2) signaling-related proteins in cells with normal miR-485- 5p expression, whereas miR-485-5p overexpression successfully suppressed OGD/R-induced upregulation of these proteins. Furthermore, the delivery of vectors overexpressing Rac1 in miR485-5p mimics-transfected cells reversed the protective effect of miR-485-5p in cells with OGD/R-induced injury.

CONCLUSION: This study showed that miR-485-5p protected cells following I/R injury via targeting Rac1/Notch2 signaling, suggesting that targeted upregulation of miR-485-5p might be a promising therapeutic option for the protection against I/R injury.

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