IntroductionMicroRNAs (miRNAs) are emerging as critical regulators in the pathological process of cerebral ischemia/reperfusion injury. miRNAs play an important role in regulating neuronal survival. miR-135b-5p has been reported as an important miRNA in regulating cell apoptosis. However, the role of miR-135b-5p in regulating neuronal survival remains poorly understood. Here, we aimed to investigate the role of miR-135b-5p in cerebral ischemia/ reperfusion using an in vitro model of oxygen-glucose deprivation and reoxygenation-(OGD/R) induced neuron injury.Material and methodsmiRNA, mRNA and protein expression was detected by real-time quantitative polymerase chain reaction and Western blot. Cell viability was detected by cell counting kit-8 and lactate dehydrogenase assays. Cell apoptosis was detected by caspase-3 activity assay. Oxidative stress was determined using commercial kits. The target of miR-135b-5p was confirmed by dual-luciferase reporter assay.ResultsWe found that miR-135b-5p expression was significantly decreased in hippocampal neurons receiving OGD/R treatment. Overexpression of miR-135b-5p markedly alleviated OGD/R-induced cell injury and oxidative stress, whereas suppression of miR-135b-5p showed the opposite effects. We observed that miR-135b-5p directly targeted the 3′-untranslated region of glycogen synthase kinase-3β (GSK-3β). We found that miR-135b-5p negatively regulates the expression of GSK-3β in hippocampal neurons. Moreover, miR-135b-5p overexpression promotes activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling. However, the restoration of GSK-3β expression significantly reversed the protective effects of miR-135b-5p overexpression.ConclusionsOverall, our results suggest that miR-135b-5p protects neurons against OGD/R-induced injury through downregulation of GSK-3β and promotion of the Nrf2/ARE signaling pathway-mediated antioxidant responses.

Recently, numerous microRNAs (miRNAs) have been considered as key players in the regulation of neuronal processes. The purpose of the present study is to explore the effect of miR‐25 on hippocampal neuron injury in Alzheimer's disease (AD) induced by amyloid β (Aβ) peptide fragment 1 to 42 (Aβ1‐42) via Kruppel‐like factor 2 (KLF2) through the nuclear factor‐E2‐related factor 2 (Nrf2) signaling pathway. A mouse model of AD was established through Aβ1‐42 induction. The underlying regulatory mechanisms of miR‐25 were analyzed through treatment of miR‐25 mimics, miR‐25 inhibitors, or small interfering RNA (siRNA) against KLF2 in hippocampal tissues and cells isolated from AD mice. The targeting relationship between miR‐25 and KLF2 was predicted using a target prediction program and verified by luciferase activity determination. MTT assay was used to evaluate the proliferative ability and flow cytometry to detect cell cycle distribution and apoptosis. KLF2 was confirmed as a target gene of miR‐25. When the mice were induced by Aβ1‐42, proliferation was suppressed while apoptosis was promoted in hippocampal neurons as evidenced by lower levels of KLF2, Nrf2, haem oxygenase, glutathione S transferase α1, glutathione, thioredoxin, and B‐cell lymphoma‐2 along with higher bax level. However, such alternations could be reversed by treatment of miR‐25 inhibitors. These findings indicate that miR‐25 may inhibit hippocampal neuron proliferation while promoting apoptosis, thereby aggravating hippocampal neuron injury through downregulation of KLF2 via the Nrf2 signaling pathway.