Abstract
Objectives To explore the role of microRNA-21-5p (miR-21-5p) in hypoxia/reoxygenation- (H/R-) induced HT22 cell damage. Methods The hypoxia/reoxygenation (H/R) model was established in mouse neuronal cells HT22. Cell Counting Kit-8 (CCK-8) and qRT-PCR were used to determine the effects of H/R treatment on cell viability and miR-21-5p expression. HT22 cells were transfected with miR-21-5p mimic or negative control (NC) followed by the induction of H/R; cell viability, apoptosis, and SOD, MDA, and LDH activities were detected. Besides, the apoptosis-related proteins including BAX, BCL2, cleaved caspase-3, and caspase-3 as well as proteins of EGFR/PI3K/AKT signaling pathways were measured by Western blot. To verify the target relation between cytoplasmic polyadenylation element binding protein 3 (CPEB3) and miR-21-5p, luciferase reporter gene experiment was performed. After cotransfection with miR-21-5p mimic and CPEB3 plasmids, the reversal effects of CPEB3 on miR-21-5p in H/R damage were studied. Results H/R treatment could significantly reduce the cell viability (P < 0.05) and miR-21-5p levels (P < 0.05) in HT22 cells. After overexpressing miR-21-5p, cell viability was increased (P < 0.05) under H/R treatment, and the apoptosis rate and the levels of apoptosis-related proteins were suppressed (all P < 0.05). Furthermore, SOD activity was increased (P < 0.05), while MDA and LDH activity was decreased (both P < 0.05). Besides, miR-21-5p could restore the activation of the EGFR/PI3K/AKT signaling pathway inhibited by H/R treatment (all P < 0.05). The luciferase reporter gene experiment verified that CPEB3 is the target of miR-21-5p (P < 0.05). When coexpressing miR-21-5p mimic and CPEB3 in the cells, the protective effects of miR-21-5p under H/R were reversed (all P < 0.05), and the activation of the EGFR/PI3K/AKT pathway was also inhibited (all P < 0.05). Conclusion This study showed that miR-21-5p may regulate the EGFR/PI3K/AKT signaling pathway by targeting CPEB3 to reduce H/R-induced cell damage and apoptosis.
Highlights
Cerebrovascular disease is the primary cause of human disability
To further study the function of miR-21-5p in H/R-treated HT22 cells, as shown in Figure 2(a), we first transfected miR-21-5p mimic to overexpress miR-21-5p in HT22 cells (P < 0:05); as shown in Figure 2(b), the Cell Counting Kit-8 (CCK-8) method results confirmed that the overexpression of miR21-5p reversed the decreases of cell viability after H/R treatment (P < 0:05)
Mouse neuronal cells HT22 were treated with hypoxia and reoxygenation to establish the H/R cell model, and we found that the H/R could induce neuronal cell damage which was accompanied by the decreased level of miR-21-5p
Summary
Cerebrovascular disease is the primary cause of human disability. It was characterized by high morbidity, disability, and mortality, which threaten human health seriously [1]. It is known that thrombolytic therapy is beneficial to the recanalization of occluded cerebrovascular and the timely recovery of ischemic brain tissue. With the continuous development of this treatment, the lives of countless ischemic stroke patients have been saved, but it has caused serious ischemia/reperfusion (I/R) injuries [2]. As of there is no clinically effective drug for the treatment of brain I/R [5]. It is of great clinical significance to actively explore and study the pathogenesis of brain I/R injury and find a treatment route
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