Department of Neurology, Affiliated Hospital of Weifang Medical College, Weifang, Shandong 261031, China.
Pain Department, Weifang People's Hospital, Weifang, Shandong 261041, China.
Anal Cell Pathol (Amst). 2021 Dec 2;2021:5543212. doi: 10.1155/2021/5543212. eCollection 2021.
To explore the role of microRNA-21-5p (miR-21-5p) in hypoxia/reoxygenation- (H/R-) induced HT22 cell damage.
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.
H/R treatment could significantly reduce the cell viability ( < 0.05) and miR-21-5p levels ( < 0.05) in HT22 cells. After overexpressing miR-21-5p, cell viability was increased ( < 0.05) under H/R treatment, and the apoptosis rate and the levels of apoptosis-related proteins were suppressed (all < 0.05). Furthermore, SOD activity was increased ( < 0.05), while MDA and LDH activity was decreased (both < 0.05). Besides, miR-21-5p could restore the activation of the EGFR/PI3K/AKT signaling pathway inhibited by H/R treatment (all < 0.05). The luciferase reporter gene experiment verified that CPEB3 is the target of miR-21-5p ( < 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 < 0.05), and the activation of the EGFR/PI3K/AKT pathway was also inhibited (all < 0.05).
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.
探讨微小 RNA-21-5p（miR-21-5p）在缺氧/复氧（H/R）诱导的 HT22 细胞损伤中的作用。
建立小鼠神经元细胞 HT22 的缺氧/复氧（H/R）模型。细胞计数试剂盒-8（CCK-8）和 qRT-PCR 用于检测 H/R 处理对细胞活力和 miR-21-5p 表达的影响。转染 miR-21-5p 模拟物或阴性对照（NC）后，诱导 HT22 细胞发生 H/R；检测细胞活力、细胞凋亡以及 SOD、MDA 和 LDH 活性。此外，通过 Western blot 检测凋亡相关蛋白，包括 BAX、BCL2、cleaved caspase-3 和 caspase-3 以及 EGFR/PI3K/AKT 信号通路相关蛋白。为了验证细胞质多聚腺苷酸化元件结合蛋白 3（CPEB3）与 miR-21-5p 之间的靶关系，进行了荧光素酶报告基因实验。共转染 miR-21-5p 模拟物和 CPEB3 质粒后，研究了 CPEB3 对 H/R 损伤中 miR-21-5p 的逆转作用。
H/R 处理可显著降低 HT22 细胞的细胞活力（<0.05）和 miR-21-5p 水平（<0.05）。过表达 miR-21-5p 后，H/R 处理下细胞活力增加（<0.05），凋亡率和凋亡相关蛋白水平降低（均<0.05）。此外，SOD 活性增加（<0.05），MDA 和 LDH 活性降低（均<0.05）。此外，miR-21-5p 可恢复 H/R 处理抑制的 EGFR/PI3K/AKT 信号通路的激活（均<0.05）。荧光素酶报告基因实验验证了 CPEB3 是 miR-21-5p 的靶标（<0.05）。当在细胞中共表达 miR-21-5p 模拟物和 CPEB3 时，miR-21-5p 在 H/R 下的保护作用被逆转（均<0.05），EGFR/PI3K/AKT 通路的激活也受到抑制（均<0.05）。
本研究表明，miR-21-5p 可能通过靶向 CPEB3 调节 EGFR/PI3K/AKT 信号通路，减少 H/R 诱导的细胞损伤和凋亡。
