Circular RNA circEfnb2 promotes cell injury after cerebral infarction by sponging miR-202-5p and regulating TRAF3 expression

IF 1.6 4区医学 Q4 IMMUNOLOGY Transplant immunology Pub Date : 2024-03-26 DOI:10.1016/j.trim.2024.102042

Limin Tu , Wei Cheng , Xudong Wang , Zhixin Li , Xing Li

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Abstract

Background

Exogenous neural cell transplantation may be therapeutic for stroke, cerebral ischemic injury. Among other mechanisms, increasing findings indicated circular RNAs (circRNAs) regulate the pathogenesis progression of cerebral ischemia. Mmu_circ_0015034 (circEfnb2) was upregulated in focal cortical infarction established by middle cerebral artery occlusion (MCAO) in mice. Our study was designed to probe the molecular mechanism of circEfnb2 in the oxygen-glucose deprivation/reperfusion (OGD/R)-induced neuronal damage in cerebral ischemia.

Methods

We established an in vitro OGD/R cell model. CircEfnb2 and microRNA-202-5p (miR-202-5p) levels were detected using real-time quantitative polymerase chain reaction (RT-qPCR). Lactate dehydrogenase (LDH), malondialdehyde (MDA), and reactive oxygen species (ROS) levels were assessed using specific kits. Tumor necrosis factor-α (TNF-α) and Interleukin-1β (IL-1β) levels were examined using an Enzyme-linked immunosorbent assay (ELISA). Flow cytometry analysis evaluated cell apoptosis. Protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), cleaved caspase 3, and Tumor necrosis factor receptor-associated factor 3 (TRAF3) were determined using Western blot assay.

Results

Overall, circEfnb2 was highly expressed whereas miR-202-5p was decreased in OGD/R-treated mouse hippocampal neuronal HT22 cells compared to normal controls (both p > 0.05). From an in vitro functional perspective, circEfnb2 knockdown attenuated an OGD/R-triggered neuronal injury compared to controls (p > 0.05). Mechanically, circEfnb2 acted as a sponge of miR-202-5p; downregulation of miR-202-5p annulled the inhibitory roles of circEfnb2 silencing in an OGD/R-caused neuronal injury model. Our analysis showed that miR-202-5p directly targeted TRAF3 as enhanced TRAF3 abolished the effects of miR-202-5p in the OGD/R-induced neuronal injury. In vivo, lentivirus with a short hairpin (sh)-circEfnb2 inhibited cerebral injury, when injected into cerebral cortex in MCAO mice (p > 0.05).

Conclusion

Our results suggest that circEfnb2 deficiency may decrease OGD/R-induced HT22 cell damage by modulating the miR-202-5p/TRAF3 axis. This explanation may provide a new direction for cerebral infarction potential therapeutic targets.

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环状RNA circEfnb2通过海绵状miR-202-5p和调节TRAF3的表达促进脑梗塞后细胞损伤。

背景：外源性神经细胞移植可治疗中风和脑缺血损伤。越来越多的研究结果表明，循环 RNA（circRNA）调节脑缺血的发病机制。Mmu_circ_0015034（circEfnb2）在小鼠大脑中动脉闭塞（MCAO）引起的局灶性皮质梗死中上调。我们的研究旨在探究circEfnb2在氧-葡萄糖剥夺/再灌注（OGD/R）诱导的脑缺血神经元损伤中的分子机制：方法：我们建立了体外 OGD/R 细胞模型。方法：我们建立了一个体外 OGD/R 细胞模型，使用实时定量聚合酶链反应（RT-qPCR）检测 CircEfnb2 和 microRNA-202-5p (miR-202-5p)的水平。使用特定试剂盒评估了乳酸脱氢酶（LDH）、丙二醛（MDA）和活性氧（ROS）水平。使用酶联免疫吸附试验（ELISA）检测肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）的水平。流式细胞术分析评估了细胞凋亡情况。用 Western 印迹法测定了 B 细胞淋巴瘤-2（Bcl-2）、Bcl-2 相关 X 蛋白（Bax）、裂解的 Caspase 3 和肿瘤坏死因子受体相关因子 3（TRAF3）的蛋白水平：总的来说，与正常对照组相比，经OGD/R处理的小鼠海马神经元HT22细胞中circEfnb2高表达，而miR-202-5p则低表达（两者的p均大于0.05）。从体外功能角度来看，与对照组相比，circEfnb2敲除可减轻OGD/R引发的神经元损伤（p > 0.05）。从机制上讲，circEfnb2充当了miR-202-5p的海绵；在OGD/R引发的神经元损伤模型中，miR-202-5p的下调取消了circEfnb2沉默的抑制作用。我们的分析表明，miR-202-5p 直接靶向 TRAF3，因为 TRAF3 的增强消除了 miR-202-5p 在 OGD/R 诱导的神经元损伤中的作用。在体内，将带有短发夹（sh）-circEfnb2的慢病毒注射到MCAO小鼠的大脑皮层中可抑制脑损伤（p > 0.05）：我们的研究结果表明，circEfnb2的缺乏可通过调节miR-202-5p/TRAF3轴来减少OGD/R诱导的HT22细胞损伤。这一解释为脑梗死的潜在治疗靶点提供了新的方向。

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来源期刊

Transplant immunology 医学-免疫学

CiteScore

2.10

自引率

13.30%

发文量

198

审稿时长

48 days

期刊介绍： Transplant Immunology will publish up-to-date information on all aspects of the broad field it encompasses. The journal will be directed at (basic) scientists, tissue typers, transplant physicians and surgeons, and research and data on all immunological aspects of organ-, tissue- and (haematopoietic) stem cell transplantation are of potential interest to the readers of Transplant Immunology. Original papers, Review articles and Hypotheses will be considered for publication and submitted manuscripts will be rapidly peer-reviewed and published. They will be judged on the basis of scientific merit, originality, timeliness and quality.