M6A甲基转移酶METTL3通过靶向铁下垂调节创伤性脑损伤。

IF 3.3 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Fei Qin, Fan Li, Wenxiao Zhao, Suqin Zhang, Jiang Shen, Xinyu Yang
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引用次数: 0

摘要

背景:外伤性脑损伤(Traumatic brain injury, TBI)是一种由外力损伤大脑结构和功能而引起的疾病。脑外伤后,铁积累和活性氧(ROS)增加脂质过氧化,促进铁下垂。甲基转移酶样3 (METTL3)通过调节相关信号通路抑制铁下垂。本研究探讨了METTL3在TBI中神经元铁下垂的作用,为TBI提供了新的见解和潜在的治疗方法。方法:建立脑外伤小鼠和神经元细胞模型,用METTL3过表达处理。Morris水迷宫(MWM)测试评估认知功能。脑组织进行组织学染色以评估脑损伤、核固缩和铁积累。免疫荧光染色检测神经元、小胶质细胞和星形胶质细胞的活化情况。使用细胞计数试剂盒8 (CCK-8)检测神经元细胞增殖。定量PCR (qPCR)和western blot检测mRNA和蛋白的表达。通过测量铁、丙二醛(MDA)、超氧化物歧化酶(SOD)和ROS的积累来评估铁下垂。采用m6A- elisa法定量细胞中n6 -甲基腺苷(m6A) RNA甲基化水平。采用甲基化RNA免疫沉淀法(MeRIP)分析GPX4 mRNA的m6A修饰。采用RNA拉下法和RNA免疫沉淀法(RIP)检测YTHDF2与GPX4 mRNA的相互作用。结果:METTL3在脑外伤脑组织中表达下调。过表达METTL3可改善认知功能和大脑恢复,同时减少铁下垂和神经炎症。METTL3过表达上调GPX4在体内和体外的表达。进一步研究表明,m6A读取器蛋白YTHDF2结合GPX4 mRNA,从而介导mettl3调控的GPX4的m6A富集和RNA稳定性。敲除GPX4和用铁下垂诱导剂治疗可消除METTL3对神经元的保护作用。结论:METTL3通过调节GPX4的m6A修饰和RNA稳定性,具有抗铁下沉作用,促进脑外伤后脑损伤恢复。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
M6A Methyltransferase METTL3 Modulates Traumatic Brain Injury by Targeting Ferroptosis.

Background: Traumatic brain injury (TBI) is a disease caused by external forces that damage brain structure and function. After TBI, iron accumulation and reactive oxygen species (ROS) increase lipid peroxidation, promoting ferroptosis. Methyltransferase-like 3 (METTL3) inhibits ferroptosis by modulating related signaling pathways. This study investigates the effects of METTL3 on neuronal ferroptosis in TBI, offering new insights and potential therapies.

Methods: TBI mouse and neuron cell models were established and treated with METTL3 overexpression. The Morris Water Maze (MWM) test evaluated cognitive function. Histological staining of brain tissues was conducted to assess brain injury, nuclear pyknosis, and iron accumulation. The activation of neurons, microglia, and astrocytes were detected using immunofluorescence staining. Neuron cell proliferation was measured using the Cell Counting Kit 8 (CCK-8). Quantitative PCR (qPCR) and western blot detected the mRNA and protein expression. Ferroptosis was assessed by measuring the accumulation of iron, malondialdehyde (MDA), superoxide dismutase (SOD), and ROS. The quantification of the N6-methyladenosine (m6A) RNA methylation levels in cells was quantified using the m6A-ELISA assay. Methylated RNA immunoprecipitation (MeRIP) assays were conducted to analyze the m6A modification on GPX4 mRNA. The interaction between YTHDF2 and GPX4 mRNA was measured using RNA pulldown and RNA immunoprecipitation (RIP) assays.

Results: METTL3 expression was downregulated in TBI-injured brain tissues. Overexpression of METTL3 improved cognitive function and brain recovery while simultaneously reducing ferroptosis and neuroinflammation. METTL3 overexpression upregulated GPX4 expression both in vitro and in vivo. Further studies indicated that m6A reader protein YTHDF2 binds to GPX4 mRNA, consequently mediating the METTL3-regulated m6A enrichment and RNA stability of GPX4. Knockdown of GPX4 and treatment with ferroptosis inducer abolished the protective effects of METTL3 on neurons.

Conclusion: METTL3 exhibits anti-ferroptosis properties and promotes brain injury recovery after TBI by regulating the m6A modification and RNA stability of GPX4.

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