目的 探讨 METTL3 对核受体 NR4A2 （Nuclear Receptor Subfamily 4, Group A, Member 2, NR4A2） mRNA的m⁶A修饰及其在帕金森病（Parkinson's disease, PD）小胶质细胞M1极化与神经炎症中的作用机制。 方法 6-羟基多巴胺（6-OHDA）单侧MFB注射构建PD小鼠模型。阿扑吗啡诱导旋转、转棒及爬杆实验评估运动功能；免疫荧光、HE染色及Western blot检测黑质多巴胺能神经元损伤、炎症因子及相关分子表达；ELISA检测组织m⁶A整体修饰水平。脂多糖（LPS）刺激BV2小胶质细胞构建体外炎症模型；siRNA及过表达质粒调控METTL3表达；RNA免疫共沉淀（RIP）验证METTL3与NR4A2 mRNA结合，SRAMP预测其结合位点；放线菌素D实验检测METTL3对NR4A2 mRNA稳定性的影响。 结果 与对照组相比，PD模型小鼠运动功能受损，黑质区神经元核固缩，酪氨酸羟化酶（TH）阳性神经元数量减少，M1表型相关指标（IL-1β、iNOS、TNF-α）表达、整体m⁶A修饰水平及METTL3表达升高，NR4A2表达降低（P<0.05）；LPS组BV2细胞变化趋势一致。敲低METTL3组BV2细胞M1表型相关指标表达降低，NR4A2 mRNA半衰期缩短（P<0.05）。RIP证实METTL3可直接结合NR4A2 mRNA（P<0.05），SRAMP预测其存在结合位点；过表达METTL3组M1表型相关指标表达升高，NR4A2 mRNA稳定性延长（P<0.05）。 结论 METTL3介导的NR4A2 m⁶A修饰通过调控小胶质细胞M1极化，在PD中发挥关键的促炎作用。
Abstract
Objective To investigate the m6A modification of nuclear receptor NR4A2 (Nuclear Receptor Subfamily 4, Group A, Member 2) mRNA by METTL3 and its mechanism of action in microglial M1 polarization and neuroinflammation in Parkinson's disease (PD). Methods A PD mouse model was established by unilateral medial forebrain bundle (MFB) injection of 6-hydroxydopamine (6-OHDA). Motor function was evaluated by apomorphine-induced rotation test, rotarod test, and pole test. Immunofluorescence, HE staining, and Western blot were used to detect dopaminergic neuron damage in the substantia nigra, expression of inflammatory factors, and related molecules. ELISA was employed to measure the global m⁶A modification level in tissues. An in vitro inflammatory model was constructed by stimulating BV2 microglia with lipopolysaccharide (LPS). METTL3 was knocked down or overexpressed using siRNA and overexpression plasmids, respectively. RNA immunoprecipitation (RIP) was used to verify the binding of METTL3 to NR4A2 mRNA, with binding sites predicted by SRAMP. The effect of METTL3 on NR4A2 mRNA stability was assessed using the actinomycin D assay. Results Compared with the control group, PD model mice exhibited impaired motor function, nuclear pyknosis in the substantia nigra, and a reduced number of tyrosine hydroxylase (TH)-positive neurons (P<0.05). Expression of M1 phenotype markers (IL-1β, iNOS, TNF-α), global m⁶A modification levels, and METTL3 expression were increased, while NR4A2 expression was decreased (P<0.05), consistent trends were observed in the LPS-stimulated BV2 cells. In the METTL3 knockdown group, expression of M1 phenotype markers decreased and NR4A2 mRNA half-life was shortened (P<0.05). RIP confirmed that METTL3 directly binds to NR4A2 mRNA (P<0.05), with potential binding sites predicted by SRAMP. In the METTL3 overexpression group, expression of M1 phenotype markers increased and NR4A2 mRNA stability was extended (P<0.05). Conclusions METTL3-mediated m⁶A modification of NR4A2 plays a crucial pro-inflammatory role in PD by regulating microglial M1 polarization.
关键词
NR4A2； /
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帕金森病； /
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神经炎症； /
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m6A RNA甲基化； /
METTL3
Key words
NR4A2; /
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Parkinson's disease; /
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Neuroinflammation; /
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m6A RNA methylation; /
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METTL3
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基金
广东省基础与应用基础研究基金项目（2025A1515010578）；深圳市科技计划项目（JCYJ20220530150212028）；深圳市宝安区高质量发展研究项目（BAGZL2024065）；深圳市“医疗卫生三名工程”项目资助项目（SZZYSM202106009）
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