Zhang Xiaomin, Du Pengyang, Bai Bo, Lian Xia, Xue Guofang
Department of Neurology, The Second Hospital of Shanxi Medical University, Taiyuan 030001, China.
Department of Neurology, The Second Hospital of Shanxi Medical University, Taiyuan 030001, China.
Brain Res Bull. 2025 Jan;220:111156. doi: 10.1016/j.brainresbull.2024.111156. Epub 2024 Nov 30.
This study explores the molecular mechanism of METTL14 regulating microglial function post ischemic stroke. A murine model was established by tMCAO. The neurological function was evaluated by mNSS. The cerebral infarct size and pathological changes were observed by TTC and H&E staining. M1 and M2 microglia in brain tissues were detected by flow cytometry. BV2 cells were subjected to OGD/R to establish an in vitro model. qRT-PCR and Western blot were used for detecting METTL14, PAX6, YTHDF2, TREM2, iNOS, and Arg1 expressions. The m6A level was quantitatively analyzed, and the binding of YTHDF2 or m6A to PAX6 was analyzed by RIP. PAX6 mRNA stability was assessed after actinomycin D treatment. ChIP was utilized for determining the enrichment of PAX6 on TREM2 promoter. The binding relationship between TREM2 and PAX6 was verified by dual-luciferase reporter assay. METTL14 was highly expressed after tMCAO, and silence of METTL14 alleviated symptoms of tMCAO mice and promoted microglial M2 polarization. METTL14 enhanced PAX6 mRNA m6A modification to promote YTHDF2 binding to PAX6 mRNA and its degradation. PAX6 bound to TREM2 promoter and facilitated its transcription and expression. In conclusion, METTL14-mediated m6A modification aggravates ischemic stroke by promoting microglial M1 polarization via YTHDF2/PAX6/TREM2 axis.
本研究探讨了METTL14在缺血性中风后调节小胶质细胞功能的分子机制。通过大脑中动脉闭塞（tMCAO）建立小鼠模型。通过改良神经功能缺损评分（mNSS）评估神经功能。通过TTC染色和苏木精-伊红（H&E）染色观察脑梗死体积和病理变化。采用流式细胞术检测脑组织中的M1和M2小胶质细胞。对小胶质细胞系BV2细胞进行氧糖剥夺/复氧（OGD/R）处理以建立体外模型。采用实时荧光定量聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法（Western blot）检测METTL14、配对盒蛋白6（PAX6）、YTH结构域家族蛋白2（YTHDF2）、触发受体表达于髓系细胞2（TREM2）、诱导型一氧化氮合酶（iNOS）和精氨酸酶1（Arg1）的表达。对m6A水平进行定量分析，并通过RNA免疫沉淀（RIP）分析YTHDF2或m6A与PAX6的结合情况。在用放线菌素D处理后评估PAX6信使核糖核酸（mRNA）的稳定性。采用染色质免疫沉淀法（ChIP）确定PAX6在TREM2启动子上的富集情况。通过双荧光素酶报告基因检测验证TREM2与PAX6之间的结合关系。tMCAO后METTL14高表达，沉默METTL14可减轻tMCAO小鼠的症状并促进小胶质细胞向M2极化。METTL14增强PAX6 mRNA的m6A修饰，以促进YTHDF2与PAX6 mRNA的结合及其降解。PAX6与TREM2启动子结合并促进其转录和表达。总之，METTL14介导的m6A修饰通过YTHDF2/PAX6/TREM2轴促进小胶质细胞向M1极化，从而加重缺血性中风。
