Zhongguo cuzhong zazhi (Jun 2024)
白N6-甲基腺苷修饰及其调控蛋在脑缺血中的表达变化及意义 Expression Changes and Significance of N6-Methyladenosine and its Regulatory Proteins in Cerebral Ischemia
Abstract
目的 本研究通过探讨N6-甲基腺苷（N6-methyladenosine，m6A）修饰及其调控蛋白在脑缺血小鼠中的表达变化，为脑缺血治疗的分子靶点研究提供参考。 方法 取60只雄性C57BL/6J小鼠，随机分为假手术组、脑缺血1 d组、脑缺血3 d组和脑缺血7 d组，每组各15只，采用线栓法制作右侧大脑中动脉梗死模型，于缺血1 h进行拔栓再灌注。通过RNA提取及斑点印迹实验检测小鼠缺血侧脑组织RNA m6A水平；使用荧光定量逆转录PCR检测小鼠缺血侧脑组织甲基转移酶3（methyltransferase 3，Mettl3）、甲基转移酶14（methyltransferase 14，Mettl14）、FTO α-酮戊二酸酯依赖性双加氧酶（FTO alpha-ketoglutarate dependent dioxygenase，Fto）、RNA去甲基化酶alkB同源基因5（alkB homolog 5，RNA demethylase；Alkbh5）、YTH N6-甲基腺苷RNA结合蛋白1（YTH N6-methyladenosine RNA binding protein 1，Ythdf1）、Ythdf2和Ythdf3的mRNA表达水平；利用免疫印迹实验检测缺血侧脑组织Mettl3、Mettl14、Fto、Alkbh5、Ythdf1、Ythdf2和Ythdf3蛋白的表达水平；借助免疫荧光染色观察缺血侧脑组织梗死周边区神经元中Mettl3、Fto、Ythdf1、Ythdf2和Ythdf3蛋白的表达变化情况。 结果 与假手术组相比，脑缺血3 d组脑组织RNA的m6A水平升高（1.620±0.339 vs. 1.000±0.192，P=0.0343）。①甲基转移酶表达情况：脑缺血7 d组Mettl3 mRNA水平降低（0.675±0.059 vs. 1.000±0.131，P=0.0331）；脑缺血1 d组Mettl3（0.548±0.107 vs. 1.000±0.056，P=0.0398）、Mettl14（0.534±0.218 vs. 1.000±0.018，P=0.0108）蛋白表达水平降低；脑缺血3 d组Mettl3（0.410±0.341 vs. 1.000±0.056，P=0.0084）、Mettl14（0.429±0.283 vs. 1.000±0.018，P=0.0026）蛋白表达水平也均下降；免疫荧光染色显示脑缺血3 d组梗死周边区神经元中Mettl3蛋白表达减少。②去甲基化酶表达情况：脑缺血1 d组（0.405±0.209 vs. 1.000±0.142，P=0.0108）、脑缺血3 d组（0.530±0.125 vs. 1.000±0.142，P=0.0412）Fto蛋白表达水平降低；免疫荧光染色显示脑缺血3 d组梗死周边区神经元中Fto表达减少。③m6A结合蛋白表达情况：脑缺血1 d组Ythdf1 mRNA水平降低（0.708±0.046 vs. 1.000±0.117，P=0.0331），Ythdf3 mRNA水平升高（1.473±0.093 vs. 1.000±0.142，P=0.0012）；脑缺血3 d组Ythdf1（0.593±0.240 vs. 1.000±0.117，P=0.0034）、Ythdf2（0.664±0.177 vs. 1.000±0.200，P=0.0100）mRNA水平降低，Ythdf3 mRNA水平升高（1.451±0.281 vs. 1.000±0.142，P=0.0018）；脑缺血1 d组Ythdf1（0.486±0.177 vs. 1.000±0.091，P=0.0197）、Ythdf3（0.536±0.107 vs. 1.000±0.125，P=0.0400）蛋白表达水平降低；脑缺血3 d组Ythdf1（0.404±0.299 vs. 1.000±0.091，P=0.0079）、Ythdf2（0.279±0.189 vs. 1.000±0.261，P=0.0136）、Ythdf3（0.450±0.220 vs. 1.000±0.125，P=0.0157）蛋白表达水平均降低；免疫荧光染色显示脑缺血3 d组梗死周边区神经元中m6A结合蛋白Ythdf1、Ythdf2、Ythdf3表达均减少。 结论 小鼠脑缺血后可能由Fto表达下调导致m6A水平升高，Ythdf1、Ythdf2、Ythdf3蛋白表达水平趋势基本一致，可能存在功能冗余。 Abstract: Objective To explore the expression changes of N6-methyladenosine (m6A) and its regulatory proteins in mouse models with cerebral ischemia and to provide a reference for finding potential molecular targets for cerebral ischemia therapy. Methods A total of 60 male C57BL/6J mice were randomly divided into 4 groups: sham-operated group, 1 day post-ischemia group, 3 days post-ischemia group, and 7 days post-ischemia group, with 15 mice in each group. The model of right middle cerebral artery occlusion was established in C57BL/6J mice by thread embolization method. Reperfusion was achieved after 1 hour of ischemia. RNA extraction and dot blotting were used to detect the levels of RNA m6A in the ischemic side of the mouse brain. Fluorescence quantitative reverse transcription PCR was used to detect the mRNA expression of methyltransferase 3 (Mettl3); methyltransferase 14 (Mettl14); FTO alpha-ketoglutarate dependent dioxygenase (Fto); alkB homolog 5, RNA demethylase (Alkbh5); YTH N6-methyladenosine RNA binding protein 1 (Ythdf1); Ythdf2; and Ythdf3 in the ischemic side of the mouse brain. Western blot was used to measure the protein expression levels of Mettl3, Mettl14, Fto, Alkbh5, Ythdf1, Ythdf2, and Ythdf3 in the ischemic side of the brain. Meanwhile, immunofluorescence staining was used to observe the expression changes of Mettl3, Fto, Ythdf1, Ythdf2, and Ythdf3 in the neurons of the ischemic side of the brain. Results Compared with the sham-operated group, the m6A level of the brain tissue RNA in 3 days post-ischemia group was increased (1.620±0.339 vs. 1.000±0.192, P=0.0343). ①Expression of methyltransferase: the mRNA level of Mettl3 decreased (0.675±0.059 vs. 1.000±0.131, P=0.0331) in 7 days post-ischemia group. The protein levels of Mettl3 (0.548±0.107 vs. 1.000±0.056, P=0.0398) and Mettl14 (0.534±0.218 vs. 1.000±0.018, P=0.0108) decreased in 1 day post-ischemia group. The protein levels of Mettl3 (0.410±0.341 vs. 1.000±0.056, P=0.0084) and Mettl14 (0.429±0.283 vs. 1.000±0.018, P=0.0026) decreased in 3 days post-ischemia group. Immunofluorescence staining revealed a reduction of Mettl3 expression in the neurons around the cerebral infarction area in 3 days post-ischemia group. ②Expression of demethylase: Fto protein levels were decreased in 1 day post-ischemia group (0.405±0.209 vs. 1.000±0.142, P=0.0108) and 3 days post-ischemia group (0.530±0.125 vs. 1.000±0.142, P=0.0412). Immunofluorescence staining showed that the expression of Fto in the neurons around the cerebral infarction area decreased after 3 days of cerebral ischemia. ③Expression of m6A binding protein: Ythdf1 mRNA level decreased in 1 day post-ischemia group (0.708±0.046 vs. 1.000±0.117, P=0.0331), while Ythdf3 mRNA level was increased (1.473±0.093 vs. 1.000±0.142, P=0.0012). The mRNA levels of Ythdf1 (0.593±0.240 vs. 1.000±0.117, P=0.0034) and Ythdf2 (0.664±0.177 vs. 1.000±0.200, P=0.0100) in 3 days post-ischemia group decreased, while the mRNA level of Ythdf3 was increased (1.451±0.281 vs. 1.000±0.142, P=0.0018). The protein levels of Ythdf1 (0.486±0.177 vs. 1.000±0.091, P=0.0197) and Ythdf3 (0.536±0.107 vs. 1.000±0.125, P=0.0400) in 1 day post-ischemia group were decreased. The protein levels of Ythdf1 (0.404±0.299 vs. 1.000±0.091, P=0.0079), Ythdf2 (0.279±0.189 vs. 1.000±0.261, P=0.0136), and Ythdf3 (0.450±0.220 vs. 1.000±0.125, P=0.0157) in 3 days post-ischemia group were decreased. Immunofluorescence staining further showed a decrease in the expression of m6A binding proteins Ythdf1, Ythdf2, and Ythdf3 in the neurons around the cerebral infarction area 3 days after ischemia. Conclusions Post-ischemic downregulation of Fto in mice may lead to an elevation of m6A levels.The expression trends of Ythdf1, Ythdf2, and Ythdf3 proteins are generally consistent, indicating the existence of functional redundancy.
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