Expression Changes and Significance of N6-Methyladenosine and its Regulatory Proteins in Cerebral Ischemia
YE Weizhen, ZHAO Shunying, JIANG Mingyu, HUANG Qiuru, WEN Shaohong, DONG Wen, CHEN Qingfang, LIU Xiangrong
2024, 19(6):
655-663.
DOI: 10.3969/j.issn.1673-5765.2024.06.007
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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.