| ObjectiveN6-methyladenine（m6A）modification can regulate the pathophysiological process and is highly distributed in the brain.CircRNAs play an important role in the disorder of cerebral infarction.However,there are no studies on the role of m6A methylated circRNAs in ischemic stroke.We investigated the potential significance of differentially m6A-methylated circRNAs after cerebral infarction.MethodWe established a mouse model of distal middle cerebral artery occlusion（MCAO）by electric coagulation The mice were divided into three groups:sham surgery,3 days after MCAO（3d）,and 7 days after MCAO（7d）.The expression level of mRNA of METTL3,METTL14,FTO,ALKBH5,YTHDF1 and YTHDF3 were tested by reverse transcription quantitative polymerase chain reaction（RT-qPCR）,ALKBH5 and YTHDF3 proteins were detected by western blot（WB）.Differentially m6A-methylated circRNAs were screened by Methylated RNA immunoprecipitation（MeRIP）and circRNA microarray,and verified by MeRIP-qPCR.Gene Ontology（GO）analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analyses were carried out for differentially m6A methylated circRNAs.Arraystar’s homemade software based on TargetScan and miRanda were used to predict its miRNAs of the three verified circRNAs and target genes of miRNAs.The interactive network was visualized by Cytoscape.Results1.We successfully induced a model of MCAO,and 2,3,5-triphenyltetrazolium chloride（TTC）and HE stain confirmed that the cerebral infarction was limited to the left cerebral cortex.2.The expression of mRNA and proteins of m6A related enzymes were significantly changed compared with the sham group.The mRNA expression of METTL3,METTL14,FTO,ALKBH5,YTHDF1 and YTHDF3 in brain tissue were significantly downregulated in the 3d group.However,compared with the 3d group,there were no significant difference in the mRNA levels of METTL3,METTL 14,and ALKBH5,while the transcriptional levels of FTO,YTHDF1,and YTHDF3 were upregulated in the 7d group.Additionally,the protein levels of ALKBH5 and YTHDF3 were significantly decreased in the 3d group while increased in the 7d group.3.The methylated RNA immunoprecipitation（MeRIP）and circRNA microarray showed a total of 12,345 m6A-methylated circRNAs were found in our research.The m6A methylation was significantly altered in 1706 circRNAs compared to the sham group,including 85 hyper-methylated and 1621 hypo-methylated circRNAs in the 3d group.However,the methylation levels increased in 57 circRNAs and decreased in 66 circRNAs in the 7d group.Among the circRNAs with different m6A methylation levels,most came from exons and a few from overlapping,intergenic,and intron.4.Bioinformatic analysis for parental genes of differentially methylated circRNAs:GO analysis indicated that the biological process,molecular function,and cellular components at 3d group mainly enriched in cell localization,the establishment of cell localization,glutamate receptor,and GTPase activator,while it shifted to the establishment of the blood-brain barrier,protein ubiquitination,ubiquitin ligase complex at 7d group.KEGG showed the transition from hippo,neurotrophic,wnt signal pathway,and axon guidance at the 3d group to phosphatidylinositol signaling pathway,ubiquitin-mediated protein hydrolysis at 7d group.5.The verification results showed that the m6A level of two circRNAs（mmucircRNA27268 and mmucircRNA20673）were significantly hypermethylated and one（mmucircRNA32905）was hypomethylated,which was consistent with the methylated RNA immunoprecipitation（MeRIP）and circRNA microarray.6.CircRNA-miRNA-mRNA interactive analysis:GO showed the biological processes（BP）,cellular components（CC）,and Molecular functions（MF）of target genes were mainly enriched in calcium ion transmembrane transport,voltage gated calcium channel complex,and voltage-gated calcium channel activity,respectively.KEGG analyses showed the pathways of the target genes were mainly enriched in MAPK signal pathway,focal adhesion,axon guidance,and glutamatergic synapse.Conclusion1.Transcriptional level of the methylated transferase,demethylase,and methylated reading protein and protein levels of ALKBH5 and YTHDF3 were changed after cerebral infarction.2.The m6A mehylation level of circRNAs was changed after cerebral infarction,and the differentially m6A-methylated circRNAs may involve in the pathophysiological process of cerebral infarction.3.In this study,we found some m6A-methylated circRNAs associated with cerebral infarction,providing a new direction for the molecular mechanism of cerebral infarction. |
