M6A Modification Regulates LMO7 And Tight Junction Proteins Expression Affecting BSCB Permeability

ObjectiveExplore the function of m6A modification on the regulation of LMO7 and tight junction-related proteins and BSCB permeability after hypoxic injury.MethodsThe BSCB model was established by co-cultivating human brain microvascular endothelial cells with human spinal cord astrocytes.After acute hypoxic injury treatment,the expression of tight junction-related proteins(ZO1,Occludin),methyltransferase Mettl3 and demethylase FTO was measured by WB,RT-qPCR.Differences in m6A modification between the injured and normal groups were detected using the m6A kit.The signaling pathways of m6A modification on the structure and function of BSCB were analyzed by ME-RIP high-throughput sequencing,and the downstream target genes of m6A modification differences normal and after damage were selected.The overexpression FTO gene plasmid and the blank vector plasmid were separately transfected into the BSCB model for hypoxic injury treatment.BSCB permeability was measured in FTO overexpression,blank vector,damage model and normal control groups by horseradish peroxidase(HRP).Target genes and TJ protein expression of each group were determined by WB and RT-qPCR.ResultsAfter hypoxia injury in the BSCB model,ZO1 and Occludin protein expression decreased,Mettl3 protein expression increased,FTO protein expression decreased,the overall m6A modification level increased compared with the normal group(P<0.05).ME-RIPSeq results showed that m6A modification is involved in the regulation of pathophysiological processes such as inflammation,autophagy and cell regeneration in BSCB after injury.LMO7,which can regulate cell adhesion function,was selected among numerous differential genes,as the target genes for m6A modification.HRP results showed that BSCB permeability increased in model group and blank vector group(P<0.05),FTO group was significantly decreased,WB and RT-qPCR showed that The expressions of LMO7,ZO1 and Occludin proteins were significantly increased in the overexpressed FTO group compared with the model group and the blank vector group.(P<0.05).ConclusionsOverexpression of FTO promoted the expression of LMO7 and ZO1,Occludin and other tight junction-related proteins by decreasing the m6A modification level,and reduced the BSCB permeability during the hypoxic injury phase.