MiR-34b-3p Inhibition Of Eukaryotic Translation Initiation Factor 4E Causes Post Stroke Depression In Adult Mice

Background:Post-stroke depression(PSD)is a common complication after stroke,which seriously affects patients’ recovery and quality of life after stroke.The clinical features of post-stroke depression include depressed mood,diminished interest,delayed thinking,and sleep disturbances.Depression seriously affects the treatment motivation,compliance and self-rehabilitation,and this vicious circle increases the disability and mortality rates in turn.The treatment for PSD is based on symptomatic supportive therapies,and there is no effective therapeutic drug available so far.Current theories on the pathogenesis of PSD involves neuroanatomy,neurotransmitters,neuroendocrine,inflammatory response,neurotrophic factors,neuropeptides,and psychosocial factors.Epigenetics is strongly associated with the occurrence of PSD,mainly through histone modification,DNA methylation and post-transcriptional regulation of miRNAs to regulate the development of ischemic stroke.MicroRNAs(miRNAs),a major class of non-coding RNAs with a length of about 22 nucleotides,are incorporated into the RNA-induced silencing complex(RISC)and bind to the 3’-UTR of the target mRNA to induce downregulation of target transcript expression through translational repression or mRNA degradation,ultimately regulating gene expression and participating in important biological processes such as apoptosis,differentiation,neurodevelopment,cell proliferation and other life activities by acting on corresponding target genes.MiRNAs are widely expressed in the brain and play a key role in the pathophysiology of various neurological diseases such as stroke,schizophrenia and depression.Objective:We established a mouse model of post-stroke depression,take the miRNAs as an entry point.Then,based on the results of transcriptome sequencing to analysis the molecular mechanism of miRNA involved in post-stroke depression.Methods:Photothrombotic-induced focal ischemic stroke or sham surgery were performed on 3-month-old adult male C57 mice.4 weeks after operation,we screened mice with poststroke depression-like(PSD)behavioral phenotype and non-post-stroke depression(NPSD)by elevated plus maze test,forced swimming test,open field test,tail suspension test,and unsupervised fuzzy clustering analysis.The location of secondary lesions in the brain of PSD mice was determined by positron emission tomography(PET).The hippocampus tissues of mice in the control group,PSD group and NPSD group were isolated for RNA sequencing and analysis the differentially expressed miRNAs,then the differentially expressed miRNAs were confirmed by qPCR.The target miRNA target genes were predicted and scored by the bioinformatics analysis method,and the target genes were determined by combining gene function enrichment analysis,western blotting experiments,immunofluorescence staining experiments and dual luciferase experiments.Adenoassociated virus(AAV2/9-eIF4E-GFP and AAV2/9-sh-eIF4E-GFP,etc.)were used to regulate the expression of eukaryotic translation initiation factor 4E(eIF4E)in the hippocampus of NPSD and PSD mice,respectively.To clarify the regulatory role of eIF4E in depressive behavior in post-stroke mice.We used a combinated techniques of whole-cell patch-clamp electrophysiological techniques,immunofluorescence experiments and qPCR experiments to further uncover the role of eIF4E on neurons and inflammatory cells in the pathogenesis of PSD.Results:PET imaging of post-stroke depression-like phenotype(PSD)and post-stroke depression-like phenotype(NPSD)and sham mice confirmed the secondary lesions of hippocampus in PSD mice.RNA sequencing analysis of hippocampus tissue found 19 differentially expressed miRNAs and the differentially expressed miRNA,miR-34b-3p,was confirmed by qPCR.The target gene of miR-34b-3p was predicted and scored by bioinformatics analysis method,and the target gene eIF4E was determined by combining gene function enrichment analysis,western blotting experiment,dual luciferase assay and immunofluorescence staining experiment.PSD-like behavior was observed in NPSD mice after overexpression of eIF4E in hippocampal neurons with adeno-associated virus.Depression-like behavior was improved in PSD mice after inhibition of eIF4E in hippocampal neurons with adeno-associated virus.Patch-clamp electrophysiological techniques,immunofluorescence experiments and qPCR experiments were used to further clarify that eIF4E induces PSD by activating microglia to increase the secretion of IL-1,IL6 and TNF-α,causing inflammatory responses.Conclusion:Downregulation of neuronal miR34b-3p in the hippocampus led to a increased expression of its target gene eIF4E,and induced neuroinflammation by microglia activation.Down-regulation of eIF4E in hippocampal neurons inhibited microglia activation,alleviated neuroinflammation,and ameliorates PSD-like symptoms.