Mechanism Of MiRNA-351-5p In Aluminum-induced Learning And Memory Impairment Via Targeted Regulation Of CPEB3

Background:The neurotoxicity of aluminum is characterized by impairment of learning and memory,and the decrease of synaptic plasticity is the main mechanism of the impairment,but the molecular mechanism is not clear.The study found that cytoplasmic polyadenylation element binding protein 3（CPEB3）is a new regulator involved in synaptic plasticity and learning and memory.The team previously sequenced the full transcriptome gene in the hippocampus of rats exposed to aluminum,and found that miR-351-5p expression was increased,and its expression was increased in Alzheimer’s disease,affecting synaptic plasticity and learning and memory.Bioinformatics methods predicted the presence of targeted binding sites for miR-351-5p and CPEB3.In this study,the role of miR-351-5p and CPEB3 in learning and memory impairment induced by aluminum will be further investigated.Objective:To explore the mechanism of miRNA-351-5p in aluminum-induced learning and memory impairment through targeted regulation of cytoplasmic polyadenylation element binding protein 3（CPEB3）.Methods:1.SD rats were subchronic intraperitoneally injected with aluminum for 3 months.The ability of learning and memory of rats was tested by Morris Water maze and New object recognition test;the expression of CPEB3 and synaptic plasticity related proteins（PRPs）（PSD95,Glu R1,Glu R2,CREB and BDNF）in hippocampus was tested by Western Blot;The mRNA expression levels of miR-351-5p,CPEB3 and PRPs were detected by q RT-PCR,and the expression levels of CPEB3 protein were detected by immunohistochemistry;Bioinformatics predicts the targeted pairing of miR-351-5p and CPEB3 genes;Double Luciferase reporter Assay verified the targeted binding of miR-351-5p and CPEB3.2.Rat adrenal pheochromocytoma cells（PC12 cells）were cultured and treated with different concentrations Al（mal）₃for different time.The cell survival rate was measured by CCK8 method,and the expression levels of CPEB3 and PRPs were detected by Western blot.The levels of miR-351-5p and CPEB3 mRNA were detected by q RT-PCR.3.Stereotaxic brain intervention experiment of AAV in SD rats:The rats received AAV brain stereotactic injection after one month of intraperitoneal injection of aluminum,then rested for one week and continued to receive aluminum for another two months.Measurement of spatial learning and memory ability in rats with Morris water maze.The expression of CPEB3 and PRPs protein in hippocampus of rats was detected by Western blot,the expression of miR-351-5p and CPEB3 mRNA was detected by q RT-PCR.The transfection of AAV-EGFP in hippocampus was detected by frozen section,the changes of hippocampal neurons were detected by HE staining and NISSL staining,immunohistochemistry and immunofluorescence were used to detect the expression and location of CPEB3 protein in rat hippocampus;electron microscopy was used to detect the ultrastructure of hippocampal neurons;and Golgi’s method was used to detect the changes of dendritic spines in rat hippocampus.Results:1.The results showed that the ability of learning and memory decreased with the increase of aluminum dose,the protein and mRNA expression of CPEB3 and PRPs decreased gradually,while the expression of miR-351-5p increased gradually.Immunohistochemical results showed that CPEB3 protein was mainly expressed in the cytoplasm of rat hippocampal neurons and decreased with the increase of Al concentration.miR-351-5p has a binding site to 3’UTR of CPEB3.There was a targeted binding between miR-351-5p and CPEB3.2.The survival rate of PC12 cells with different concentrations of Al（mal）₃for 24h was more than 80%and there were differences.The time of 24h exposure was determined.The expression levels of CPEB3 protein,mRNA and PRPs protein in infected PC12 cells were lower than those in the control group,while the expression levels of miR-351-5p were higher than that in the control group.3.The green fluorescence of AAV-EGFP was found in hippocampal neurons in frozen sections of rat brain tissue.Compared with the Control group,the learning and memory ability of the rats in the Al（mal）₃+AAV-miR-351-5p inhibitor group was improved,the expression levels of CPEB3,PRPs protein and CPEB3 mRNA in the hippocampus were increased,the expression level of miR-351-5p was decreased,and the structure of hippocampal neurons was improved.The morphological damage of neurons decreased,the expression of CPEB3 protein in hippocampal CA1 region increased,the ultrastructural damage of neurons decreased,the number of synapses increased,and the number and density of dendritic spines in hippocampal CA1 region increased.Conclusion:1.Spatial learning and memory decline in subchronic aluminum-exposed rats.Rno-miR-351-5p targets CPEB3,which is the target gene of miR-351-5p.2.The expression of miR-351-5p was increased and the expression of CPEB3,PRPs and mRNA was decreased in subchronic aluminum-exposed rats and aluminum-exposed PC12 cells.3.Aluminum may modulate the expression of CPEB3 by mediating miR-351-5p,affect the expression of PRPs,induce the decrease of hippocampal synaptic plasticity and impair the ability of learning and memory in rats,miR-351-5p/CPEB3-PRPs pathway may be an important mechanism of aluminum-induced learning and memory impairment.4.Stereotactic cerebral injection of AAV inhibition of miR-351-5p expression in the hippocampal CA1 region can reverse the impairment of spatial learning and memory,decrease the expression of miR-351-5p and increase the expression of CPEB3,PRPs and mRNA in the hippocampus of rats exposed to subchronic aluminum exposure,ameliorating the injury of hippocampal neurons.