The Role Of CPEB3 Regulated By MiRNA-351-5p In Aluminum-induced Synaptic Structure Plasticity In Rat Neurons

Objective:1.To investigate the effects of morphology and the expression of miR-351-5p,CPEB3 and synaptic plasticity related proteins after rat cortical neurons and rat adrenal gland pheochromocytoma（PC12 cells）exposed to aluminum maltolate.2.To investigate the regulation mechanism of miR-351-5p on the expression of CPEB3 during the neuronal cell synaptic structure plasticity impairment induced by aluminum maltolate through establishing a low expression of miR-351-5p cell model.Methods:1.The cortex of newborn SD rats（≤24h）was cultured for primary neurons.Primary neurons were exposed to different doses of Al（mal）₃and including control group,10μmol/L,20μmol/L and 40μmol/L Al（mal）₃groups.After 24h exposure,Neuron-specific classⅢβ-tubulin was used to detect neuron purity by immunohistochemical assay;CCK-8 method was used to measure cell viability;real-time fluorescent quantitative RT-PCR method to detect the expression of miR-351-5p and CPEB3 m RNA.The high-content analysis was used to detect the morphological changes of neurons,CPEB3 and the fluorescence intensity of GluR1,GluR2,and PSD95.2.The PC12 cells was cultured and treated with different doses of Al（mal）₃including the control group,100μmol/L,200μmol/L and 400μmol/L Al（mal）₃groups.After 24h exposure,CCK-8 was taken to assay cell viability;RT-PCR was applied to evaluate the expression of miR-351-5p and CPEB3 m RNA;Western blot was employed to evaluate the expression of CPEB3,GluR1,GluR2 and PSD95.3.The lentiviral infection technique was used to transfect miR-351-5p inhibitor into PC12 cells,including blank control group,200μmol/L Al（mal）₃group,the negative transfection group,the negative transfection+200μmol/L Al（mal）₃group,the miR-351-5p inhibitor transfection group and the miR-351-5p inhibitor transfection+200μmol/L Al（mal）₃groups.The expression of miR-351-5p and CPEB3 m RNA were evaluated by RT-PCR;the level of CPEB3,GluR1,GluR2,and PSD95 were detected by Western blot;the Luciferase reporter assay was employed to find out the targeting relationship.Results:1.The results of rat cortical neuronsThe results of immunohistochemical assay showed that,the purity of neurons reaching 96%.Compared with the control group,the expression level of miR-351-5p increased in the 10μmol/L Al（mal）₃,20μmol/L Al（mal）₃and 40μmol/L Al（mal）₃groups（P<0.05）;Compared with the group,the expression level of CPEB3 m RNA in the10μmol/L Al（mal）₃group was no statistical difference（P>0.05）,and the CPEB3expression level in the 20μmol/L Al（mal）₃and 40μmol/L Al（mal）₃groups decreased（P<0.05）.The high-content analysis indicated that,compared with the control group,the number of cells were no statistical difference in the 10μmol/L Al（mal）₃and 20μmol/L Al（mal）₃groups（P>0.05）,and the amount of cells in 40μmol/L Al（mal）₃group was lessen（P<0.05）.The mean outgrowth per cell in the 10μmol/L Al（mal）₃group was no obvious difference（P>0.05）.The mean outgrowth per cell of the 20μmol/L Al（mal）₃and40μmol/L Al（mal）₃groups was shortened（P<0.05）;there was no significant change in the mean processes per cell in the 10μmol/L Al（mal）₃group（P>0.05）,but the mean processes per cell in the 20μmol/L Al（mal）₃and 40μmol/L Al（mal）₃groups decreased（P<0.05）.The mean branches per cell in the 10μmol/L Al（mal）₃group did not change significantly（P>0.05）,and the mean branches per cell in the 20μmol/L Al（mal）₃and40μmol/L Al（mal）₃groups were decreased（P<0.05）.The high-content analysis demonstrated that,compared with the control group,the expression of CPEB3,GluR2and PSD95 were decreased in the 10μmol/L Al（mal）₃,20μmol/L Al（mal）₃and 40μmol/L Al（mal）₃groups（P<0.05）;compared with the control group,the intensity of GluR1 in the 10μmol/L Al（mal）₃groups no significant change（P>0.05）,but there were statistically significant in both the 20μmol/L Al（mal）₃and 40μmol/L Al（mal）₃groups（P<0.05）.2.The results of PC12 cell exposed to aluminum maltolateRT-PCR results demonstrated that,compared with the control group,the expression level of miR-351-5p was increased in 100μmol/L Al（mal）₃,200μmol/L Al（mal）₃and400μmol/L Al（mal）₃groups（P<0.05）;compared with the control group,the expression level of CPEB3 m RNA in the 100μmol/L Al（mal）₃,200μmol/L Al（mal）₃and 400μmol/L Al（mal）₃was decreased（P<0.05）.Western blot results displayed that,compared with the control group,the expression levels of CPEB3 and PSD95 in the 100μmol/L Al（mal）₃group had no significant changes（P>0.05）,the expression levels of CPEB3 and PSD95in the 200μmol/L Al（mal）₃and 400μmol/L Al（mal）₃groups were decreased（P<0.05）;compared with the control group,the expression levels of GluR1 and GluR2 were decreased in 100μmol/L Al（mal）₃,200μmol/L Al（mal）₃and 400μmol/L Al（mal）₃group（P<0.05）.3.The results of transfection in PC12 cellAfter PC12 cells were transfected with miR-351-5p inhibitor,it was found that,compared with the blank group,the expression of both CPEB3 m RNA and CPEB3 in the negative transfection group were no significant change（P>0.05）.The expression of CPEB3 m RNA and CPEB3 were increased in the miR-351-5p inhibitor transfection group（P<0.05）;compared with 200μmol/L Al（mal）₃group,the expression of CPEB3m RNA and CPEB3 was no significant change in negative transfection+200μmol/L Al（mal）₃group（P>0.05）,however,there were both increased in the miRNA-351-5p inhibitor transfection+200μmol/L Al（mal）₃group（P<0.05）.The results of dual luciferase reporter gene experiment showed that,miR-351-5p and CPEB3 was targeted binding.Compared with the blank group,the expression of GluR1,GluR2 and PSD95 in the negative transfection group did not change significantly（P>0.05）,and the expression of GluR1,GluR2 and PSD95 in the miR-351-5p inhibitor transfection group were increased（P<0.05）;Compared with the 200μmol/L Al（mal）₃group,the negative transfection+200μmol/L Al（mal）₃group GluR1,GluR2 and PSD95 expression did not change significantly（P>0.05）,the expression of GluR1,GluR2 and PSD95 were both increased in the miRNA-351-5p inhibitor transfection+200μmol/L Al（mal）₃group（P<0.05）.Conclusion:1.Aluminum maltolate damages the synaptic structure plasticity of rat cortical neurons and PC12 cells.2.miR-351-5p reduces the damage of the synaptic structure plasticity of PC12 cells induced by aluminum via targeting to increase CPEB3.