MiR-497-5p Regulates Wnt3a/β-catenin/GSK-3β Signaling Pathway And Participates In Learning And Memory Impairment Induced By Aluminum

Objective:To investigate the mechanism of miR-497-5p regulating Wnt3a/β-catenin/GSK-3βsignaling pathway in aluminium-induced learning and memory impairment.Methods:In this study,60 SD rats were randomly divided into four groups:blank control group（normal saline group）,10μmol/kg Al（mal）₃,20μmol/kg Al（mal）₃ and 40μmol/kg Al（mal）₃.Intraperitoneal injection was used every other day for 3 months.After exposure,Morris water maze test was used to detect the learning and memory ability of rats,and RT-qPCR was used to detect the expression of miR-497-5p and Wnt3a genes in hippocampus of rats.The expressions of Wnt3a,β-catenin,GSK-3β,tau and p-tau（Ser396）in rat hippocampus were detected by Western blotting.The changes of the same indexes were further observed in aluminum-stained PC12 cells.Then the targeting relationship between miR-497-5p and Wnt3a was proved by double luciferase gene reporting assay and transfection with miR-497-5p inhibitor.Finally,the expression of miR-497-5p,Wnt3a,β-catenin,GSK-3β,tau and p-tau was observed in aluminium-stained PC12 cells.Results:1.The effect of chronic aluminum exposure on learning and memory ability of rats:from the third to the fifth day,the escape latency of rats in each group was significantly different（P<0.05）;O n the sixth day,there were significant differences in the time of staying in the NE quadrant and the number of crossing the platform among the groups（P<0.05）.2.The effect of chronic aluminum exposure on the expression of miR-497-5p and Wnt3a in the hippocampus of rats:with the increase of aluminum exposure dose,the relative expression of miR-497-5p increased gradually,and the relative expression of Wnt3a decreased gradually,and the differences between groups were statistically significant（P<0.05）.Compared with the control group and the 10μmol/kg Al（mal）₃ group,the 20μmol/kg Al（mal）and 40μmol/kg Al（mal）₃ groups had a significant increase in the relative expression of miR-497-5p and a significant reduction in the relative expression of Wnt3a（P<0.05）.Compared with 20μmol/kg Al（mal）₃ group,the relative expression of miR-497-5p in 40μmol/kg Al（mal）₃ group was increased,and the relative expression of Wnt3a was decreased,and the differences were statistically significant（P<0.05）.3.Effects of chronic aluminum exposure on Wnt3a/β-catenin/GSK-3βpathway proteins in rat hippocampus:The relative expression levels of Wnt3a,β-catenin and p-GSK-3（Ser9）proteins were gradually decreased,and the relative expression level of GSK-3βprotein was gradually increased with the increase of aluminum exposure dose,and the differences between groups were statistically significant（P<0.05）.Compared with the control group,the relative expression of GSK-3βprotein in 10μmol/kg,20μmol/kg and 40μmol/kg Al（mal）₃groups increased by 1.45,1.65 and 1.83 times,respectively,and the differences were statistically significant（P<0.05）.4.The effect of chronic aluminum exposure on the expression of tau/p-tau protein in rat hippocampus:with the increase of aluminum exposure dose,the relative expression of total tau and p-tau（Ser396）protein increased gradually,and the differences among gro ups were statistically significant（P<0.05）.5.The effect of aluminum exposure on miR-497-5p and Wnt3a in PC12 cells:with the increase of aluminum exposure dose,the relative expression of miR-497-5p was gradually increased,and the relative expression of Wnt3a was gradually decreased,and the differences between groups were statistically significant（P<0.05）.6.Effects of aluminum exposure on Wnt3a/β-catenin/GSK-3βpathway proteins in PC12cells:The relative expression levels of Wnt3a,β-catenin and p-GSK-3（Ser9）proteins were gradually decreased,and the relative expression level of GSK-3βprotein was gradually increased with the increase of aluminum exposure dose,and the differences between groups were statistically significant（P<0.05）.Compared with the control group,the relative expression of GSK-3βprotein in the other three groups increased,and the differences were statistically significant（P<0.05）.7.The abnormal phosphorylation of tau p rotein in PC12 cells exposed to aluminum:with the increase of aluminum dose,the relative expression of total tau and p-tau（Ser396）protein increased gradually,and the differences among groups were statistically significant（P<0.05）.8.Compared with NC mimics/r-Wnt3a-3UTR-wt group,the luciferase activity of rno-miR-497-5p/r-Wnt3a-3UTR-wt group was significantly decreased（P<0.05）;Compared with NC mimics/r-Wnt3a-3UTR-mut group,the luciferase activity of Rno-miR-497-5p/r-Wnt3a-3UTR-mut group did not change significantly（P>0.05）.These results indicated that rno-miR-497-5p and r-Wnt3a-3UTR-wt had complementary binding sites.9.After transfection with miR-497-5p inhibitor,there were significant differences between groups（P<0.05）.Compared with the control group,the expression of miR-497-5p in the miR-497-5p inhibitor group decreased,and the expression of Wnt3a increased,and the differences were statistically significant（P<0.05）,indicating that miR-497-5p and Wnt3a had a targeted regulatory relationship.10.Effects of inhibiting miR-497-5p on Wnt3a/β-catenin/GSK-3βpathway proteins in PC12 cells exposed to aluminum:Compared with the control group,the expression levels of Wnt3a,β-catenin and p-GSK-3β（Ser9）protein in the 200μmol/L Al（mal）₃ group were significantly decreased（P<0.05）.Compared with the 200μmol/L Al（mal）₃group,The relative expression levels of Wnt3a,β-catenin,and p-GSK-3β（Ser9）protein in the200μmol/L Al（mal）₃+miR-497-5p inhibitor group were up-regulated by 1.97,1.22,and1.27 times,respectively,and the differences were statistically significant（P<0.05）.There was no significant difference in the relative expression of GSK-3βprotein among the groups（P>0.05）.11.The effect of inhibiting miR-497-5p on the abnormal phosphorylation of tau protein in PC12 cells exposed to aluminum:compared with the control group,the relative expression levels of tau and p-tau（Ser396）protein in 200μmol/L Al（mal）₃ groups increased,and the difference was statistically significant（P<0.05）.Compared with 200μmol/L Al（mal）₃ group,the relative expression of tau and p-tau（Ser396）protein in 200μmol/L Al（mal）₃+miR-497-5p inhibitor group decreased by 32%and 23%,respectively,and the differences were statistically significant（P<0.05）.Conclusion:1.Both animal and cell experiments confirmed that abnormal phosphorylation of tau protein after aluminization was associated with increased expression of miR-497-5p.2.The target gene of miR-497-5p was Wnt3a.3.Wnt3a/β-catenin/GSK-3βsignaling pathway is involved in the abnormal phosphorylation of tau protein induced by aluminum.