Aluminum Exposure Induces The Apoptosis Of PC12 Cells Via MiR-665 Targeting GNB3

Objective:Aluminum exerts neurotoxic effects through various pathways,mainly manifested as cognitive impairment,resulting in decreased learning and memory function.In this study,by confirming the activation of PI3K/AKT signaling pathway by mi R-665 targeting combined with GNB3,we revealed the effect of aluminum exposure on apoptosis of PC12 cells,and the mechanism of neurotoxicity of aluminum was elaborated,providing a new research idea for the prevention and treatment of aluminum toxicity.Methods:1.Rat adrenal pheochromocytoma cells（PC12 cells）were used as study vectors,and PC12 cells were treated with Al（mal）₃at different concentrations of 0μM,100μM,200μM and 400μM（control group,low dose group,medium dose group and high dose group）.Follow-up experiments were performed 24 hours after exposure.CCK8 was used to detect cell viability and determine the concentration of maltol aluminum-stained venom used in subsequent experiments.The apoptosis of PC12 cells was detected by flow cytometry.RT-q PCR was used to detect the relative expression level of RNA,and Western blotting was used to detect the expression level of GNB3/PI3K/AKT signaling pathway and the protein expression level of apoptotic protein Caspase-3.2.The targeted binding relationship between mi R-665 and GNB3 was determined by double luciferase assay in 293T cells.3.PC12 cells were transfected and divided into four groups（Control group,200μM Al（mal）₃groups,200μM Al（mal）₃+inhibitor NC group,200μM Al（mal）₃+mi R-665inhibitor group）.The apoptosis of PC12 cells was detected by flow cytometry.RT-q PCR was used to detect the relative expression level of RNA.Western blotting was used to detect the expression level of GNB3/PI3K/AKT signaling pathway and the protein expression level of apoptotic protein Caspase-3.Results:1.Results of maltol aluminum dose group（1）Cell morphology:With the dose of maltol aluminum increased,the number of cells and intercellular connections decreased,and the cell state deteriorated.（2）Cell viability:Compared with the control group,the cell viability of aluminum maltol at concentrations of 100,200 and 400μM decreased gradually,and the difference was statistically significant in the medium-dose group.（3）Apoptosis:Compared with the control group,With the dose of maltol aluminum increased,the premature and total mortality rates of PC12 cells increased gradually.The expression of Caspase-3 protein,which is related to apoptosis,increased gradually.（4）Cell RNA expression:relative expression level of mi R-665:Compared with the control group,with the dose of maltol aluminum increased,the relative expression level of mi R-665 showed an increasing trend（P<0.05）.Relative expression level of GNB3,Compared with the control group,with the dose of maltol aluminum increased,the relative expression level of GNB3 m RNA showed a downward trend（P<0.05）.（5）Cell protein expression:PI3K/AKT pathway protein expression level:compared with the control group,PI3K/AKT protein expression level decreased with the dose of maltol aluminum increased,and the statistical results is significant（P<0.05）.and the expression level of GNB3 protein showed a downward trend（P<0.05）.2.Double luciferase report experimental resultsCompared with the NC group,rno-mi R-665 significantly down-regulated the luciferase activity of r-GNB3-3’UTR-wt（P<0.001）,indicating that mi R-665 had a targeted binding relationship with GNB3.3.Results of mi R-665 inhibitor transfection experiment（1）Cell morphology:compared with maltol aluminum exposure group,the number of dead cells in transfection inhibitor NC group was slightly increased,but there was no significant difference in cell number and intercellular connectivity.After transfection with mi R-665 inhibitor,the number of cells was increased,and the number of cells was tight and the number of connections was increased.（2）Cell apoptosis:Compared with Control group,the early and total apoptosis rates of Al（mal）₃and Al（mal）₃+inhibitor NC group were increased,while the early and total apoptosis rates of Al（mal）₃+mi R-665 inhibitor group were decreased compared with Al（mal）₃inhibitor group.All the differences were statistically significant（P<0.05）.Compared with the Control group,the expression of apoptosis-related protein Caspase-3in Al（mal）₃group was increased.Compared with Al（mal）₃inhibitor group,the expression of apoptosis-related protein Caspase-3 in Al（mal）₃+mi R-665 inhibitor group was decreased,and the difference was statistically significant（P<0.05）.（3）Cell RNA expression:relative expression of mi R-665:PCR results showed that transfection with mi R-665 inhibitor effectively inhibited the expression of mi R-665.Compared with Control,the relative expression of mi R-665 in Al（mal）₃and Al（mal）₃+inhibitor NC group was increased（P<0.05）.Compared with Al（mal）₃,mi R-665 expression was decreased in Al（mal）₃+mi R-665 inhibitor group（P<0.05）.Relative expression of GNB3:Compared with Control,GNB3 relative expression of Al（mal）₃and Al（mal）₃+inhibitor NC group decreased（P<0.05）.Compared with Al（mal）₃inhibitor,GNB3 relative expression of both groups decreased（P<0.05）.GNB3expression of Al（mal）₃+mi R-665 inhibitor group was increased（P<0.05）.（4）PI3K/AKT pathway protein expression levels:Compared with the Control group,GNB3/PI3K/AKT pathway protein expression levels in Al（mal）₃groups decreased,and the difference was statistically significant.Compared with Al（mal）₃inhibitor group,the expression levels of pathway protein GNB3/PI3K/AKT in Al（mal）₃+mi R-665 inhibitor group were increased,and the difference was statistically significant.Conclusion:By increasing the expression level of mi R-665,aluminum exposure can reduce the expression level of GNB3,and reduce the activity of PI3K/AKT signaling pathway,leading to apoptosis of PC12 cells.This study provides a new idea for exploring the mechanism of neurotoxicity of aluminum.