Study On The Mechanism Of HnRNP U-Mediated Lead Exposure On REST

Objective:Lead is a non-degradable environmental pollutant that can accumulate in the body and cause damage to multiple systems of the body.Studies have shown that the Restrictor element silencing transcription factor(REST)is a zinc finger protein with transcriptional regulation,which can specifically bind to neuron-restrictive silencer element(NRSE),thus affecting the expression of some special genes.hnRNP U(heterogeneous nuclear ribonucleoprotein U,hnRNP U)is a member of the nuclear heterogeneous ribonucleoprotein family and is involved in transcriptional and post-transcriptional regulation as an RNA-binding protein.Recent studies have shown that hnRNP U specifically binds to REST.Therefore,this study explores the effects of lead exposure on the expression of REST and hnRNP U by establishing animal models and cell models,and provides clues to reveal the mechanism of neurotoxicity caused by lead exposure.Methods:1.Effects of lead exposure on REST and hnRNP U expression in rat hippocampus: pregnant rats were randomLy divided into control group(CON),low lead exposure group(LLG)and high lead exposure group(HLG),CON drinking double steamed water,LLG drinking 0.05% lead acetate aqueous solution,HLG drinking 0.2% lead acetate aqueous solution.Lead exposure was performed using free drinking water until weaning(postnatal 3 weeks).Morris water maze system was used to detect learning and memory ability of rats,ICP-AES was used to detect lead content of whole blood and hippocampus tissue in rat,reverse transcriptase polymerase chain reaction(RT-PCR),western blot(WB)and immunofluorescence(IF)were used to detect the expression of REST and hnRNP U in rat hippocampus.2.The effects of lead exposure on the expression of REST and hnRNP U in PC12 cells: 0 ?M,1 ?M and 100 ?M lead acetate were added to PC12 cells,respectively.The mRNA and protein expressions of REST and hnRNP U in PC12 cells were detected by RT-PCR and WB,the expression of hnRNP U in PC12 cellswas further detected by IF.3.The effect of hnRNP U intervention on REST expression in lead exposure environment: PC12 cells were divided into blank group,control group and experimental group.blank group does not add lead acetate,100 ?M lead acetate was added to the control group and the experimental group.The blank group and control group was transfected with DDW and the experimental group was transfected with hnRNP U-siRNA.RT-PCR was used to detect the mRNA expression of hnRNP U and REST,and IF was used to detect the protein expression of REST.Results:1.Morris water maze experiment: lead exposure can damage the learning and memory ability of rats.(1)Escape latency: Compared with CON,the escape latency of LLG and HLG was significantly prolonged,and the difference was statistically significant(P<0.05).(2)The number of crossing platforms: compared with CON,LLG and HLG was significantly reduced(P<0.05);there was also a significant difference between the LLG and HLG(P<0.05).2.Lead content in whole blood and hippocampus tissues: Lead content was not detected in CON,blood lead and hippocampal lead content in HLG were higher than LLG group,and the difference was statistically significant(P<0.05).3.The effect of lead exposure on REST expression in rat hippocampus:(1)RT-PCR analysis showed that compared with CON,the mRNA expression of REST in hippocampus at LLG and HLG was significantly increased(P<0.05),there was also a significant difference between the LLG and the HLG(P<0.05).(2)WB experiments showed that compared with CON,the expression of REST protein in hippocampus of LLG and HLG was significantly increased(P<0.05),and there was also significant difference between LLG and HLG(P<0.05).(3)IF experiment showed that compared with CON,the protein expression of REST in CA3,CA1 and DG areas in hippocampus of LLG and HLG increased significantly(P<0.05),and there were significant differences between LLG and HLG in DG area(P<0.05).4.The effect of lead exposure on hnRNP U expression in rat hippocampus:(1)RT-PCR showed that there was no significant change in hnRNP U expression levelin LLG and HLG compared with CON(P>0.05).(2)WB experiment showed that there was no significant change in the total hnRNP U protein expression level in the hippocampus of LLG and HLG rats compared with the CON(P>0.05),but the nuclear protein expression level was significantly increased(P<0.05).(3)IF experiment showed that the distribution of hnRNP U nucleus in CA1 area of hippocampus increased,the distribution of cytoplasm decreased and the level of nuclear protein expression increased significantly in LLG and HLG compared with CON(P<0.05),and there were significant differences between LLG and HLG(P<0.05).5.Effects of lead exposure on REST expression in PC12 cells:(1)RT-PCR experiments showed that REST mRNA expression levels were significantly increased in the 1 ?M group and 100 ?M group compared with the 0 ?M group(P<0.05),there were also significant differences between the 1 ?M group and 100 ?M group(P<0.05).(2)WB experiments showed that the REST protein expression levels were significantly increased in the 1 ?M group and the 100 ?M group compared with the 0?M group(P<0.05),there was also a significant difference between the 1 ?M group and 100 ?M group(P<0.05).6.The effect of lead exposure on hnRNP U expression in PC12 cells:(1)RT-PCR assay showed that there was no significant change in hnRNP U expression level in 1?M group and 100 ?M group compared with 0 ?M group(P<0.05).(2)WB experiment showed that there was no significant change in the expression of hnRNP U protein in 1 ?M group and 100 ?M group compared with 0 ?M group(P>0.05).(3)IF experiment showed that the distribution of hnRNP U in nucleus increased significantly,the distribution of cytoplasm decreased significantly and the expression of nuclear protein increased significantly in the 1 ?M group and the 100 ?M group compared with the 0 ?M group(P<0.05),there was significant difference between 1?M group and 100 ?M group(P<0.05).7.Intervention of hnRNP U on REST expression:(1)RT-PCR experiment showed that the expression level of hnRNP U in the control group had no significant change compared with the blank group(P>0.05),and the expression level of hnRNP U in theexperimental group decreased significantly compared with the control group(P<0.05);compared with the blank group,the expression level of REST in the control group increased significantly(P<0.05),and the expression level of REST in the experimental group decreased significantly compared with the control group(P<0.05).(2)IF experiments showed that compared with the blank group,the expression level of REST protein in the control group increased significantly(P<0.05);compared with the control group,the expression level of REST protein in the experimental group decreased significantly(P<0.05).Conclusion:Lead exposure promotes nuclear translocation of hnRNP U,up-regulates REST mRNA expression levels,and increases its protein expression,further affecting the expression of downstream genes regulated by REST,thereby impairing learning and memory.This provides a theoretical basis for exploring the neurotoxic mechanism of lead,and a new idea for finding new drugs to treat lead poisoning.