The Alternative Expression Of MicroRNA Induced By Lead Exposure And Its Regulatory Effect On BACE1 And SIRT1

ObjectiveA time series mice model of the early-life lead（Pb）exposure was established to screen and verify the expression changes of specific microRNA and their target genes like SIRT1 and BACE1 at different time points.In order to preliminarily investigate the regulation effect of specific microRNA on relevant genes of of lead-exposed mice.An in-vitro experiments was conducted to explore the regulatory mechanism of specific microRNA on target genes,so as to provide new ideas for the study on the mechanism of the influence of lead on cognitive function from the perspective of epigenetics.MethodsThe C57BL/6 male mice were employed to conduct a free drinking water lead exposure time series model with free access to Pb（Ac）₂.The changes in the expression of SIRT1 and BACE1 genes related to cognitive function after the lead exposure were detected by Western Blot and RT-qPCR at nodes of different ages.Bioinformatics was used to predict the microRNAs that might be related to the target genes,and then the changes of selected microRNAs expression in vivo were detected to explore the correlation between lead exposure,microRNA expression and genes related to learning and memory.In vitro experiments were conducted to observe the expression of specific microRNAs and their target molecules in SH-SY5Y cells with lead exposure.Then an in-vitro model of miR-124-3p activation was constructed to detect the role of miR-124-3p and its targets in the lead-induced nerve injury process.Results1.Effects of lead exposure on the expression of SIRT1 and BACE1 molecules in the brain tissue of aging mice were detected,in the hippocampus and cortex of mice exposured to lead,the BACE1 protein and mRNA levels at age of 4 months and 16months were increased（P<0.05）compared with the control group,while SIRT1 protein levels were decreased at age of 16 months（P<0.05）.2.Bioinformatics was used to predict the microRNAs,TargetScan,miRDB,DIANA,miRand,starBace and other online software were used to screen microRNAs that might be related to SIRT1 and BACE1,and HMDD database was used to find the microRNA related to neurodegeneration and AD.Combined with literature reports,six miRNAs including miR-124-3p,miR-138-5p,miR-34c-3p,miR-29b-3p,miR-135a-5p,miR-141-3p were preliminarily screened for subsequent verification.3.RT-qPCR was used to verify the changes in the expression of six miRNAs in the brain tissues of mice exposured to lead.The results showed that the levels of miR-29b-3p,miR-124-3p,miR-135a-5p and miR-34c-3p in the hippocampal and cortical tissues of mice exposured to lead were remarkably lower than the control groups,whereas the levels of miR-138 were increased compared with the control groups at the age of 4months（P<0.05）.The levels of miR-124-3p and miR-135a-5p in the 16-month-old mice were reduced by lead exposure,while the levels of miR-138-5p and miR-141-3p were increased（P<0.05）.4.In vitro,the expression of miR-29b-3p,miR-124-3p as well as miR-135a-5p were decreased,while the expression of miR-138-5p and miR-141-3p increased（P<0.05）after the SH-SY5Y cells treated 48 hours with Pb（Ac）₂ at the concentrations above 5μM,which was consistent with the expression trend o of mice exposured to lead.5.After transfection with the miR-124-3p mimic in SH-SY5Y cells,the expression of miR-124-3p increased significantly（P<0.001）,and the mRNA and protein expression levels of the corresponding target gene BACE1 decreased.After 6h transfection with miR-124-3p mimic and 25μM of lead for 48h,the BACE1 protein expression level was significantly decreased（q=8.280,P<0.01）and mRNA level was also decreased（q=10.855,P<0.001）.Conclusions1.Lead exposure can induce the differential expression of miR-29b-3p,miR-124-3p,miR-135a-5p,miR-138-5p and miR-141-3p in brain tissues of mice aged 4 months and 16 months,and the miRNA mentioned above have potential targeted regulation effect on the cognitive function related molecules BACE1 and SIRT1 of lead-stained mice..2.Lead exposure inhibited the levels of miR-29b-3p,miR-124-3p and miR-135a-5p in SH-SY5Y cells,and induced the expressions of miR-138-5p and miR-141-3p.Overexpression of the miR-124-3p significantly down-regulated the expression of BACE1 in SH-SY5Y cells exposed to lead.