Role Of MiR-380-3p In Paraquat-induced Neurotoxicity And Its Potential Interaction With Nrf2

Part ?.The influence PQ and Nrf2 cast on expression level of mi R-380-3p in Neuro-2a cellsObjective Previously we found that exposure to PQ altered the mi R-380-3p level in mice Substantia nigra,and the mice of wild type Nrf2 seemed to maintain a higher level of mi R-380-3p expression than the Nrf2 knock-out ones.In this part,we treated Neuro-2a cells with PQ or specific activator of Nrf2,so that we can if and how PQ or Nrf2 have an influence on mi R-380-3p abundance in neurons.Methods The Neuro-2a cells were treated to PQ at 100 or 200 ?M and t BHQ,a specific activator of Nrf2,was applied at 40 ?M.By q RT-PCR we measured the mi R-380-3p abundance in the cells after treatments.To locate potential Nrf2 binding site,we searched the 2000 bp upstream of the micro RNA cluster originating mi R-380 by binding-site-motif-based sequence analysis.Results The mi R-380-3p level in Neuro-2a cells was basically decreased by PQ(P<0.05)and increased by activation of Nrf2(P<0.001),which was also consistent with what the gene array analysis had found in our earlier study.The binding-site-motif-based sequence analysis revealed a possible Nrf2 binding site located at about 200 bp upstream of the cluster and the result is bioinformatically reliable(P<0.0001).Conclusion Exposure to PQ downregulates the expression of mi R-380-3p in neurons while transcription fator Nrf2 upregulates the expression of mi R-380-3p.Part ?.The role of mi R-380-3p in PQ-induced nerve cell damageObjective The fuction of mi R-380-3p is hitherto unknown to us,so we decided to look into the infulences mi R-380-3p may have in neurons,besides if and how it's going to get involved in PQ-induced nerve cell damage.Mtehods In this part we conducted transient transfection in Neuro-2a cells with mi R-380-3p mimic or inhibitor,to produce models of nerve cell overexpressing or in depletion of mi R-380-3p.And on these models we applied CCK8 cell vitality detection,Annexin V-FITC/PI staining detection and cell cycle assay to verify the influence mi R-380-3p may have on cell proliferation,apoptosis and cell cycle pregersssion when the cells are exposed to PQ at different at different concentrations.Results Vitality of Neuro-2a cells was whittled by transient transfection of mi R-380-3p mimic(P<0.05),which was also quite invariable through groups exposed to PQ at different doses.And transient transfection of mi R-380-3p mimic also induced a higher apoptosis rate in Neuro-2a cells exposed to PQ at different concentrations(P<0.05).Furthermore,transient transfection of mi R-380-3p mimic kept more cells in S stage(P<0.01)and reduced the proportion of G2/M stage(P<0.001).When the cells were also treated with PQ,mi R-380-3p mimic brought down the scale of G2/M stage(P<0.001)and kept more cells in G0/G1 stage(P<0.001).Anyway,transient transfection of mi R-380-3p inhibitor didn't bring up obvious alterations in those aspects.Conclusion Overexpression of mi R-380-3p reduces vitality of nerve cells and causes apoptosis and cell cycle arrest in them,whereas depletion of mi R-380-3p doesn't seem to cast critical impact.Part ?.The relationship between mi R-380-3p and its target gene Sp3Objective By sequence-based prediction we found that Sp3 might well be a target gene of mi R-380-3p.In this part we sought to verify the mi R-380-3p regulation on Sp3 expression and reveal the mechanism.Methods In this part we adopted qRT-PCR and Western-blot to measure the m RNA level and protein level in mi R-380-3p overexpression models and mi R-380-3p depletion models produced by transient transfection of mi R-380-3p mimic and mi R-380-3p inhibitor.To find the binding site on which mi R-380-3p binds to Sp3 m RNA,we praticed dual luciferase assay to testify the binding site we predicted in sequence-based analysis.Results Abundance of Sp3 m RNA was not altered by transient transfection of mi R-380-3p mimic or inhibitor,whereas the protein level of Sp3 in Neuro-2a cells was downregulated by mi R-380-3p mimic(P<0.05)and upregulated by mi R-380-3p inhibitor(P<0.05).The dual luciferase vector containing original predicted binding site showed a decrease in renilla luciferase expression after overexpression of mi R-380-3p(P<0.05),and mutation in the predicted binding site abolished the alteration induced by mi R-380-3p.Conclusion Sp3 is a target gene of mi R-380-3p and mi R-380-3p is likely to bind to Sp3 m RNA via the predicted binding site.Anyway,mi R-380-3p may not induce degeneration of Sp3 m RNA but block the translation of Sp3 m RNA.