The Role Of LncRNA LOC680225 Targeting Regulation Of Slc5a7 Through MiR-194-5p In Fluoride-induced Neurotoxicity

Impaired transmission of cholinergic neurotransmitte induced by fluoride is associated with decreased learning and memory ability,but its mechanism has not been fully elucidated.In the cholinergic system,high affinity choline transporter 1(Cht1)is a major rate-limiting factor in the transport of choline to synthesise for acetylcholine(ACh).The dysfunction of ACh synthesis and release caused by abnormal expression of Cht1 is closely related to cognitive deficits.In recent years,studies have found that long non-coding RNA(lncRNA)can not only directly participate in the regulation of gene expression,but also serve as competitive endogenous ribonucleic acid(ceRNA)that interacts with micro RNA(miRNA)to regulate the expression of target genes,which plays an important role in neurotoxicity caused by exogenous substances.Part 1.The role of lncRNA LOC680225 / miR-194-5p /Slc5a7 in fluoride-induced neurotoxicityObjective: To investigate the role of lncRNA LOC680225/miR-194-5p /Slc5a7 regulatory network mediated cholinergic system dysfunction in fluoride induced neurotoxicity,the NaF treated Sprague-Dewley(SD)rat model and the PC12 cell model treated with fluoride and intervention were established.Methods: In vivo experiment: Forty SD female rats(8 weeks old)were randomly divided into control group and NaF groups with the concentration of 10mg/L,50 mg/L and 100 mg/L NaF exposure,10 rats in each group.After 6 months of NaF treatment,Morris Water Maze system was used to detect the learning and memory ability of rats,and the changes of hippocampal neurons were observed by Nissl staining.RNA from hippocampal tissues of rats was extracted for whole transcription sequencing(mRNA,lncRNA and miRNA).Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways enriched analysis of differential genes mediated signaling pathways,screened out target genes.The target lncRNA interacting with target gene was screened out by bioinformatics analysis.The co-binding miRNA between target lncRNA and target gene was screened by software according to ceRNA regulation mechanisms.Quantitative real-time polymerase chain reaction(qRT-PCR)was further used to verify the expression of target gene,target lncRNA and miRNA in the hippocampus of rats,and the mRNA expression of choline acetyltransferase(ChAT),acetylcholinesterase(AChE),M1 muscarinic acetylcholine receptor(M1mAChR),M3 muscarinic acetylcholine receptor(M3mAChR)and α7nicotinic acetylcholine receptor(α7nAChR)in rat hippocampus were also detected by qRT-PCR.The protein expressions of protein encoded by target gene,ChAT,and AChE were detected by western blot,and the content of ACh in serum was detected by enzyme-linked immunosorbent assay(ELISA).In vitro experiment: PC12 cells were divided into control group and NaF(20,40 and 80 mg/L NaF)exposure group for 24 h.The effect of fluoride on synaptic linkage of PC12 cells was detected by scanning electron microscopy(SEM),the content of ACh in the supernatant of PC12 cells was detected by ELISA.qRT-PCR was used to detect the RNA expression of target gene,target lncRNA,target miRNA,ChAT,AChE,post-synaptic density protein-95(PSD-95),synapsin-1,M1 mAChR,M3 mAChR and α7nAChR.The protein expression level of protein encoded by target gene,ChAT,AChE,PSD-95,synapsin-1 and cleaved cysteinyl aspartate specific proteinase 3(cleaved caspase 3)were detected by western blot.The distribution and expression of protein encoded by target gene,target lncRNA and target miRNA in PC12 cells were detected by fluorescence in situ hybridization(FISH).PC12 cells were treated with miRNA mimic,miRNA inhibitor and adenovirus expressing target lncRNA(Ad-target lncRNA).The protein expression level of Cht1 and cleaved caspase 3 and cell viability were detected.Double luciferase reporter assay was used to verified the combination of target miRNA with target lncRNA and target gene.Results: In vivo experiment: compared with the control group,the times of crossing the platform and the ratio of time staying in target quadrant to total time in NaF group were significantly decreased(P < 0.05).The results of Nissl staining showed that the hippocampal neurons in the control group were closely arranged and the number of Nissl bodies in the cytoplasm was abundant.However,with increasing NaF exposure the arrangement of neurons was gradually loosened,the color of Nissl bodies became lighter and the number was decreased.Transcriptome sequencing results showed that 7 mRNAs were up-regulated and 37 mRNAs were down-regulated in rat hippocampus due to fluoride exposure;KEGG pathway enrichment analysis showed that cholinergic synapse involved in the differential genes were associated with neurotoxicity.We found that the down-regulated gene solute carrier family 5member 7(Slc5a7)encodes Cht1.LncRNA sequencing results showed that 3lncRNAs were up-regulated and 13 lncRNAs were down-regulated due to fluoride exposure;the lncRNA interacting with Slc5a7 was found to be LOC680225.miRNA sequencing results showed that 42 miRNAs were up-regulated and 9 miRNAs were down-regulated due to fluoride exposure;the co-binding miRNAs between LOC680225 and Slc5a7 were rno-miR-194-5p and rno-miR-511-3p;it was further found that miR-194-5p had the same sequence in human and rats,so we screened miR-194-5p for further study.qRT-PCR results showed that the RNA expression levels of LOC680225,Slc5a7,ChAT,M1 mAChR,M3 mAChR and α7nAChR were decreased with increasing NaF exposure,miR-194-5p and mRNA expression levels of AChE were increased with increasing NaF exposure(P < 0.05).Western blot results showed that the expression levels of Cht1 and ChAT were decreased with increasing NaF exposure,the protein expression level of AChE was increased(P < 0.05).With increasing NaF exposure,the content of ACh in serum of rats was decreased(P <0.01).In vitro experiment: The results of SEM showed that with increasing NaF exposure,the number of dendritic spine was decreased,the synaptic connections between PC12 cells were decreased and broken,and the cell became shinking.With increasing NaF exposure,the content of ACh in supernatant of PC12 cells was decreased.qRT-PCR results showed that the RNA expression levels of LOC680225,Slc5a7,ChAT,M1 mAChR,M3 mAChR and α7nAChR were decreased,miR-194-5p and mRNA expression levels of AChE were increased with increasing NaF exposure(P < 0.05).Western blot results showed that the expression levels of Cht1 and ChAT were decreased,and the protein expression level of AChE and cleaved csapase-3 were increased with increasing NaF exposure(P < 0.05).In addition,the protein and mRNA expression levels of PSD-95 and synapsin-1 were decreased with increasing NaF exposure(P < 0.05).FISH results showed that Cht1 and LOC680225 were distributed both in cytoplasm and nucleus,mainly in cytoplasm,and miR-194-5p was evenly distributed in cytoplasm and nucleus.Compared with control group,the expression of miR-194-5p was increased,and the expression of Cht1 was significantly decreased in miR-194-5p mimic group(P < 0.05),miR-194-5p expression was significantly decreased,the expression of Cht1 was significantly increased in miR-194-5p inhibitor group(P < 0.05);the expression of LOC680225 and Cht1 were increased in Ad-LOC680225 group(P < 0.05).Compared with the NaF group,the expression of Cht1 was decreased,the expression of cleaved caspase3 was increased and cell viability was decreased in miR-194-5P mimic+NaF group(P < 0.05);the expression of Cht1 was increased,the expression of cleaved caspase3 was decreased and cell viability was increased in miR-194-5p inhibitor+NaF group(P < 0.05);the expression of Cht1 was increased,the expression of cleaved caspase3 was decreased and cell viability was increased in Ad-LOC680225 +NaF group(P < 0.05).Double luciferase reporter assay results showed that miR-194-5p could targeted bind to LOC680225 and Slc5a7.Conclusions: Fluoride exposure can reduce the expression of LOC680225,increase the expression of miR-194-5p and decrease the expression of Slc5a7 in the hippocampus of SD rats and PC12 cells,leading to dysfunction of the cholinergic system and ultimately neurotoxicity.LOC680225,founction as ceRNA,mediates the role of cholinergic system dysfunction in fluoride neurotoxicity by competitively binding to miR-194-5p and co-regulating Slc5a7 expression.Part 2.Association analysis between low-to-moderate fluoride exposure,cholinergic system and intelligence in childrenIn the first part of the study,we found that the cholinergic system is involved in the development of fluoride neurotoxicity,lncRNA LOC680225 and miR-194-5p were involved.A large number of epidemiological studies have shown that the Intelligence Quotients(IQ)of children in high-fluoride exposure areas is significantly lower than that in the corresponding low-fluoride areas,but the mechanism of the reduction in IQ caused by fluoride has not been clarified.Objectives: In this cross-sectional study,we investigated the relationship between low-to-moderate fluoride exposure,cholinergic system and Intelligence Quotients(IQ)in children.Methods: 329 and 380 school children aged 7～13 years were selected from fluorosis areas(water fluoride concentration > 1.0 mg/L)and control areas(water fluoride concentration ≤ 1.0 mg/L)by multistage random sampling method of Baodi District in Tianjin.Spot urine and peripheral blood samples were collected,and fluoride ion selective electrode method was used to determine the content of fluoride ion in drinking water and urine.Combined Raven test was used to measure IQ score of the children.ChAT,Cht1,AChE and ACh levels were measured by ELISA.Multiple linear regression and logistic regression models were used to evaluate the relationship between low-to-moderate fluoride exposure,cholinergic system and IQ.Mediating effect analysis was used to explore the mediating effects of cholinergic system in the relationship between fluoride exposure and IQ in children.Results: Multiple linear regression analysis showed that the concentration of fluoride in water was negatively association with the content of ChAT,Cht1 and ACh in serum of children,and positively association with the concentration of AChE.There was a dose-response relationship between the increase of fluoride concentration in water and the decrease of ChAT,Cht1,ACh and increase of AChE concentrations in serum(P for trend were 0.008,<0.001,<0.001,0.017).In adjusted models,urinary fluoride concentration was negatively association with the content of ChAT and Cht1,and positively association with the concentration of AChE.There was a dose-response relationship between the increase of urinary fluoride concentration and the decrease of ChAT,Cht1 and increase of AChE concentrations in serum(P for trend were 0.013,< 0.001,0.013).The correlation between water fluoride and children’s IQ scores was negative,so was urinary fluoride.Logistic regression analysis showed that with every 1 mg/L increase of fluoride concentration in water fluoride,the probability of children having dull normal and below IQ was increased by 43%(OR = 1.43,95% CI: 1.08,1.88),and the probability of children having excellent IQ level was decreased by 49%(95% CI: 0.12,0.70).For each 1 mg/L increase of urinary fluoride concentration,the risk of having excellent IQ level was decreased by 66%(95% CI: 0.15,0.86).Multiple linear regression showed that children’s IQ score was negatively associated with the concentration of AChE.Compared with the children in the reference group of AChE concentration(≤ 133.66nmol/L),those in the fourth quartile(≥ 184.03 nmol/L)were 2.25-fold more likely to have dull normal and below IQ level(OR = 3.25,95% CI: 1.22,8.70),and 87% less likely to have excellent IQ level(95% CI: 0.02,0.72).Mediation analysis showed that the proportion of mediation by AChE between water fluoride and IQ scores were 6.8%(95% CI: 2.2%,18.9%),and 7.7%(95% CI: 2.6%,20.5%)between urinary fluoride and IQ scores.Conclusions: Low-to-moderate fluoride exposure was associated with the disorder of cholinergic system and lower IQ scores in children,and AChE was associated with lower odds of children having excellent IQ level.AChE partly mediated the association between fluoride exposure and the decrease of IQ scores.