Mechanism Of MiR-1a-3p/Twinfilin 1 In Lung Injury Induced By Exposure To Tire Wear Particles Inhalation

BackgroundWith the continuous increase in the number of motor vehicles and the gradual replacement of fossil fuel-powered vehicles by new energy vehicles,the composition of particulate matter from transportation sources is also changing dramatically.The content of Tire wear Particles（TWP）in the environment is increasing year by year.However,the current research on tire wear particles mainly focuses on the ocean,fresh water and other water bodies,and the atmosphere is an important way of transmission of tire wear particles,and its impact on health is still lack of clear evidence.ObjectiveAt present,the influence of tire wear particles in the atmospheric environment may have been included in the morbidity and mortality caused by total particulate matter(PM₁₀,PM_2.5)pollution.The purpose of this study was to investigate the respiratory toxicity induced by inhalation exposure of TWP and the role of epigenetic mechanisms in this process.Methods1.The tires are cleaned and made of TWP for experiments by filing grinding,liquid nitrogen quick-freezing,grinding instrument grinding,ultrasonic dispersion,and filter filtration,and the physical and chemical properties of the particles are determined for follow-up research.40 7-8 weeks old C57BL/6 mice,male and female,were randomly divided into control groups and low（0.125 mg/kg）,medium（0.5 mg/kg）,high（1 mg/kg）exposure groups;the aerosol produced by TWP was evenly dispersed into the lungs of mice using a handheld liquid aerosol lung delivery.2.Lung tissue was ground,total RNA was extracted by Trizol method,miRNA sequencing was performed and downstream targeted mrnas were predicted.The direct binding site between miRNA and m RNA was verified by dual luciferase reporter gene.Quantitative real-time PCR was used to detect the expression levels of related genes.At the cell level,after transfection with miRNA mimic/inhibitor,real-time quantitative PCR and western blot were used to investigate the regulation of miRNA on its target gene Twinfilin1 at RNA and protein levels,respectively.3.The expression of E-cadherin in mouse lung tissue and Twinfilin 1 in human bronchial epithelial cells（BEAS-2B）treated with TWP were detected by Immunofluorescence（IF）staining.The change of migration function of BEAS-2B cells after TWP treatment was detected by cell scratch assay.The morphological changes of cytoskeleton after TWP treatment were observed by Phalloidin staining.Results1.In dry powder state,tire wear particles are long and flocculent,with sharp edges.The hydrated particle size dispersed in the solution is concentrated at 100 nm,and the Zeta potential is-18.41 m V,which may agglomerate into larger particles suspended in the air.2.In TWP-infected mice,PEF,FVC and FEV_0.1 were significantly down-regulated,while FEV_0.1/FVC had no significant change.In the exposed group,obvious inflammatory cell infiltration,lung parenchymal area enlargement and abnormal accumulation of collagen secretion were observed.This suggested restrictive ventilatory dysfunction,lung injury,and fibrotic pathologic changes in TWP exposed mice.3.miRNA sequencing results suggested that the down-regulation of miR-1a-3p was involved in TWP-induced lung injury.4.It was predicted by Targetscan,miRDB and Starbase database that Twinfilin 1 was highly expressed in the targeted m RNA of miR-1a-3p,and there was a significant negative correlation between Twinfilin 1 and Twinfilin 1.5.Dual luciferase reporter assay confirmed the existence of targeted binding sites between cytoskeleton regulatory gene Twinfilin 1 and miR-1a-3p.Cell transfection,immunofluorescence and cell scratches further confirmed that miR-1a-3p/Twinfilin 1involved in cytoskeletal rearrangement plays an important regulatory role in lung injury induced by TWP exposure.ConclusionsExposure to tire wear particles by inhalation can lead to restrictive ventilatory dysfunction and fibrotic pathologic changes in mice.During this process,miR-1a-3p inhibits F-actin formation by targeting the cytoskeletal regulatory protein Twinfilin 1,which leads to fibrotic lung injury.Our study describes a possible mechanism by which epigenetics is involved in the regulation of lung injury induced by tire wear particles,a finding that may lead to a better understanding of the key mechanisms underlying the inhalation toxicity of tire wear particles.