The Regulatory Mechanism Of Exosomal MiR-4687-5p In Alleviating Silica-induced EMT Of Lung Epithelial Cells

Objective:Silicosis is an interstitial lung disease caused by long-term exposure to silica dust,and is characterized by silicotic nodules and diffuse pulmonary fibrosis.Macrophages-derived exosomes play an important role in the development of silicosis based on existing studies.And micro RNA（mi RNA）,a common cargo of exosomes has received much attention in the study on pulmonary fibrosis diseases.Epithelial-mesenchymal transition（EMT）is a process by which epithelial cells acquire mesenchymal cell properties and plays an important role in fibrotic disease.It has been found that exosomes might play a role in regulating fibrotic diseases by carrying mi RNAs and regulating EMT signaling pathways.This study aimed to investigate the role of exosomal mi R-4687-5p in silicosis and lung epithelial cell injury induced by Si O₂.The delayed effect of mi R-4687-5p by regulating wnt/β-catenin signaling pathway on EMT process in epithelial cells was checked.Methods:In this study,the low expression of mi R-4687-5p in the peripheral blood serum exosomes of silicosis patients was screened out by performing transcriptome sequencing as well as bioinformatics analysis.Realtime-PCR was used to double check the expression of those screened mi RNAs in Si O₂ treated macrophages in vitro.To demonstrate the inhibitory effect of macrophages derived exosomal mi R-4687-5p on the EMT phenotype of epithelials,the cultured supernatant of THP-1 with different treatments was added into A549.And the levels of EMT-related factors were detected by Realtime-PCR and Western blot.To further identify the regulatory role of mi R-4687-5p on the EMT process of A549,mimic-mi R-4687-5p was applied to overexpress mi R-4687-5p.Western blot,Realtime-PCR,and fluorescence staining experiments were used to check the effect of mi R-4687-5p on wnt/β-catenin signaling pathway.Results:1.Mi R-4687-5p expression levels were down-regulated in the serum exosomes of silicosis patients.A significantly down-regulated mi RNA,mi R-4687-5p,was screened by whole-transcriptome sequencing.The expression level of this mi RNA in macrophages was detected by realtime-PCR.The results showed that Si O₂ treatment significantly inhibited the expression level of mi R-4687-5p in macrophages.2.Macrophage-derived exosomes inhibit the progression of EMT induced by Si O₂ in lung epithelial cells.The human macrophage line THP-1 was first treated with DMSO and GW4869,then the cells and supernatant were collected by stimulating the cells with Si O₂,co-cultured with A549 lung epithelial cells for 24h,and the cells and supernatant were collected for use.The EMT-related markers as well as mi R-4687-5p expression levels were detected by westrn-blot technique and realtime PCR.It was found that mi R-4687-5p expression was elevated in the Si O₂ group,EMT markers E-cadherin expression was elevated and Vimentin expression was decreased.The expression of mi R-4687-5p was significantly decreased in the Si O₂ group after exosome inhibition,the expression of EMT marker E-cadherin was significantly decreased,and the expression of Vimentin was significantly increased.The differences were statistically significant（P<0.05）.3.Mi R-4687-5p can regulateβ-catenin to suppress the EMT phenotype in pulmonary lung epithelial cells induced by Si O₂.Lung epithelial cell line A549 was treated separately and divided into the saline group,Si O₂ group,mimic-NC Si O₂group and Si O₂+mimic 4687-5p group.Mi R-4687-5p direct target gene was predicted by Targetscan,mi RDB and mi RTa Base bioinformatics to beβ-catenin,and mi R-4687-5p can bind toβ-catenin by double luciferase.A significant decrease inβ-catenin entry into the nucleus was observed in the effect of mi R-4687-5p by immunofluorescence staining technique.Changes in mi R-4687-5p,CTNNB1 and EMT-related markers in epithelial cells in Realtime-PCR.It was found that the mi R-4687-5p level was significantly decreased,CTNNB1 expression level was significantly decreased,the Vimentin expression level was significantly increased and the E-cadherin expression level was significantly decreased in the Si O₂ group.When mi R-4687-5p was overexpressed,mi R-4687-5p levels increased,CTNNB1 and E-cadherin expression levels increased significantly,and Vimentin expression levels decreased significantly in the Si O₂+mimic 4687-5p group.At the protein level,the total protein level ofβ-catenin decreased significantly,the expression level of Vimentin increased significantly,and the expression level of E-cadherin decreased significantly in the Si O₂ group;when overexpressing mi R-4687-5p,the expression ofβ-catenin and E-cadherin levels were significantly increased and Vimentin expression levels were significantly decreased in the Si O₂+mimic 4687-5p group（P<0.05）.4.Mi R-4687-5p/β-catenin regulatory axis inhibits the migration of pulmonary epithelial cells induced by Si O₂.Using the scratch test,it was observed that the migration ability of A549 was significantly enhanced in the Si O₂ group compared to the saline group,whereas the migration ability of A549 was significantly reduced in the Si O₂+mimic 4687-5p group（P<0.05）.Conclusions:Mi R-4687-5p expression was significantly downregulated in serum exosomes from silicosis patients.Overexpression of mi R-4687-5p delayed Si O₂-induced EMT phenotype in lung epithelial cells by inhibitingβ-catenin.