| Objective:To investigate the role of exosome-derived miRNA-21-5p in the epithelialmesenchymal transdifferentiation of dust-induced rat lung tissue.Method:Sixty healthy male SD rats were randomly divided into control group,silica group,and coal dust group according to body weight,with 20 rats in each group.First,we injected 1 ml of coal dust suspension(50mg/ml)and 1ml of silica suspension(50 mg/ml)into the trachea of rats in the silica group and coal dust group by non-exposed endotracheal contamination,and injected the same volume of normal saline into the trachea of rats in the control group.After 4 weeks and 16 weeks of molding,10 rats were sacrificed in each group.The left lung lobe was fixed in paraformaldehyde,the pathological changes of the lung were observed,and the remaining lung tissue was frozen in the-80 °C refrigerator for backup.Lung histopathological changes in rats after dust contamination were detected by HE and Masson staining,and the hydroxyproline(HYP)content reflected the deposition of collagen in the lungs.Exosomes were extracted by kit method,morphological identification by transmission electron microscopy(TEM),nanotracking analysis(NTA)to detect the particle size of exosomes,and western blot to detect exosome marker proteins.In order to observe the correlation between exosome-derived miRNA-21-5p and EMT in rat alveolar lavage fluid after silica dust and coal dust exposure,we used RT-PCR and Westernblot to detect exosomederived miR-21-5p gene levels and EMT protein in lung tissue,identified smad7 as the target gene of miR-21-5p by double luciferase reporter gene experiment.We used Westernblot to detect the effect of dust on the molecular protein levels of the Smad7/Snail pathway.Results:1.Detection results of pulmonary fibrosis in a rat model of pneumoconiosis(1)HE and Masson results showed that HE staining showed that the alveolar structure of the control group was intact,and there was no obvious infiltration of inflammatory cells.When the dust was stained for 4 weeks,round or irregular coal dust fiber stoves were visible in the coal dust group,and obvious coal silicon nodules could be seen at 16 weeks.At 16 weeks of dust contamination,the alveolar wall of the silica group thickened,and there were more pronounced silicon nodules.Masson staining showed an increase in blue-stained collagen fibers at 16 weeks compared to 4weeks,and the degree of pulmonary fibrosis was aggravated.(2)HYP content in lung tissue: The results showed that compared with the control group,the expression of HYP in the lung tissues of rats in the silica group and the coal dust group was increased(P<0.05),and the content of hydroxyproline in the lung tissues of rats in the coal dust group was not significantly different from that in the silica group compared with the silica group(P>0.05).2.Exosome-derived miR-21-5p detection results in pneumoconiosis rats(1)Exosome identification results: transmission electron microscopy(TEM)observed the teatray-like structure scattered in the visible field of view in the middle depression with a diameter range of 50-140 nm.Nanotracking analysis(NTA)showed that the average particle size of exosomes was 95.8 nm and the concentration was 4.7E+10/m L.Western Blot results showed that the expression of exosome marker proteins CD9 and CD63 could be detected.(2)expression of miR-21-5p: RT-PCR test results showed that dust contamination could lead to upregulation of exosome-derived miR-21-5p expression in coal dust group and silica group;Compared with the control group,the expression of exosome-derived miR-21-5p in the coal dust group and the silica group was upregulated(P<0.05),and the expression of miR-21-5p in the coal dust group was higher than that in the silica group at 4 weeks,and the difference was statistically significant(P<0.05).At 16 weeks,the expression level of miR-21-5p in the silica group was elevated compared with the coal dust group(P<0.05).3.Signal path determination(1)Diluciferase targeting relationship: The results of double luciferase reporter gene experiments showed that the activity of diluciferase decreased significantly(P<0.05)in the experimental group co-transfected with miR-21-5pmimics and Smad7-3’UTR wild-type plasmids,while the luciferase activity did not change significantly in the experimental group co-transfected with miR-21-5pmimics and Smad7-3’UTR mutant plasmids,indicating that Smad7 was a target of miR-21-5p.(2)Expression of Smad7/Snail signaling pathway-related proteins: Western blotting results showed that compared with the control group for 4 weeks and 16 weeks,the expression of Smad7 in the silica group and the coal dust group was significantly reduced(P<0.05),and the expression level of Smad7 in the silica group was reduced compared with the coal dust group(P <0.05).Dust contamination could lead to upregulation of p-Smad2,p-Smad3 and Snail expression in silica group and coal dust group,and the expression of pSmad2 in the silica group was higher than that in the coal dust group at 16 weeks,and there was no significant difference in the expression level of p-Smad2 in the silica group compared with the coal dust group at 4 weeks(P>0.05).Compared with the coal dust group,the expression level of p-Smad3 in the silica group was increased(P<0.05)at 4 weeks and 16 weeks.At 4 weeks,the expression of Snail protein in the silica group increased by(P<0.05).compared with the coal dust group,and at 16 weeks,there was no significant difference in the expression level of Snail in the silica group compared with the coal dust group(P>0.05).4.EMT-related protein determinationDust contamination could lead to downregulation of E-cad expression in the silica group and coal dust group,and the expression of E-cad protein in both silica group and coal dust group decreased(P<0.05)compared with the control group after 4 weeks and 16 weeks of dust exposure.Compared with the control group,the expression of Vimentin and α-SMA in the silica group and the coal dust group was upregulated(P<0.05),and the expression of Vimentin and α-SMA in the coal dust group and the silica group was increased.compared with the control group,and the expression of Vimentin protein in the silica group was increased(P<0.05).At 16 weeks,the expression of α-SMA in the coal dust group was lower than in the silica group(P<0.05).Conclusion:1.Both silica dust and coal dust can lead to increased expression of exosome-derived miRNA-21-5p in rat alveolar lavage solution,and are related to dust causing EMT in lung tissue.2.miRNA-21-5p may affect dust-induced lung tissue EMT through the Smad7/Snail signaling pathway... |
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