The Role Of LncRNAUCA1/miR-204-5p/ZEB1 Axis In Epithelial-mesenchymal Transition Of Silicosis Associated Lung Epithelial Cells

ObjectiveAs one of the most common types of pneumoconiosis,silicosis is mainly manifested as pulmonary fibrosis.At present,silicosis is a serious hazard,and it is still the weak point in the prevention and control of occupational diseases.The reason is that its pathogenesis is not yet clear.It is generally believed that the key link of pulmonary fibrosis is the proliferation and transdifferentiation of fibroblasts.The type II alveolar epithelial cells can be transformed into lung fibroblasts through epithelial-mesenchymal transition（EMT）,which promotes pulmonary fibrosis.Previous studies have shown that Lnc RNA UCA1 is involved in the EMT process of a variety of cancers.It can competitively adsorb miR-204-5p and promote the target gene zinc finger E-box binding homeobox 1（ZEB1）to promote the EMT process,thereby enhancing the invasion and migration ability of cancer cells.However,the role of UCA1 in the related research of silicosis fibrosis EMT has not been reported yet.Therefore,this study established SiO2 treatment of silicosis model in mice and human lung epithelial cells（A549 cells）EMT models to observe the changes and related effects of UCA1 and miR-204-5p.Intervene with UCA1 and miR-204-5p respectively,and further explore their mechanism in this process,in order to provide new ideas for the exploration of the mechanism of silicosis fibrosis process.Method1 Establishment of silicosis model in mice treated with SiO₂C57BL6/N mice were randomly divided into 8 groups:1 day,7 days,28 days and56 days silicosis group and corresponding control group.The silicosis group was treated with non-exposed tracheal dripping method of 50μL SiO₂suspension（50mg/m L）,and the control group was treated with equal volume of normal saline.The silicosis model was established in mice.Lung tissues of mice were collected,and changes in lung tissues and fibrosis degree of mice were observed by HE and Masson staining.Relevant markers of EMT,E-cad and Vimentin,were detected by immunohistochemistry,RT-qPCR and Western blot.2 Establishment and observation of EMT model in vitroIn vitro EMT model was established by stimulating A549 cells with 5 ng/m L of TGF-β1.The experiment was divided into TGF-β1 stimulation group and blank control group.The changes of E-cad,Vimentin andα-SMA before and after TGF-β1stimulation of A549 were detected by RT-qPCR and Western blot.Meanwhile,the changes of UCA1,miR-204-5p and target gene ZEB1 were detected.3 Effect of knockdown UCA1 on EMT model in vitroDual luciferase reporter gene assay was used to verify the targeted binding relationship between UCA1 and miR-204-5p.UCA1 si RNA was transfected into A549cells,experiments were divided into NC group,NC+TGF-β1 stimulation group,si-UCA1 group and si-UCA1+TGF-β1 stimulation group.The changes of cell migration ability were obtained by scratch test.The expression changes of EMT markers and UCA1,miR-204-5p and target gene ZEB1 were detected by RT-qPCR and Western blot.4 Effect of intervention of miR-204-5p level on EMT model in vitroDual luciferase reporter gene assay was used to verify the targeted binding relationship between miR-204-5p and ZEB1.Mi R-204-5p mimics/inhibitor was transfected into A549 cells to specifically increase or decrease the expression level of miR-204-5p.The experiments were divided into NC group,NC+TGF-β1 stimulation group,miR-204-5p mimics/inhibitor group and miR-204-5p mimics/inhibitor+TGF-β1stimulation group.The changes of cell migration ability were obtained by scratch test.The changes of EMT markers,miR-204-5p and target gene ZEB1 were detected by RT-qPCR and Western blot.5 Statistical analysisThe experimental data are statistically analyzed using SPSS21.0.The measurement data were compared equally using two independent sample t-tests andαone-factor variance analysis methods,the test levelα=0.05.Results1 SiO₂-treated silicosis model in mice1.1 Pathological changes of silicosis miceHe and Masson staining results showed that compared with the corresponding control group,lung tissue in silicosis group at different time points showed different degrees of inflammatory aggregation,alveolar wall destruction or thickening,some serious parts of the cell nodules;In contrast,the alveolar structure of the control group was relatively intact,and the alveolar wall was monolayer with no cellular nodules.In addition,the mice treated at 56 days had the most severe alveolar destruction and cell nodules.1.2 Changes of EMT-related markers in lung tissues of miceImmunohistochemical results showed that,compared with the corresponding control group,the expression of epithelial marker E-cad was decreased and the expression of stromal marker Vimentin was increased in the lung tissues of mice in silicosis group（P<0.05）;The results of RT-qPCR and Western blot showed the same trend as the results of immunohistochemistry.2 A549 cell EMT model identification and related gene changesThe results of RT-qPCR and Western blot showed that the expression of E-cad was down-regulated and the expression of Vimentin andα-SMA were up-regulated in TGF-β1 stimulation group.The model of EMT in vitro was established successfully.Meanwhile,UCA1 and ZEB1 expression were up-regulated,and miR-204-5p expression was down-regulated（P<0.05）.3 Changes of related genes in the EMT process of A549 cells after UCA1knockdown3.1 Dual luciferase reporting assay verifies the target binding between UCA1 and miR-204-5pDual luciferase report assay showed that the activity of dual luciferase was decreased in the co-transfection group of miR-204-5p mimics and wild-type UCA1-3’UTR plasmid,there was a targeted binding relationship between them（P<0.05）.3.2 Changes of EMT-related genes after si-UCA1 transfectionThe silence of UCA1 significantly increased the level of miR-204-5p.The results of RT-qPCR and Western blot showed that the expression of miR-204-5p and E-cad were up-regulated in the si-UCA1+TGF-β1 stimulation group compared with the NC+TGF-β1 stimulation group,and the expression of Vimentin,α-SMA and ZEB1were down-regulated（P<0.05）.The scratch experiment showed that the migration ability of A549 cells was reduced after si-UCA1 transfection（P<0.05）.4 The changes of related genes in the EMT process of A549 cells after intervention of miR-204-5p4.1 Dual luciferase reporting assay verified the targeted binding between miR-204-5p and ZEB1Dual luciferase reporting assay showed that the activity of dual luciferase was decreased in the co-transfection group of miR-204-5p mimics and wild-type ZEB1-3’UTR plasmid,there was a targeted binding relationship between them（P<0.05）.4.2 Changes of EMT-related genes after transfection with miR-204-5p mimicsThe overexpression of miR-204-5p reduced the level of ZEB1.The results of RT-qPCR and Western blot showed that compared with the NC+TGF-β1 stimulation group,the miR-204-5p mimics+TGF-β1 stimulation group increased the expression of E-cad,and the expression of Vimentin,α-SMA and ZEB1 was down-regulated（P<0.05）.Scratch experiments showed that the migration ability of A549 cells was reduced after miR-204-5p was overexpressed（P<0.05）.4.3 Changes of EMT-related genes after transfection with miR-204-5p inhibitorWhen miR-204-5p was inhibited,the expression level of ZEB1 increased.RT-qPCR and Western blot analysis showed that miR-204-5p inhibitor+TGF-β1 group had a decreased expression of E-cad and an increased expression of Vimentin,α-SMA and ZEB1 compared with NC+TGF-β1 stimulated group（P<0.05）.The scratch test showed that the migration of A549 cells was enhanced when miR-204-5p was inhibited（P<0.05）.ConclusionThe occurrence of pulmonary fibrosis was related to EMT,in the process of EMT,UCA1 may reduce the level of free miR-204-5p through competitive absorption of miR-204-5p,and cause the release of the target gene ZEB1 of miR-204-5p,thus exerting its regulatory role.