| Background and objectiveSilicosis is a systemic disease characterised by diffuse fibrosis of the lung tissue caused by long-term inhalation of free silica dust during the production process.The pathogenesis of silicosis is not fully understood.Alveolar type II can partially transform into myofibroblasts through epithelial-mesenchymal transdifferentiation,and it can also secrete cytokines,exosomes or other signal factors through EMT to promote the transformation of fibroblasts into myofibroblasts,which is consistent with The occurrence of pulmonary fibrosis is closely related.The ZEB1 is a key transcription factor in the process of epithelial mesenchymal transdifferentiation.It can target the E-box box that can bind to the E-cadherin promoter region to inhibit its expression and initiate EMT.LncRNA regulates the expression of ZEB1 through a competitive endogenous RNA and then affects the process of EMT in organ fibrosis diseases,which has been reported by related studies.LncRNA XIST and miR-101-3p have been shown to be associated with both the EMT process and fibrotic disease in a variety of diseases.Therefore,Si O2-induced mouse silicosis-related fibrosis model and alveolar epithelial cell EMT model were established to observe the expression of lncRNA XIST,miR-101-3p and ZEB1 in this study.In vitro transfection technology interfered with the expression of LncRNA XIST and miR-101-3p to explore its influence on ZEB1 gene and EMT.Our research hopes to provide new research ideas and reference basis for the EMT study of alveolar epithelial cells in the process of silicosis-related pulmonary fibrosis.Methods1.Establish mouse silicosis model and detection of lncRNA XIST,miR-101-3p,ZEB1,and EMT-related transdifferentiation genesForty male C57BL/6N mice were randomly divided into a control group and a Si O2dust-stained group,with 20 mice in each group for subsequent experiments.The mice in the Si O2 dust group were instilled 50μL of Si O2 suspension（concentration of50 mg/m L）by non-exposure tracheal instillation,and the mice in the normal saline group were instilled 50μL of normal saline in the same way.Five mice in each group were sacrificed on day 1,7,28,and 56 days.The lung tissues of the mice were collected.HE and Masson staining of lung tissue sections were used to determine the degree of fibrotic lesions in mouse lung tissue.RT-q PCR,WB,and immunohistochemistry were used to detect the expression of ZEB1 and EMT-related transdifferentiation genes in mouse lung tissue.RT-q PCR was used to detect the expression of lncRNA XIST and miR-101-3p in mouse lung tissue.2.Establishment of A549 cell-related EMT model and detection of LncRNA XIST,miR-101-3p,ZEB1,and EMT-related transdifferentiation genesA549 cells were plated into a 6-well plate at a density of 2×105 cells,and 2 m L of complete medium was added to each well.After the cells were stably cultured in the incubator for 24 hours,2 m L of TGF-β1（concentration 5 ng/m L）complete medium stimulated the cells to establish an in vitro lung epithelial cell EMT model.The inverted phase contrast microscope was used to observe changes in cell morphology,RT-q PCR and WB were used to detect the expression of EMT-related transdifferentiation genes and ZEB1 gene expression.RT-q PCR was used to detect the expression of LncRNA XIST and miR-101-3p.3.In vitro transfection of small interfering RNA（siRNA）for LncRNA XIST and detection of related genesSi-XIST and si XIST-NC were transfected into A549 cells.The experimental groupings were set to 4 groups.The experimental groups are as follows:si XIST-NC group,si XIST-NC+TGF-β1 group,si-XIST group,and si-XIST+TGF-β1 group.RT-q PCR and WB were used to detect the expression of E-cadherin,Vimentin,α-SMA,and ZEB1 in each group.The cell scratch test was used to determine the migration of A549 cells after transfection with si-XIST.Fluorescence in situ hybridization experiment was used for the subcellular localization of LncRNA XIST.RT-q PCR was used to detect the expression of miR-101-3p after transfection of si-XIST.4.Transfection of miRNA-101-3p mimics and inhibitor and detection of related genesMiRNA-101-3p mimics,miRNA-101-3p inhibitor,mimics-NC,and inhibitor-NC were transfected into A549 cells.The mimics are set to 4 groups.Experiments were grouped as follows:mimic-NC group,mimics-NC+TGF-β1 group,miR-101-3p mimics group,and miR-101-3p mimics+TGF-β1 group.The inhibitor set-up experiments were grouped into 4 groups:inhibitor-NC group,inhibitor-NC+TGF-β1 group,miR-101-3p inhibitor group,and miR-101-3p inhibitor+TGF-β1group.RT-q PCR and WB were used to detect the expression of E-cadherin,Vimentin,α-SMA and ZEB1 in each group.The cell scratch test was used to determine the migration of A549 cells after transfection of miRNA-101-3p mimics and inhibitors.The dual luciferase report was used to verify the targeting relationship between miRNA-101-3p and ZEB1.5.Statistical analysisSPSS 21.0 was used for statistical analysis.Student’s t test was used for pairwise comparisons between two independent sample means.One-way ANOVA（One-way ANOVA）was used to compare the means of multiple independent samples.If the sample means of multiple groups were statistically different,the LSD-t method was used for pairwise comparisons between groups.The test levelα=0.05 and P<0.05were considered as statistically significant.Results1.Establishment of a mouse silicosis model and expression of LncRNA XIST,miR-101-3p,ZEB1 and EMT-related genes in mouse lung tissuesThe results of HE staining showed that the lung tissue of the mouse had no cellular nodules and intact alveolar structures in the control group.In the dust-exposed group,the alveoli of the mice were destroyed and the alveolar wall thickened for 7 days.Obvious cellular nodules appeared in mouse lung tissue on 28days,and cellular nodules were increasing in size in the lung tissue of mice at 56 days.The results of Masson staining showed that there was obvious collagen deposition in the lung tissue of mice at 28 days compared with the control group.The collagen deposition in the lung tissues of mice at 56 days was more severe compared with the control group.Decreased expression of E-cad and increased expression of VIM and ZEB1 were observed by immunohistochemistry,RT-q PCR and WB in lung tissue of mice on day 56 after dusting compared to control（P<0.05）.Increased expression of LncRNA XIST and decreased expression of miR-101-3p in mouse lung tissue on day56 after dusting were observed by RT-q PCR compared to controls（P<0.05）.2.Establishment of EMT model and expression of LncRNA XIST,miR-101-3p,ZEB1 and EMT transdifferentiation genes in A549 cells in EMT modelThe cells were transformed from pavement-like epithelial cells to long spindle-like mesenchymal cells after 48 h of TGF-β1 stimulation under inverted phase contrast microscopy.Decreased E-cad expression and increased expression of VIM,α-SMA and ZEB1 were detected in the TGF-β1 stimulated group by RT-q PCR and WB compared to the control group（P<0.05）.Increased expression of LncRNA XIST and decreased expression of miR-101-3p in the TGF-β1 stimulated group was detected by RT-q PCR compared to the control group（P<0.05）.3.In vitro transfection of LncRNA XIST with small interfering RNA（siRNA）and observation of its resultsAll three siRNAs targeting LncRNA XIST were able to down-regulate LncRNA XIST expression as verified by RT-q PCR.Si XIST-2 had the highest specific siRNA sequence silencing efficiency（P<0.05）.Reduced E-cad expression and increased VIM,α-SMA and ZEB1 expression were detected in the si NC+TGF-β1 group by RT-q PCR and WB compared to the si-NC group（P<0.05）.Increased E-cad expression and decreased VIM,α-SMA and ZEB1 expression were observed in the si XIST-2+TGF-β1 group compared to the si NC+TGF-β1 group（P<0.05）.Reduced cell migration in the si XIST-2+TGF-β1 group was observed by cell scratching assay compared to the si NC+TGF-β1 group.The experimental results of fluorescence in situ hybridization indicated that LncRNA XIST was mainly localized in the cytoplasm.Reduced expression of miR-101-3p in the si NC+TGF-β1 group was detected by RT-q PCR compared to the si-NC group（P<0.05）.Increased miR-101-3p expression was detected in the si XIST-2+TGF-β1 group compared to the si NC+TGF-β1 group（P<0.05）.4.Transfection of miRNA-101-3p mimics and inhibitor and its result observationReduced E-cad expression and increased VIM,α-SMA and ZEB1 expression were detected by RT-q PCR in the m-NC+TGF-β1 group compared to the m-NC group（P<0.05）.Increased E-cad expression and decreased VIM,α-SMA and ZEB1expression were observed in the miR-101-3p mimics+TGF-β1 group compared to the m-NC+TGF-β1 group（P<0.05）.Decreased E-cad expression and increased VIM,α-SMA and ZEB1 expression in the i-NC+TGF-β1 group were detected by RT-q PCR compared to the i-NC group（P<0.05）.Decreased E-cad expression and increased VIM,α-SMA and ZEB1 expression in the miR-101-3p inhibitor+TGF-β1 group were detected by RT-q PCR compared to the i-NC+TGF-β1 group（P<0.05）.Cell migration was diminished in the miR-101-3p mimics+TGF-β1 group as observed by cell scratch assay compared to the m-NC+TGF-β1 group.Cell migration was enhanced in miR-101-3p inhibitor+TGF-β1 group compared to i-NC+TGF-β1 group.Dual luciferase reporter assays showed a significant decrease in dual luciferase activity when cotransfected with miR-101-3p mimics and ZEB1 wild-type plasmids（P<0.05）,while dual luciferase activity was unchanged when cotransfected with ZEB1 mutant plasmids（P>0.05）.ConclusionsIn the EMT process of silicosis related pulmonary fibrosis,LncRNA XIST expression was upregulated,miR-101-3p expression was downregulated,and ZEB1 expression was upregulated.Up-regulated LncRNA XIST promotes EMT in alveolar epithelial cells by regulating miR-101-3p to up-regulate the expression of ZEB1. |
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