| ObjectiveAt present,the pathogenesis of silicosis has not been elucidated,and the prevention and control of silicosis is one of the important tasks of occupational disease prevention in china.It is generally believed that under the influence of many factors such as silica dust,alveolar Ⅱ type transformation of epithelial cells through the epithelial-mesenchymal transition(EMT)into myfibroblasts,a large number of proliferation and secretion of collagen and other related proteins,is an important biological event in the development of pulmonary fibrosis.Previous studies have indicated that miR-34a-5p and its target gene SMAD4 are involved in the EMT process of cancer and liver fibrosis,which may be an important link in the EMT process and a potential intervention target.However,its role in silicosis related EMT process not to see the document report yet at present.Therefore,this study construct mouse model of pulmonary fibrosis induced by silica and EMT model of lung epithelial cells to observe the existence and change of miR-34a-5p and SMAD4.And the intervention of miR-34a-5p and SMAD4 by liposome transfection was carried out to further explore the biological role in EMT,so as to provide a new thought and reference for the further exploration of pathogenesis of silicosis.Methods1.Experimental observation of the existence of miR-34a-5p in EMT of silicosis miceThe C57BL/6N mice were randomly divided into experimental groups at different time points(inducing 1 day,14days,28 days,56 days)and corresponding normal saline control group.The pulmonary fibrosis model of mice was established by non-exposed tracheal instillation of SiO2 suspension(50mg/mL)or volume saline.HE and Masson staining were used to observe the degree of pulmonary fibrosis in mice.RT-qPCR,Western blot and Immunofluorescence were used to detect the expression of EMT-related markers(epithelial markers:E-cad,mesenchymal markes:a-SMA and Vimentin),and the expression of miR-34a-5p and SMAD4 were also detected.2.Experimental observation of the effect of miR-34a-5p in EMT model of A549 cellsHuman adenocarcinoma epithelial cell line A549 was cultured in RPMI 1640 medium.In this study,A549 cells were stimulated with 5ng/mL TGF-β1 to construct an EMT cell model.A549 cells were transfected with miR-34a-5p mimics/inhibitor to over-expression/inhibition its level.A549 cells were transfected with siRNA to silence the expression of SMAD4.RT-qPCR,Western blot and Immunofluorescence were used to detect the expression of EMT-related markers,miR-34a-5p and SMAD4 Dual luciferase reporter gene assay detected the targeted binding relationship between miR-34a-5p and SMAD4.3.Statistical analysisThe resulting data were statistically analyzed using SPSS 21.0 software.Independent sample t test and one-way ANOVA were used for statistical analysis of experiment data.Check level=0.05.The bar charts were all completed by GraphPad 8.0 software.Results1.The existence of miR-34a-5p during EMT in silicosis mice1.1 Establishment of silicosis model induced by silicaHE and Masson staining showed that the alveolar wall structure was intact in the control group at different time points.In the experimental group,the alveolar wall was thickened,some alveolar structures were destroyed and there were cell nodules.And the degree of pulmonary fibrosis was the most serious in experimental group at 56 days.1.2 EMT related gene expression in lung tissues of silicosis miceThe results of RT-qPCR and Western blot showed that the expression of E-cad was down-regulated in the lung tissues of experimental group,while the expression of the mesenchymal markers Vimentin and a-SMA was up-regulated(P<0.05).Immunofluorescence results showed that E-cad was down-regulated in the lung tissues of experimental group.1.3 Expression of miR-34a-5p and SMAD4 in lung tissues of silicosis miceRT-qPCR results showed that the expression of miR-34a-5p was down-regulated in the lung tissues of experimental,while the expression of SMAD4 was up-regulated(P<0.05).Immunofluorescence results showed that SMAD4 was down-regulated in the lung tissues of experimental group.2.Effect of miR-34a-5p in EMT model of A549 cells2.1 The identification and detection of TGF-β1 induced EMT model in vitroAfter the A549 cells were induced by TGF-β1,the cell morphology changed from the typical epithelial morphology to spindle fibroblast morphology under the microscope.RT-qPCR and Western blot results showed that after TGF-β1 stimulation,the expression of E-cad decreased and the expression of a-SMA and Vimentin increased.Meanwhile,the expression of miR-34a-5p decreased and the expression of SMAD4 increased(P<0.05).2.2 The changes of EMT related indicators after the intervention of miR-34a-5pTransfection of miR-34a-5p mimics could significantly increase the expression of miR-34a-5p in A549 cells.The over-expression of miR-34a-5p down-regulated the expression level of SMAD4.RT-qPCR and Western blot results showed that compared with the negative control group,E-cad,α-SMA,Vimentin,and SMAD4 significantly changed in the miR-34a-5p mimics group,the same as the miR-34a-5p mimics+TGF-β1 stimulation group compared with the TGF-β1 stimulation group(P<0.05).Transfection of miR-34a-5p inhibitor could significantly decrease the expression of miR-34a-5p in A549 cells.The low expression of miR-34a-5p up-regulated the expression of SMAD4.RT-qPCR and Western blot results showed that compared with the negative control group,E-cad,a-SMA,Vimentin,and SMAD4 significantly changed in the miR-34a-5p inhibitor group,the same as the miR-34a-5p inhibitor+TGF-β1 stimulation group compared with the TGF-β1 stimulation group(P<0.05).2.3 Dual luciferase reporter gene assay verified the targeting relationship between miR-34a-5p and SMAD4The result of Dual Luciferase Reporter Assay showed that co-transfection of miR-34a-5p mimics or m-NC with the SMAD4 3’-UTR of wild type could significantly decrease the luciferase activity level(P<0.05).But co-transfection of miR-34a-5p mimics or m-NC with the mutated SMAD4 3’-UTR failed to reduce the luciferase activity level when compared with its control(P>0.05).2.4 The expression of EMT-related indicators after silencing the SMAD4 geneRT-qPCR and Western blot results showed that three specific sequences designed for SMAD4 could all reduce the expression of SMAD4,among which siR-3 specific sequence had the highest silencing efficiency(P<0.05),which was used in subsequent experiments.RT-qPCR and Western blot results showed that compared with the negative control group,E-cad,α-SMA,Vimentin,and SMAD4 significantly changed in the siR-SMAD4 group,the same as the siR-SMAD4+TGF-β1 stimulation group compared with the TGF-β1 stimulation group(P<0.05).ConclusionsIn the EMT of silicosis mice and A549 cells,the expression of miR-34a-5p was down-regulated,and miR-34a-5p may play the potential role in inhibiting EMT by targeting SMAD4. |
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