| Objective:(1)To investigate the mechanism by which TGF-β1 down-regulates miR-92b-3p expression in respiratory epithelial cells via SMAD3;(2)To investigate the specific mechanism of miR-92b-3p targeting ITGAV and clarify the role of miR-92b-3p in mediating epithelial-mesenchymal transition and pulmonary fibrosis.Methods: We established an in vitro model of pulmonary fibrosis by stimulating the respiratory epithelial cell line A549 with TGF-β1.Lung tissues were collected from 9healthy adults and 7 patients with idiopathic pulmonary fibrosis.By means of q RT-PCR,we were able to detect the expression of miR-92b-3p in both vivo and in vitro models.We divided A549 cells and MLE12 cells into three groups: the Control group,SMAD3 siRNA group,and NC group.The Control group was a blank control,and the cells were cultured without any intervention.The SMAD3 siRNA group was added with TGF-β1 and SMAD3 siRNA to silence the expression of SMAD3.After transfection,q RT-PCR detected the content of miR-92b-3p in the NC group,which had been supplemented with negative control sequences of SMAD3 siRNA and TGF-β1.Using bioinformatics technology,we predicted the target gene of miR-92b-3p,and A549 and MLE12 cell lines were transfected with miR-92b-3p mimics to create cell lines that overexpressed miR-92b-3p.The regulatory effect of miR-92b-3p on its target gene ITGAV was detected by western blot.An in vitro pulmonary fibrosis cell model was constructed using A549 cells,and western blotting was employed to evaluate the influence of miR-92b-3p on the expression of EMT-related genes,which are regulated by TGF-β1.Forty mice were randomly split into four groups: Control group,PBS group,miR-92b-3p group and NC group.The mice in the Control group received routine feeding,the mice in the PBS group were injected with PBS through the tail vein,and the mice in the miR-92b-3p group were injected with r AAV-miR-92b-3p vector through the tail vein.In NC group,r AAV-GFP negative control vector was injected into the tail vein of mice.After three days,the PBS group,miR-92b-3p group and NC group were treated with BLM to induce pulmonary fibrosis mice model.HE staining revealed the structural and morphological alterations in the lung tissue of mice,and Masson staining revealed the collagen deposition therein.Results: The expression of miR-92b-3p in A549 cells stimulated by TGF-β1 was notably lower than other miRNAs that were differentially expressed,and in the lung tissues of IPF patients,it was around 1/4 of that of healthy adult controls.In contrast,transfection of SMAD3 siRNA significantly increased the expression of miR-92b-3p in respiratory epithelial cells stimulated by TGF-β1,compared to the negative control group.ITGAV protein levels in A549 and MLE12 cell lines were down-regulated by miR-92b-3p mimic transfection.Overexpression of miR-92b-3p in A549 cells significantly increased the expression level of epithelial marker E-Cadherin,while decreased the expression levels of mesenchymal markers such as N-Cadherin,Vimentin and Fibronectin.Fibrosis was observed in the lung tissue of mice in the PBS group,and the NC group’s lung fibrosis was comparable to that of the PBS group,yet the miR-92b-3p group’s lung fibrosis was significantly diminished in comparison to the PBS and NC groups.Conclusion: The expression of miR-92b-3p is down-regulated in both pulmonary fibrosis in vitro models and IPF patients.TGF-β1 activation down-regulates the expression of miR-92b-3p through SMAD3 to inhibit the negative regulatory effect of miR-92b-3p on ITGAV and promote the development of IPF.Overexpression of miR-92b-3p can inhibit EMT process and reduce pulmonary fibrosis. |
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