The Regulation Of Mesenchymal Transition By ZC3H4 In Pulmonary Fibrosis Of Silicosis

Aim:Silicosis is a systemic disease caused by long-term inhalation of free silicon dioxide?SiO₂?particles,characterized by pulmonary fibrosis.The fibrogenic factors released by alveolar macrophages act on pulmonary fibroblasts,resulting in increased cell proliferation and migration ability during irreversible pulmonary fibrosis.Myofibroblasts derived from epithelial-mesenchymal transition?EMT?and endothelial-mesenchymal transitions?EndoMT?are involved in this pathological process.Previous study from our lab showed that MCPIP1?ZC3H12A?,a member of CCCH zinc finger protein family,plays an important role in EndoMT of silicosis.While the role of ZC3H4,another member from the same family,in EMT/EndoMT of silicosis is still unknown.In addition,recent studies have found that stem cells have advantages on promoting re-epithelialization,reducing inflammation and delaying pulmonary fibrosis process.However,the effects of stem cells on EndoMT are poorly understood.Therefore,this project will focus on studying the effects of ZC3H4 on EMT/EndoMT and exploring whether human bone marrow mesenchymal stem cells?hBMSC?regulate EndoMT by targeting ZC3H4 expression during silicosis.Methods:1)To explore the mechanisms by which ZC3H4 mediated EMT in silicosis.a)The silicosis models of C57BL mice were established,and sirius red staining were used to detect pulmonary fibrosis lesions.b)Tissue immunofluorescence staining was used to observe the EMT phenomenon and ZC3H4 expression in lung tissues of silicosis patients and mice.c)Western Blot and cellular immunofluorescence staining were utilized to observe the expression of epithelial markers,mesenchymal specific markers and related functional protein in mouse lung epithelial cell lines MLE12,human lung epithelial cell lines A549 and BEAS-2B.d)qRT-PCR,Western Blot and immunofluorescence staining were used to observe the effects of SiO₂ on the expression of circZC3H4 and ZC3H4 in MLE12 cells.e)ZC3H4 was specificly down-regulated using CRISPR/Cas9 to explore whether ZC3H4regulated EMT.f)circZC3H4 was knocked down by small interference RNA to examine whether circZC3H4 regulated ZC3H4 expression and EMT.g)Cell scratch test was used to verify the effects of ZC3H4 and circZC3H4 on the migration function of MLE12 cells.h)microRNA-212 mimics and antagonists?microRNA anti?were transfected to assay the regulation of microRNA-212 on ZC3H4 expression.i)Co-transfection,in-situ and pull down techniques were used to explore the effects of circZC3H4 and microRNA-212 on ZC3H4expression in MLE12 cells.2)To explore the mechanisms by which ZC3H4 mediated EndoMT in silicosis.a)Chest computed tomography?CT?assay,sirius red staining and serum hydroxyproline assay were used to detect pulmonary fibrosis lesions.b)Tissue immunofluorescence staining was used to observe the EndoMT phenomenon and ZC3H4 expression in lung tissues of silicosis patients and mice.c)Western Blot and cellular immunofluorescence staining were utilized to verify the expression of epithelial markers,mesenchymal specific markers in HUVEC and HPAEC.d)CRISPR/Cas9 gene knockout techniques combined with pharmacological methods were designed to explore the mechanisms by which ZC3H4 regulated EndoMT.3)To explore whether BMSC regulated EndoMT by targeting ZC3H4 expression during silicosis.a)CT imaging experiment and immunohistochemistry were designed to analyze the temporal and spatial distribution of hBMSC double-labeled with SPIO and Dil in pulmonary and the protective effects of BMSC on pulmonary vessels.b)Western Blot and cellular immunofluorescence staining were used to detect the effects of SiO₂ and hBMSC on the expression of ZC3H4 and the biomarkers of EndoMT in vitro.c)Elisa assay was used to detect the vascular endothelial growth factor-A?VEGF-A?concentration of conditioned medium from stem cells.d)Pharmacological inducer or inhibitors were applied to evaluate the role of VEGF-A in EndoMT.Results:1)SiO₂ increased ZC3H4 expression and prompted EMT.The circZC3H4/miR-212/ZC3H4axis regulated the process of EMT and the migration of epithelial cells.The expression of ZC3H4 was increased significantly in epithelial cells of silicosis patients.2)SiO₂ stimulated EndoMT and up-regulated ZC3H4 expression in endothelial cells significantly.EndoMT was attenuated by specific knock-down of ZC3H4.ZC3H4 regulated EndoMT via activating endoplasmic reticulum stress and autophagy.The expression of ZC3H4 was increased significantly in endothelial cells of silicosis patients.3)BMSC double-labeled with SPIO and Dil were found in the lungs after 28 days since endotracheal injection.Pulmonary fibrosis,vascular damage,ZC3H4 expression were attenuated by BMSC.Conditioned medium from BMSC reversed EndoMT and ZC3H4expression via VEGF-A.Conclusion:Our study clarified the roles of zinc finger protein ZC3H4 on EMT and EndoMT,as well as the great impact of hBMSC on the progression of SiO₂-induced EndoMT via ZC3H4,which represented a key target for the pathogenesis and the treatment of silicosis.