Role Of ZC3H4 In Anoikis Resistance In Pulmonary Fibrosis

Objective:Pulmonary fibrosis is a lung disease developed in susceptible individuals due to abnormal healing of wounds after repeated injury of the alveoli,mainly manifested in chronic inflammation,the deposition of extracellular matrix（collagen）,which eventually leads to lung function injury,an ultimate fate of a variety of lung diseases.This process has the characteristics of continuity and non-reversibility,and involves a variety of effect syphonating cells such as pulmonary fibroblasts,pulmonary epithelial cells and pulmonary endothelial cells,so far there is no clear evaluation of pulmonary fibrosis.Moreover,the excessive proliferation of cells in the process of fibrosis is similar to the uncontrolled proliferation of cells caused by anoikis resistance in tumor invasion and migration/skin injury repair.Therefore,the study mainly explores the anokis resistance in pulmonary fibroblasts,alveolar epithelial cells and pulmonary endothelial cells during the pulmonary fibrosis process,pays special attention to the role of ZC3H4,a member of the zinc finger protein family involved in the pulmonary fibrosis process,and then explores possible molecular mechanisms to provide new ideas for the diagnosis and treatment of fibrosis diseases and the prognosis.Methods:1)The anoikis resistance phenomenon in pulmonary fibroblast/alveolar epithelial/pulmonary microvascular endothelial cells during the fibrosis process.a)The Si O₂ suspension with a final concentration of 50μg/cm² was applied to stimulate macrophage cell line THP-1.The conditional media（referred to CM）was collected to simulate the environment of pneumonia.CCK8 was applied to detect vitality changes in pulmonary fibroblasts（HPF-a）,pulmonary epithelial cells（BEAS-2B）and pulmonary microvascular endothelial cells（HPMEC）;b)Western Blot was used to detect the anoikis changes after CM stimulation,such as the anoikis resistance marker Trk B.c)After induction of CM,WB and flow cytometry was applied to detect the change of anoikis resistance;d)Cell counting was employed to detect the effect of CM on the anoikis resistance of MLG cells culture in the lung tissue skeleton.e)CCK8,cell count and WB experiment were applied to study the cell viability,cell number,and apoptosis after induction of anoikis in vitro.2)The molecular mechanism of ZC3H4 in the anoikis resistance in pulmonary fibroblasts.a)Western Blot and q RT-PCR were used to detect the changes of ZC3H4 protein,m RNA and circ ZC3H4 after CM stimulation in HPF-a.b)Immunofluorescence staining was applied to confirm the expression pattern of ZC3H4and Trk B in HPF-a cells.c)Western Blot was applied to detect Trk B protein after CRISPR/Cas9 knock down of ZC3H4.d)Changes in MAPK/PI3K signaling pathways after CM stimulation were detected by Western Blot in HPF-a.e)The effect of signaling pathway inhibitors on anoikis resistance was also detected by Western Blot.f)The effect of signaling pathway inhibitors on ZC3H4 was detected by Western Blot.Results:1)CM increased the activity of HPF-a,BEAS-2B and HPMEC cells.While only HPF-a showed siginificant increase in Trk B protein,a molecular marker of anoikis resistance,which was confirmed by WB results from mouse silicosis tissue.CM promoted the anoikis resistance of MLG cells cultured in the extracellular matrix of mouse lung tissue.After induction of anoikis by cell detachment in vitro,the effects of CM on increasing HPF-a viability and anoikis resistance were reversed.2)CM induced expression of ZC3H4 in HPF-a cells.Knock-down of ZC3H4 with CRISPR/Cas9 reversed the CM-induced anoikis resistance in HPF-a cells.CM activated PI3K and MAPK signaling pathways in HPF-a cells.PI3K and MAPK signaling pathway inhibitors can reverse CM-induced ZC3H4 expression and anoikis resistance.Conclusion:Current study reveals that the inflammatory microenvironment activates the PI3K/MAPKs signaling pathway in pulmonary fibroblasts,which in turn,induces the expression of ZC3H4 protein,and specifically causes the anoikis resistance,followed by pulmonary fibrosis.The above results suggest that targeting the anoikis resistance in pulmonary fibrosis may effectively delay the process of pulmonary fibrosis,providing new ideas for the clinical diagnosis and treatment of pulmonary fibrosis.