The Role Of ZC3H4 In Macrophage Activation Induced By Silica

Aims:Phagocytosis of silicon dioxide?SiO₂?into lung cells causes an inflammatory cascade which results in fibroblast proliferation and migration followed by fibrosis.Pulmonary fibroblast act as a vital effect cells,which can synthesize and release amounts of extracellular matrix.The activation of alveolar macrophages?AMs?plays a crucial role in this pathological process.MCPIP1?ZC3H12A?,a CCCH type zinc finger protein,attracted much attention in recent years.Tissue expression profile of CCCH type zinc finger proteins showed that the high expression of those proteins are associated with the organs full of AMs.Previous study in our laboratoary showed that MCPIP1 was involved in AM activation after SiO₂ treatment.ZC3H4,a new member of CCCH family proteins,have shown a role in some pathological setting.However,whether ZC3H4 involved in pulmonary fibrosis remains unclear.Current study mainly focused on the association between ZC3H4 and SiO₂-induced AM activation,as well as the mechanism involved in this process.Methods:RAW264.7 macrophage cell line was used to explore whether ZC3H4involved in SiO₂-induced AM activation.Western blot,immunocytochemistry and immunohistochemistry staining were used to detect ZC3H4 and biomarkers of AM activation.ZC3H4 was specificly down-regulated using CRISPR/Cas9 to explore the involvement of ZC3H4 in AM activation.The effect of ZC3H4-induced AM activation on PFB was investigated using 3D migration model.To depict the effect of circZC3H4 on ZC3H4 and AM activation,q-PCR were conducted to test the expression of circZC3H4,while siRNA were used to knock-down circZC3H4.Both anti-miR-212 and mimic-miR-212 were applied to explore the regulation of miR-212on ZC3H4.To further confirm the relationship between circZC3H4 and miR-212,FISH and RNA pull-down assay were applied.To validate our in vitro finding,the expression of ZC3H4 was also measured in U937 and macrophages derived from bronchoalveolar lavage fluid?BALF?of healthy donors and silicosis patients.Results:1)SiO₂ increased ZC3H4 expression significantly,associated with upregulation of AM activation biomarkers.2)The AM activation was attenuated by specific knock-down of ZC3H4.3)The fibroblast migration induced by the conditional medium from AM treated with SiO₂ was inhibited by the specific knock-down of ZC3H4 in AMs.4)The expression of circZC3H4 increased after SiO₂exposure.Moreover,siRNA of circZC3H4 decreased the level of ZC3H4 and AM activation.5)Regulation of miR-212 on ZC3H4,as well as interaction between circZC3H4 and miR-212,indicated that the effect of circZC3H4 on ZC3H4 maybe via ceRNA mechanism.6)The expression of ZC3H4 was upregulated in both U937cells and macrophages from bronchoalveolar lavage fluid?BALF?from silicosis patients.Conclusions:circZC3H4 may regulate ZC3H4 via miR-212.The AM activation asscoiates the increase of ZC3H4,which induces migration of PFB.Our study elucidated a link between SiO₂-induced macrophage activation and the circZC3H4/ZC3H4 pathway,thereby providing novel insight into the potential use of ZC3H4 to develop novel therapeutic strategies for silicosis.