The Role Of CircHECTD1/HECTD1 In Silica-induced Pulmonary Endothelial-mesenchymal Transition

Aims:Silicosis is a disease caused by inhalation of large amounts of free silica（SiO₂）,which is characterized by pulmonary inflammation and advanced fibrosis.After being activated,fibroblasts exposed to SiO₂ proliferate and transform into myofibroblasts that secrete large amounts of extracellular matrix migration（ECM）.As the increase of myofibroblasts,fibroblast foci form and gradually replace normal lung tissue in the lung,causing abnormal remodeling of the lung structure and triggering pulmonary fibrosis.Although it is well known that fibroblast foci comes from the proliferation and transition of myofibroblasts,recent studies have shown that other lung cells may be the source of some fibroblast foci in the process of pulmonary fibrosis.For example,alveolar epithelial cells and vascular endothelial cells can transform into mesenchymal cells（fibroblasts,myofibroblasts）by epithelial-mesenchymal transition（EMT）and endothelial-mesenchymal transition（EndMT）respectively,which are considered to be the one of the important sources of local fibroblasts.However,the mechanism of EMT and EndMT remains unclear.EndMT,a special form of EMT,results in the loss of an endothelial phenotype and the acquisition of a mesenchymal phenotype,which play an important role in the process of the pulmonary fibrosis.Current study mainly focused on the role of circHECTD1/HECTD1 in EndMT induced by SiO₂.Methods:Mouse silicosis model was established using C57BL and STK TEK-GFP 287 Sato/JNju（Tie2-GFP）mice.Mouse endothelial cells（MML1）was exposed to SiO₂（50μg/cm²）for detecting the changes of HECTD1 and EndMT markers.In addition,to valuate the clinical significance of this study,tissue samples were collected from silicosis patients to detect the changes of both endothelial and mesenchymal markers.Both immunofluorescence staining and Western Blot（WB）were applied to detect the levels of specific endothelial and mesenchymal markers in the MML1 exposed to SiO₂,elucidating the mechanism of SiO₂ induced-EndMT.Sirius scar staining assay was used to observe the changes of collagen in endothelial cells of the Tie2-GFP mouse exposed to SiO₂.MTT assay was used to analyze the changes of cell viability.Cell scratch assay and three-dimensional migration assay were applied to detect the change of cell migration ability,reflecting the influence of SiO₂ on cell functions.To further explore the mechanism of circHECTD1 and HECTD1 in EndMT,Real-time Quantitative PCR Detection System（qPCR）and Fluorescence in situ hybridization（FISH）were used to detect the changes of hectd1 and circHECTD1 expression.Lung tissue samples from both Tie2-GFP mice exposed to SiO₂ and silicosis patients were collected to confirm the observations of in vitro experiments.Results:SiO₂ increased the expression of mesenchymal markers-type I collagen（COL1A1）,type III collagen（COL3A1）and alpha smooth muscle actin（α-SMA/Acta2）,decreased the expression of endothelial markers-vascular endothelial cadherin（VE-cad/Cdh 5）and platelet endothelial cell adhesion molecule-1（PECAM 1/CD31）,indicating the occurrence of the EndMT in response to SiO₂ exposure both in vivo and in vitro.SiO₂ concomitantly increased circHECTD1 expression,which,in turn,inhibited HECTD1 protein expression.SiO₂-induced increases in cell proliferation,migration,and changes in marker levels were restored by either a small interfering RNA（siRNA）targeting circHECTD1 or overexpression of HECTD1 via the CRISPR/Cas9 system,confirming the involvement of the circHECTD1/HECTD1pathway in the EndMT.Moreover,tissue samples from SiO₂-exposed mice and silicosis patients confirmed the EndMT and change in HECTD1 expression.These results suggested that:（1）SiO₂ induced acquisition of a mesenchymal phenotype;（2）SiO₂ exposure induced the changes of circHECTD1 expression and HECTD1 level;（3）circHECTD1 and HECTD1 were involved in SiO₂-induced EndMT;（4）circHECTD1 regulated HECTD1 protein levels via non-transcriptional mechianism.Conclusion:This study reveals a potentially new function for the circHECTD1/HECTD1pathway and suggests a possible mechanism of fibrosis in patients with pulmonary silicosis.