| Aims:The inhalation of silicon dioxide(SiO2)particles causes pneumoconiosis,an untreatable pulmonary disease characterized by alveolar inflammation at the early stage and progressive lung fibrosis at the late stage,in which the cause of this often devastating disease remains elusive.Circular RNAs(circRNAs)are a subclass of non-coding RNAs involved in diseases;however,researchers have not determined whether circRNAs are involved in the pathophysiological process of silicosis.The project was designed to study the effects of circHECTD1 on inflammatory response and subsequent pulmonary fibrosis,to provide a new target and strategy for the development of therapeutic for treatment of silicosis.Methods:1)The silicosis models were established to investigate the expression of circRNAs.a)Microarray was used to detect circRNAs expression in the lung of mouse exposed to SiO2.b)qRT-PCR combined with fluorescence in situ hybridization(FISH)assay were utilized to observe the expression of circHECT1 in RAW 264.7 and MLE12.c)The circHECTD1 in macrophages of mouse BALF were detected by qRT-PCR.2)The role of circHECTD1/HECTD1 in macrophage activation induced by silica was investigated,a)Lentivirus was used to uprgulate circHECTD1 expression in RAW 264.7.b)The cell polarization,cell viability and migration in RAW 264.7 induced by silica were evaluated by WB,MTT and 3D migration model,respectively.c)WB,qRT-PCR combined with immunocytochemistry were utilized to investigate HECTD1 level in RAW 264.7 after exposure to silica,d)CRISPR/Cas9 system was used to regulate HECTD1 level in RAW 264.7.e)The role of HECTD1 and MCP-1 induced protein(MCPIP1/ZC3H12A)on polarization was assessed by WB in RAW 264.7 induced by silica,f)The interaction between HECTD1 and ZC3H12A was demonstrated by Co-IP assay.g)RIP assay was used to test the interaction between circHECTD1 and ZC3H12A.f)The effects of conditioned medium from RAW 264.7 on fibroblasts migration were evaluated by cell scratch assay.3)The roles of HECTD1 in epithelial-mesenchymal transition(EMT)induced by silica was explored,a)Sirius Red staining,WB combined with immunocytochemistry were utilized to confirm EMT induced by silica,b)CRISPR/Cas9 system was used to regulate HECTD1 level in MLE12.c)The effects of HECTD1 on cell viability and EMT induced by silica were respectively assessed by MTT and WB in MLE12.d)The effects of HECTD1 on cell migration were investigated by cell scratch assay and 3D migration model,e)The role of MAPKs and PI3K/Akt pathways in EMT were evaluated by pharmacological blockers in MLE12 after exposure to silica,f)The effects of conditioned medium from MLE12 on fibroblasts activation and migration were evaluated by cell scratch assay combined with WB.Results:1)120 circRNAs were differentially expressed in the silica-treated group compared with the Saline group,73 of these circRNAs were upregulated,and 47 were downregulated in the lung of mouse silicosis model.The expressions of circHECTD1 were decreased in macrophages,but upregulated in MLE12 induced by silica,and mostly expressed in cytoplasm.2)SiO2 concomitantly decreased the circHECTD1 levels and increased the expression of the HECTD1 protein in RAW 264.7.circHECTD1 and HECTD1 initiated SiO2-induced macrophage activation via ubiquitination,which,in turn,promoted fibroblast proliferation and migration in RAW 264.7.The samples from silicosis patients confirmed the the upregulation of HECTD1.3)SiO2 concomitantly increased the expression of HECTD1 protein and induced EMT in MLE12.The EMT,cell viability and migration were enhanced by HECTD1 in MLE12 after exposure to silica.The MAPKs and PI3K/Akt pathways were involved in EMT in MLE12 induced by silica.SiO2-activated MLE12 promoted fibroblast activation and migration via HECTD1.Conclusion:Current study elucidated a link between SiO2-induced macrophage activation,EMT and the circHECTD1/HECTD1 pathway,thereby providing a novel insight into the potential use of HECTD1 to develop novel therapeutic strategies for treating silicosis. |
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