The Regulatory Mechanism Of Exosomal MiR-23a-3p On The Pathogenesis Of Silicosis By Targeting CUL3

Objective: Silicosis is an irreversible lung disease characterized by diffuse pulmonary fibrosis and silicotic nodule due to exposure and inhalation of a large amount of free silicon dioxide(Si O2)in the process of occupational production.Si O2 dust cannot be degraded in the lung and can damage alveolar epithelial cells,expand the range of inflammatory response,and form local granulomas in the lung interstitial.At the same time,lung epithelial cell apoptosis is aggravated under the stimulation of various inflammatory factors,and lung epithelial cell apoptosis is one of the important causes of pulmonary fibrosis.Mi RNAs are highly conserved single-stranded non-coding RNAs that exist widely in nature.In recent years,the role of miRNA in pulmonary diseases has been gradually revealed,but the mechanism of miRNA played in the progression of silicosis remains to be further explored.This study aims to investigate the regulatory mechanism of exosomal miR-23a-3p in Si O2-induced lung inflammation and fibrosis.The effect of miR-23a-3p targeting CUL3 on apoptosis and reduction of fibrosis was explored at the cellular level,and verified in mouse silicosis models.Methods: In this study,the peripheral blood serum exosomes of silicosis patients and healthy people were collected for transcriptome sequencing.Bioinformatics analysis was performed to screen differentially expressed miR-23a-3p.THP-1 macrophage cell line was cultured in vitro,macrophages were activated by Si O2,exosome released from macrophages was blocked by GW4869,and the culture supernatant of macrophages in different treatment groups was added to A549 epithelial cell line stimulated by Si O2.The communication role exosomes played between macrophages and epithelial cells was verified by realtime-PCR.Si O2 was used to stimulate epithelial cells and miR-23a-3p mimic was added for overexpression.Dual luciferase assay was used to verify the targeting relationship between miR-23a-3p and CUL3.The effects of miR-23a-3p on apoptosis of lung epithelial cells and the expression of fibrosis factors were verified by western blot and realtime-PCR.Male C57BL\6 mice,aged 6-8 weeks,were randomly divided into 4 groups according to body weight,the control group,the Si O2 group,the Si O2+agomir-miR-23a-3p group and the Si O2+agomir-NC group,the mice in each silicosis group were unexposed.Silicosis mouse model was established by intratracheal instillation of Si O2.The overexpression effect of miR-23a-3p was detected by realtime-PCR.H&E staining was used to observe the pathological conditions of each group.Lung tissue apoptosis was observed by Tunel staining.Westernblot and realtime-PCR were used to detect the gene and protein levels of apoptosis or inflammatory fibrosis factors.The effect of miR-23a-3p overexpression on collagen deposition in lung tissue of mice was observed by hydroxyproline content detection and Masson staining.The above experiments demonstrated the regulatory effect of miR-23a-3p on lung tissue apopt osis and fibrosis induced by Si O2.Results: 1.63 differentially expressed miRNAs were screened by transcriptome sequencing,among which 14 were up-regulated and 49 were down-regulated.4 differentially expressed miRNAs were selected by orthogonal partial least squares regression and discriminant analysis(OPLS-DA),among which miR-23a-3p had the highest VIP value.2.The target gene of miR-23a-3p was CUL3.3.The results showed that in the Si O2+GW4869 group,the expression of miR-23a-3p was significantly decreased,while the gene expression levels of PAI1 and TGF-β1 were significantly increased.4.The expression level of miR-23a-3p in Si O2 group was significantly decreased,and the m RNA transcription level of its target gene CUL3 was significantly increased,with statistical significance(P<0.05).The expression level of miR-23a-3p in Si O2+ mimic-miR-23a-3p group was significantly increased,while the m RNA transcription level of CUL3 was significantly decreased(P<0.05).Western blot results showed that the protein levels of CUL3 and cleaved CASPASE3 in the Si O2 group were increased significantly,and the protein level of BCL2 was decreased significantly.Thehprotein levels of CUL3 and cleaved-CASPASE3 were decreased significantly in the Si O2+ mimi-mir-23a-3p group,and protein levels of BCL2 were increased significantly.5.Western blot results showed that the protein levels of PAI1 and TGF-β1 in Si O2 group were significantly Increased.The protein levels of PAI1 and TGF-β1 in Si O2+ mimic-miR-23a-3p group were significantly decreased.6.Realtime-PCR results showed that the m RNA transcription level of CUL3 in lung homogenate of mice in Si O2 group was significantly increased.ACUL3 in lung homogenate of mice in Si O2+agomir-miR-23a-3p group was significantly decreased.Western blot results showed that CUL3,cleaved-CASPASE3 protein levels were significantly increased,and BCL2 protein levels were significantly decreased in lung of mice in Si O2 group.In the lung homogenate of mice in Si O2+ agomir-miR-23a-3p group,CUL3 and cleaved-CASPASE3 protein levels were significantly decreased,and BCL2 protein levels were significantly increased.Tunel staining showed that lung tissue apoptosis was aggravated in Si O2 group.The apoptosis of lung tissue in Si O2+ agomir-miR-23a-3p group was significantly reduced.7.The inflammatory cell count results showed that the total cell numb-er,macrophages,lymphocytes andlneutrophils in the alveolar lavage fluid(BALF)of Si O2 group were significan-tly increased,whilej Si O2+ agomir-miR-23a-3p group was significantly decreased.H&E staining showed that the inflammatory cell in Si O2 group was aggravated.The inflammatory cell infiltration of lung tissue in Si O2+ agomir-miR-23a-3p group was reduced.Realtime-PCR results showed that the m RNA transcription levels of inflammatory cytokines Il-1β and Tnf-α in lung homogenate of mice in Si O2 group were significantly increased.Si O2+ agomir-miR-23a-3p group were significantly decreased.8.Masson staining showed that the collagen deposition in lung tissue of Si O2 group was significantly ag-gravated.The collagen deposition of lung tissue in Si O2+ agomir-miR-23a-3p groupwas significantly reduced.Hydroxyproline measurement results showed that the con-tent of hydroxyproline in lung tissue of mice in Si O2 group was significantly increased.Si O2+agomir-miR-23a-3p group was significantly decreased.Tgf-β1 and Pai1 in lung homogenate of mice in Si O2 group were significantly increased.Si O2+agomir-miR-23a-3p group were significantly decreased.Western blot results showed that the levels of fibrosis-related proteins TGF-β1 and α-SMA in lung of mice in Si O2 group were significantly increased.The protein levels of TGF-β1 and α-SMA in lung of mice in Si O2+ago mir-miR-23a-3p group were significantly decreased.Conclusion: 1.The expression of miR-23a-3p in the serum exosome of silicosis patients was down-regulated.2.Overexpressing miR-23a-3p alleviated the Si O2-induced lung inflammation and fibrosis through inhibiting the apoptosis of epithelial by targeting CUL3.