The Study On The Mechanism Of Macrophage-derived MiR-7219-3p-containing Exosomes Mediate Fibroblast Activation In Silicosis

Background and objective:Silicosis is one of the most serious occupational diseases with the main feature of inflammatory cell infiltration,fibroblasts activation,and large deposition of extracellular matrix in the lung.Increasing evidence indicates that macrophage-derived exosomes may play an important role in the development of silicosis by transferring its loaded micro RNAs（miRNAs）.Here we confirmed the pro-fibrotic role of exosomes from silica-exposed macrophages and further explored the potential mechanisms of exosomal miR-7219-3p in fibroblast to myofibroblast activation and silicosis,which may provide a potential new insight for the treatment of silicosis.Methods:（1）The detection of macrophage-derived exosomal miRNAs and their target gene prediction:macrophages were treated with200ug/ml SiO₂ and 0ug/ml SiO₂ respectively for 48h,and exosomes were extracted by ultracentrifugation,then detected by transmission electron microscope（TEM）,nanoparticle tracking and analysis technology（NTA）and Western blotting,the expression of exosomal miRNAs were detected by RT-q PCR to verify the sequencing data.Potential target genes of miRNAs were predicted through the online database（Targetscan）.Functional annotation of target genes was conducted via Diana online tools mir Path-v3.The expression of miR-7219-3p in vitro and in vivo:RT-q PCR was used to measure the expression levels of miR-7219-3p in macrophages and their exosomes,fibroblasts,and silicotic mice.（2）The effect of miR-7219-3p in vivo silicotic mouse model:Mice were performed in control（saline）,SiO₂（50mg/kg）,SiO₂（50mg/kg）+NC-antagomir,SiO₂（50mg/kg）+miR-7219-3p-antagomir group,then lung tissues were collected after 28 days.The expression levels of miR-7219-3p in each group were determined by RT-q PCR,and lung tissues were observed by HE staining andα-SMA immunohistochemistry.The expression levels of Collagen-1 were determined by Western blotting.（3）The effect of miR-7219-3p on fibroblast activation in vitro:Fibroblasts were treated in the Control group,NC-mimic group,miR-7219-3p-mimic group,and TGF-βgroup;NC-mimic containing exosome group,miR-7219-3p-mimic containing exosome group,and PBS group,SiO₂+NC-inhibitor-containing exosome group,and SiO₂+miR-7219-3p-inhibitor-containning exosome group.then the expression of myofibroblast markers（α-SMA,Collagen I）was measured by Western blotting.Cell scratch assay and proliferation assay were conducted to explore the effect of miR-7219-3p on the migration and proliferation of fibroblasts.（4）The molecular mechanism of miR-7219-3p in regulating fibroblasts activation:the potential target gene Sprouty1（SPRY1）of miR-7219-3p was predicted by the Targetscan target gene prediction website,and the target-targeting relationship of SPRY1 and miR-7219-3p was verified by double luciferase reporter experiment.The expression of ERK/MAPK and its phosphorylated proteins were determined by Western blotting.（5）The function and mechanism of SPRY1 in the fibroblast activation induced by miR-7219-3p:co-transfected with miR-7219-3p and SPRY1plasmid,the expression of myofibroblast markers（α-SMA,Collagen I）,ERK and its phosphorylated protein were detected by Western blotting.Results:（1）Transmission electron microscopy（TEM）revealed a typical saucer-like vesicle with a diameter under 100nm.Nanoparticle tracking analysis（NTA）showed vesicles diameter ranging from 30-110 nm in both conditions.Moreover,macrophages secreted more exosomes apparently after silica-exposed compared to normal conditions.Western blot analysis further confirmed that the expression level of exosomal specific protein markers:HSP70,TSG101,and CD63 was significantly up-regulated in the silica-exposure group.These results suggested that silica-exposure facilitated macrophage secreting of more exosomes.Furthermore,14 significantly differentially-expressed miRNAs were verified by RT-q PCR:miR-219c-3p、miR-365-3p、miR-93-3p、let-7d-3p、miR-7219-3p、miR-328-3p were elevated,miR-378d、miR-664-3p were down-regulated.The most enriched GO terms include cell differentiation,cell cycle,cell morphogenesis,and cell-cell organization were presented.KEGG pathway analysis showed number of genes were enriched in the TGF-βsignaling pathway,ECM-receptor interaction,m TOR signaling pathway,Focal adhesion,etc,which were critical in silicosis development.In the vitro and vivo experiments,miR-7219-3p is highly expressed in vivo,as well as in silica-exposed macrophages and their exosomes,which can be transferred to fibroblasts.（2）Pathological changes showed an obvious inflammatory reaction,the proliferation of myofibroblasts,nodules formation,and collagen deposition in the silica group and silica+NC-antagomir group,while it was attenuated in the miR-7219-3p antagomir group.In addition,the inhibition of miR-7219-3p also downregulated the expression of collagen 1 in vivo.（3）To further investigate the affection of miR-7219-3p in fibroblasts,we up-regulated the expression level of miR-7219-3p by transfection miRNA mimics or miR-7219-3p containing exosomes in NIH-3T3 cells,an obvious elevation of fibrotic markers（α-SMA,Collagen I）were detected by western blotting in fibroblasts.The results of the CCK8 assay and cell scratch assay showed increased proliferation and migration ability of fibroblasts induced by miR-7219-3p.Furthermore,the inhibition of miR-7219-3p also downregulated the level ofα-SMA,Collagen I.（4）The dual-luciferase reporter experiment results indicated an obvious reduction in SPRY1-wt&miR-7219-3p mimics co-transfection group compared with SPRY1-mut&miR-7219-3p mimics group,and no difference in NC mimics group.The up-regulation or downregulation of miR-7219-3p promoted or inhibited the expression level of p-ERK.（5）As the results showed:knock-down and over-expression of SPRY1promoted or inhibited the expression level of p-ERK,α-SMA,Collagen I respectively,with no influence on ERK.The above conclusions demonstrated that SPRY1,as a target of miR-7219-3p,played a role in regulating fibroblast activation through the RAS/ERK/MAPK pathway.Conclusions:（1）The exposition of SiO₂ induced the change of exosomal miRNAs,these miRNAs were closely related to silicosis.（2）MiR-7219-3p is highly expressed in vivo,as well as in silica-exposed macrophages and their exosomes,which can be transferred to fibroblasts,and induced fibroblasts activation.（3）Exosomal miR-7219-3p regulates ERK/MAPK activation in fibroblasts by targeting SPRY1,thus affect fibroblast’s activation.