Mechanisms Of MiR-122 Regulating ADAM10 In Silica-induced Pulmonary Fibrosis

Silicosis,a kind of pneumoconiosis,is a worldwide occupational lung disease caused by long-term inhalation of silica dusts.It is characterized by damaged alveolar epithelial cells,abnormal proliferation of pulmonary fibroblasts,massive deposition of extracellular matrix and eventually extensive fibrosis in the lungs.Even the exposure of silica dust ceases,the process of fibrosis will still continue which seriously affects the health of dust exposed workers causing a huge social burden.The existing studies on silicosis could not provide effective treatment methods to block or reverse pulmonary fibrosis,so it is of great significance to further study the pathogenesis of silicosis.Fibroblasts are key effector cells in fibrotic disease.Silica activates macrophages and damages alveolar epithelial cells after entering lung tissue,resulting in the secretion and release of a large number of inflammatory factors and profibrotic factor.Transforming growth factor-β1(TGF-β1)is a critical profibrotic factor,which can induce activation of pulmonary fibroblasts.Upon activation,resting fibroblasts obtain a myofifibroblast phenotype,which is characterized by expression of contractile proteins and enhanced release of extracellular matrix.Excessive deposition of a large amount of extracellular matrix in the alveolar space gradually leads to the formation of scarring and nodular lesions,resulting in abnormal remodeling of the lung tissue structure.Micro RNAs(miRNAs)are a class of small,non-coding RNAs with within 25 nucleotides in length.They could bind to the 3’-UTR region of target m RNA through base pairing,regulate gene expression post-transcriptionally.Growing evidences have found multiple dysregulated miRNAs in a variety of diseases and play an important role in the occurrence and development of human diseases,including pulmonary fibrosis.In our previous miRNA microarray analysis study,the expression of miR-122 was decreased in fibrotic murine lung tissues induced by silica.This observation was further demonstrated by the finding that miR-122 was down-regulated in two fibroblast lines treated with TGF-β1 in vitro and silica-induced pulmonary fibrosis mouse model in vivo.Therefore,we hypothesize that miR-122 may play an important regulatory role in TGF-β1-medicated fibroblast activation and silica-induced pulmonary fibrosis.Objectivewe constructed mice silicosis model,TGF-β1-induced pulmonary fibroblast activation model and an intervention mice model with high expression of miR-122,to investigated the molecular mechanisms of miR-122 in the regulation of silicosis process,aimed to provide a new basis for prevention and treatment prevention and treatment of silicosis.Methods(1)Animal level: the model of silica-induced pulmonary fibrosis in mice and the model of miR-122 agomir intervention in pulmonary fibrosis were constructed,HE staining was used to observe the pathological changes of lung tissue and to score the severity of fibrosis.the expression of miR-122 in mouse lung tissue was verified by q RT-PCR and the expression of fibrosis markers was detected by western blot,hydroxyproline content was used to detect the degree of pulmonary fibrosis,to verify the intervention effect on silicosis of miR-122 in vivo.(2)Cell level: fibroblast activation models were constructed by treating lung fibroblasts(MRC-5 and NIH/3T3cells)with TGF-β1,the expression level of miR-122 was detected by q RT-PCR,fibrosis markers were detected by western blot;bioinformatics software was used to predict that ADAM10 was a potential target gene of miR-122,and the combination of them was verified by double luciferase reporter assay;lung fibroblasts were transfected with miR-122 mimic,ADAM10 si RNA,respectively.Then the levels of downstream molecules and fibrosis markers were detected by western blot and immunofluorescence.Besides,the Ed U cell proliferation assay kit was used to detect the proliferation of lung fibroblasts;miR-122 mimic and ADAM10 plasmid were co-transfected into TGF-β1 treated MRC-5 cells,to demonstrate that miR-122 regulates the activation of lung fibroblasts by ADAM10;the downstream molecular mechanism of ADAM10 was further validated by Ephrin-B2 extracellular recombinant protein treating the MRC-5 cells with ADAM10 si RNA or plasmids.Results1.miR-122 was decreased in silica-induced mouse pulmonary fibrosis modelHE staining results of pathological sections showed that the severity of pulmonary fibrosis increased gradually with the prolonged exposure time.The formation of silica nodules was observed in the lung tissue of the dust-stained 28 d mice.The protein expression levels of fibrosis markers including Fibronectin 、Collagen I、α-SMA、Vimentin increased gradually,while miR-122 expression levels in lung tissue decreased.2.miR-122 alleviates silica-induced pulmonary fibrosisThe intervention effect of miR-122 on pulmonary fibrosis was observed after increasing the expression of miR-122 in mice by injecting miR-122 agomir through tail vein.HE staining and fibrosis score of mice showed that the severity and distribution of pulmonary fibrosis in the intervention group were significantly reduced compared with that in the miR-NC agomir group;Western blot and hydroxyproline content test results also indicated that the expression of fibrosis markers in the intervention group decreased significantly.Above results indicate that elevated miR-122 expression can alleviate the pulmonary fibrosis induced by silica dust.3.miR-122 inhibits TGF-β1-induced activation of fibroblastsThe expression level of miR-122 decreased and the protein expression level of fibrosis markers(Fibronectin、Collagen I、α-SMA、Vimentin)increased in TGF-β1treated lung fibroblasts,which was consistent with results of mice lung tissue.After the transfection of miR-122 mimic into lung fibroblasts,the expression level of fibrosis markers and the proliferation ability of lung fibroblasts were significantly inhibited.These results showed that miR-122 could inhibit TGF-β1 induced pulmonary fibroblast activation.4.ADAM10 is the target gene of miR-122Through prediction in bioinformatics website(Targetscan),we found ADAM10 might be a potential target gene of miR-122 which was proved in existing studies.We further confirmed the combination in activated fibroblasts induced with TGF-β1 by double luciferase reporter gene experiments.ADAM10 expression levels were elevated in mouse silicosis and TGF-β1-treated pulmonary fibroblasts,while ADAM10 expression was inhibited in vivo and in vitro,further suggesting a regulatory relationship between miR-122 and ADAM10.5.miR-122 affects the activation of lung fibroblasts by regulating ADAM10On the basis of confirmed combination between miR-122 and ADAM10,we further explored whether miR-122 could affect lung fibroblast activation through regulating ADAM10.Firstly,we transfected ADAM10 si RNA into lung fibroblast,the results showed that both fibrotic markers protein expression and cell proliferation were inhibited.Then we co-transfected miR-122 mimic and ADAM10 plasmid into lung fibroblast finding that overexpression of ADAM10 could restore the inhibitory effect of miR-122 on fibrosis markers.6.ADAM10 affects pulmonary fibroblasts activation via Ephrin-B2/EPHB3/EPHB4/STAT3 signaling axisPrevious studies have confirmed that ADAM10 could shear the transmembrane proteins Ephrin-B2 extracellular domain on the surface of lung fibroblasts,producing soluble s Ephrin-B2,and promote the activation of lung fibroblasts through its receptors EPHB3/4.Interestingly,studies also found that both EPHB3 and EPHB4 promoted STAT3 phosphorylation.Therefore,we further explored whether ADAM10 affected the phosphorylation of STAT3 by regulating Ephrin-B2.We observed increased protein levels of fibrosis markers and STAT3 phosphorylation after treatment of MRC-5 cells using Ephrin-B2 extracellular recombinant protein,however,it could be inhibited by EPHB3/4 si RNA.Then we co-transfected ADAM10 plasmid and Ephrin-B2 si RNA into MRC-5 cells,results showed that knockdown of Ephrin-B2 expression significantly inhibited ADAM10 induced elevated protein expression levels of both p-STAT3 and fibrosis markers.In addition,the results of functional recovery experiments showed that Ephrin-B2-Fc could reverse the inhibitory effect of ADAM10 si RNA on protein expression of p-STAT3 and fibrosis markers.Finally,after combined transfection of miR-122 mimic and ADAM10 plasmids into MRC-5 cells,we observed that ADAM10 could revert the inhibition of miR-122 to p-STAT3.These results suggest that miR-122 regulated ADAM10 inhibition of pulmonary fibroblast activation,at least,partly depend on Ephrin-B2/EPHB3/EPHB4/STAT3 pathways.ConclusionIn this study,in vivo and in vitro experiments demonstrated that miR-122 played an anti-fibrotic role by targeting ADAM10 to regulate Ephrin-B2,influencing pulmonary fibroblast activation through EPHB3/EPHB4/STAT3 pathways in silica-induced pulmonary fibrosis.