Effect Of Exosomal MiR-107 On Transdifferentiation Of Pulmonary Fibroblast In Silicosis And Its Mechanism

Background and ObjectivePulmonary fibroblast transdifferentiation plays a key role in the development of silicosis.Previous studies have suggested that fibroblasts exposed to silicon dust are activated by cytokines,mechanical factors and extracellular matrix,etc.Then they transdifferentiate into myofibroblasts which can synthesize and secrete excessive collagen to cause silicosis.In recent years,studies have shown that exosomes and their contents,especially miRNAs,represent a new type of intercellular signal transmission mediator related to angiogenesis,antigen presentation,intracellular homeostasis,inflammation,etc.Exosomes participates in biological processes and regulate the occurrence and development of various diseases including cancer and fibrosis.Our previous study revealed the change of serum exosomal miRNA profile in silicosis patients using high-throughput miRNA sequencing.However,the actual level of exosomal miRNAs,the signal transmission of exosomal miRNAs and its mechanism during silicosis are remains unclear.Therefore,the serological investigation of silicosis patients and the systematic observation of mouse silicosis models were conducted to explore the levels and distribution characteristics of exosomes miRNAs in peripheral blood.We investigated the effect of exosomal miRNAs derived from macrophages on pulmonary fibroblasts and found the potential targets and signal transduction pathways to reveal its exact regulatory mechanism.The present research provides a new insight to extent the explanation of pathogenesis of silicosis,and gives reference to the discovery of potential silicosis biomarkers and intervention targets.Methods1.Detection of serum exosomal miRNAs and prediction of target genes of miRNAs in patients with silicosis1.1 PatientsFifty male patients with silicosis and 50 male normal controls were recruited.Their basic information and venous blood were collected.1.2 Exosomes isolationExosomes were isolated using different methods including ultracentrifugation,ultracentrifugation with 30%sucrose pad,8%PEG precipitation plus wash step,8%PEG precipitation plus 5%PEG precipitation and the commercial kit.Exosomes were characterized by transmission electron microscopy,nanoparticle tracking analysis as well as Western blot.With the reference to yield and purity,exosomes were compared and the method was selected for serum exosome isolation.1.3 Serum exosomal miRNAs level in silicotic patientsMiRNAs were extracted from serum exosomes.Levels of serum exosomal miR-27b-5p,miR-30c-2-3p,miR-107,miR-122-5p,miR-125a-5p,miR-126-5p,and miR-335-5p of the subjects were examined using RT-qPCR.1.4 Prediction of target genes of the miRNAsMiRWalk website was used to predict target genes of the miRNAs.2.Temporal level of miR-107 and its function in silicosis mouse2.1 Establishment of silicosis model in mouseFifty C57BL/6N mice were randomly but equally divided into control group and SiO2 group,with 25 mice in each group.The silicosis mouse model with silicosis was established by non-exposed tracheal instillation of silica particles.After silica exposure,mice were sacrificed on day 1,7,14,28,56.Their peripheral blood and lung tissue were collected.The alveolar structure and collagen deposition in mice lung tissues of the mice were observed by HE and Masson staining.The expression of fibrosis hallmarks and CDK6 were examined using Western blot.2.2 Levels of miR-107 in serum exosomes and lung tissues in silicosis mouseLevels of miR-107 in serum exosomes and lung tissues of the mice were detected by RT-qPCR,and the correlation between them was analyzed.2.3 Effects of miR-107 antagomir on SiO2-induced pulmonary fibrosis in mouseMiR-107 antagomir was used to interfere the SiO2-induced pulmonary fibrosis in mouse.Twenty mice were randomly divided into the SiO2 group,SiO2+anta-miR group,SiO2+anta-NC group and control group,with 5 mice in each group.After silica exposure for 28 days,the mice were sacrificed and the pathological changes in the lungs were observed.The levle of miR-107 and expression of related proteins in the lung tissues were detected.3.Effects of exosomes of macrophage-derived exosomes and exosomal miR-107 in the transdifferentiation of pulmonary fibroblasts3.1 Characterization of exosomes derived from silica-exposed macrophageRAW264.7 cells were treated with silica particles to establish the in vitro cell model.Exosomes from macrophages both silica-exposed or not were isolated and characterised.3.2 Effects of exosomes and exosomal miR-107 on the transdifferentiation of pulmonary fibroblastsIsolated exosomes were incubated with pulmonary fibroblasts.Uptake of exosomes was observed by laser confocal microscopy.MiR-107 level in macrophages and their secreted exosomes and pulmonary fibroblasts were detected by RT-qPCR.Expression of a-SMA,Collagen I,Fibronectin and CDK6 protein of pulmonary fibroblasts were examined by Western blot.MiR-107 synthetic mimics were transfected into macrophages to elevate the level of miR-107 in exosomes,then the exosomes were co-cultured with pulmonary fibroblasts,and the expression of transdifferentiation hallmarks and CDK6 were examined.3.3 Role of miR-107 in the transdifferentiation of pulmonary fibroblasts induced by TGF-βThe dual luciferase gene report assay was used to confirm the interaction between miR-107 and CDK6.MiR-107 mimic or mimic-NC was co-transfected with CDK63’UTR wild or mutant type plasmid into 293T cells,and then the luciferase activity level was examined.The level of miR-107 and CDK6 in NIH-3T3 cells treated with a series of concentrations of TGF-β was detected by using RT-qPCR.MiR-107 mimic,miR-107 inhibitor,CDK6 overexpression plasmid and CDK6 siRNA were transfected into NIH-3T3 cells respectively.Then the expression of transdifferentiation hallmarks and signaling pathway related proteins were detected by Western blot and the cell cycle was detected by flow cytometry.MiR-107 mimic,mimic-NC,miR-107 inhibitor and inhibitor-NC were transfected into MRC-5 cells respectively,then the expression of transdifferentiation hallmarks and CDK6 was detected.4.Statistical analysisSoftware SPSS 21.0 statistical software was used for statistical analysis.The data under normal distribution were expressed as mean±standard deviation,t test was used to compare two groups while ANOVA was used for the comparison of multiple groups.The skewed distributional data was represented by the median,the first quartile and the third quartile.The rank sum test was used for the comparison of the samples.P values<0.05 were considered statistically significant,unless otherwise indicated.Results1.Detection of serum exosomal miRNAs in patients with silicosis and prediction of target genes of miRNAs1.1 Comparison of different methods for the isolation of serum exosomeThe ultracentrifugation method has low yield and purity of exosomes.The method of ultracentrifugation with 30%sucrose pad has high purity but low yield of exosomes.The kit method has high yield but low purity of exosomes.Compared with method of 8%PEG precipitation plus wash step,the exosomes purity of the 8%PEG precipitation plus 5%PEG precipitation method is slightly lower but the yield is more abundant.Among them,8%PEG precipitation plus 5%PEG precipitation method is the most appropriate method.1.2 Serum exosomal miRNAs levels in patients with silicosisWhile age did not significantly differ between the normal controls and the patients,neither age nor dust exposure time showed significant difference among patients at different stages of silicosis.Levels of miR-27b-3p,miR-107,miR-122-5p,miR-125 a-5p,miR-126-5p and miR-335-5p in the case group were significantly higher than those in the control group(P<0.05).Further comparison revealed that compared with patients with silicosis at stage Ⅰ,levels of five miRNAs in patients at stage II were decreased,the difference were statistically significant(P<0.05).1.3 Target genes of miRNAs were identified by bioinformatics methodMiRWalk online website was used to predict the target genes of miR-107,miR-122-5p,miR-125a-5p,miR-126-5p from human and mouse origins.Five miRNA-target gene interactions were predicted:miR-107～CDK6,miR-122-5p～TFDP2,miR-125a-5p～TRAF6,miR-125a-5p～CBX7 and miR-125a-5p～VSIR.Among them,the interaction of miR-107 and CDK6 acquired the highest prediction score.2.Temporal level of miR-107 and its function in silicosis mouse2.1 Establishement of silicosis mouse modelThe histopathological findings showed that mice exposed to silica showed mainly lung inflammation during the first 14 days,and afterwards followed the formation of lung fibrosis.2.2 Levels of miR-107 in serum exosomes and lung tissuesCompared with the control group,the level of serum exosomal miR-107 was decreased on the 7th day,while increased on the 14th,28th,and 56th day.The difference was statistically significant(P<0.05).Consistently,miR-107 level in the lung tissue was decreased on the 7th day after silica exposure,and increased on the 28th and 56th day.The difference was statistically significant(P<0.05).There was a positive correlation of miR-107 level between in serum exosomes and in lung tissue(P<0.05).2.3 Effect of inhibiting miR-107 on SiO2-induced pulmonary fibrosis in mouseCompared with the SiO2+anta-NC group,the pathological changes of pulmonary fibrosis in SiO2+anta-miR group were significantly reduced;the level of miR-107 and expression fibrosis hallmarks was decreased,while CDK6 was increased.3.Effects of exosomes of macrophage-derived exosomes and exosomal miR-107 in the transdifferentiation of pulmonary fibroblasts3.1 Characterization of exosomes derived from macrophagesExosomes were observed to be saucer-like structure under TEM with size rangeing from 30 to 150 nm under TEM.The number of exosomes secreted by macrophages was increased after silica exposure.The exosomal expression of ALIX,TSG101,and CD63 was also increased with statistical significance(P<0.05).3.2 Effect of exosomes derived from silica-exposed macrophages on the transdifferentiation of pulmonary fibroblastsAfter co-incubation of exosomes and NIH-3T3 cells,red spindle-shaped NIH-3T3 cells could be seen under a laser confocal microscope.Green dots representing exosomes were fluorescently concentrated around the cells or fuse with the cell membrane(yellow),which indicating the uptaken of exosomes by the NIH-3T3 cells.Silica treatment resulted in an incrase of miR-107 level in macrophages,exosomes and activated fibroblasts.With exosomes derived from silica-exposed macrophage treated,expression of transdifferentiation related-proteins was significantly increased,and CDK6 was decreased.The miR-107 level was increased in exosomes derived from macrophages with miR-107-mimic treated,and the expression of transdifferentiation makers in NIH-3T3 cells also increased after co-culctured with the above exosomes.3.3 The mechanism of miR-107 in activating the transdifferentiation of lung fibroblasts induced by TGF-βThe results of the double luciferase gene report assay revealed that co-transfection of miR-107 mimic with the CDK6-3’UTR wild-type plasmid reduced the luciferase activity level when compared with its control(P<0.05).While co-transfection of miR-107 mimic with the mutated CDK6-3’UTR plasmid failed to diminish the luciferase activity level.With the increasing concentration of TGF-β,miR-107 level was increased while the expression CDK6 was downregulated.2 ng/mL has proved to be an effective concentration chosen for subsequent studies.With the addition of miR-107 mimic,miR-107 level was increased,CDK6 expression was decreased,and transdifferentiation related-genes and-proteins were also increased.Oppositely,the addtion of miR-107 inhibitor downregulated the level of miR-107 but upregulated the expression of CDK6.Meanwhile,transdifferentiation related-genes and-proteins was decreased,the proportion of cells in G1 phase was decreased,the proportion of cells in S phase was increased,and the expression of downstream genes cyclin D,pRb,E2F1 was decreased.The difference was statistically significant(P<0.05).In NIH-3T3 cells,CDK6 overexpression from plasmid resulted in the decrease of transdifferentiation markers,and knock-down via siRNA led to the increase of those marker proteins with statistically significant difference(P<0.05).Consistantly,the expression of CDK6 and transdifferentiation markers was also negatively correlated in both the mimic-miR group and the inhibitor-miR group in MRC-5 cells.ConclusionThe level of miR-107 in pulmonary tissue and serum exosomes was increased after the inflammatory phase in the pathogenesis of silicosis and inhibition of miR-107 could significantly reduce pulmonary fibrosis.In vitro,exosomal miR-107 derived from silica-exposed macrophages can promote lung fibroblast transdifferentiation,and may associate to the induction of cell cycle arrest.