| Objective:To investigate the effect of dust on the expression profile of exosome-derived miRNAs in rat alveolar lavage fluid and to explore the temporal changes in the expression of some exosome-derived miRNAs(miRNA-21-5p,miRNA-29a-3p,miRNA-26a-5p,miRNA-34a-5p)during pulmonary fibrosis.Methods:Sixty healthy male SD rats were randomly divided into three groups(20 rats per group):control,coal dust and quartz groups.The rats were injected with 1 ml of coal dust suspension(50 mg/ml)and 1 ml of quartz suspension(50 mg/ml)into the trachea of the coal dust and quartz groups respectively by means of disposable non-exposed intratracheal dusting,and the control rats were injected with the same volume of saline,and were anaesthetized,sampled and executed after 2,4,8 and 16 weeks of dusting,respectively,for subsequent experiments.Routine HE staining and Masson staining were used to evaluate the extent of lung tissue damage and fibrotic lesions in rats at the pathological level.The total collagen level of rat lung tissue was analysed by the hydroxyproline method(HYP)to quantitatively evaluate the extent of lung tissue fibrosis.Exosomes were extracted by the dose box method(PEG precipitation method),observed by transmission electron microscopy,detected by Nano Tracking Analysis(NTA)for exosome concentration,and detected by Western Blot for exosome surface marker proteins.Three 4-week-old rats were selected from the quartz group and the control group,and the bronchoalveolar lavage fluid was extracted for highthroughput sequencing to screen out the differentially expressed genes,and GO enrichment analysis and KEGG pathway prediction were performed.The sequencing results were validated by quantitative real-time fluorescence polymerase chain reaction(q RT-PCR)and the temporal patterns of the target exosome-derived miRNAs were observed.Results:1.Histopathological and fibrotic findings: HE staining showed that the alveoli of the control rats were structurally intact,with no obvious inflammatory cells in the alveoli or in the alveolar septa;whereas in the coal dust and quartz groups,foci of coal dust fibres and a large number of inflammatory cell infiltrates,as well as obvious silica nodules,were evident in the late lungs as time progressed.collagen fibres;whereas the late collagen accumulation in the lung tissue of the rats in the coal dust and quartz groups was very severe.Hydroxyproline results: Compared with the control group,the 8-week quartz group,the 16-week coal dust group and the quartz group had significant increases in HYP content in lung tissue(P<0.05).2.The results of electron microscopy(TEM)showed that the exosomes derived from rat alveolar lavage fluid extracted by PEG co-precipitation kit were round in shape,about 50-150 nm in diameter,and had an obvious complete bilayer membrane structure.Particle size identification(NTA)results showed that the main peak size of exosomes was 95.82 nm,and the particle concentration was 4.78E+10/m L.Surface protein detection(WB)showed that the exosome marker protein CD9 and CD81 were positive.3.High-throughput sequencing and bioinformatics analysis: 66 different expressed miRNAs including 37 upstream genes and 29 downstream genes were analysed in experimental and control groups(P<0.05).GO functional analysis showed that most of the differential genes were enriched in redox processes,protein phosphorylation,transmembrane transport,mitochondria,protein binding,transferase activity,etc.KEGG pathway analysis showed that most of the differential genes were enriched in cancer pathway,MAPK signaling pathway,Fox O signaling pathway,etc.4.PCR validation of differential exosomes-derived miRNAs: Compared with the control group,the levels of miRNA-21-5p and miRNA-29a-3p in the coal dust group and the quartz group were increased(P<0.05),while the levels of miRNA-26a-5p and miRNA-34a-5p were decreased(P<0.05),which was consistent with the previous sequencing results.5.Temporal changes in the expression of exosome-derived target miRNAs:(1)miRNA-21-5p: The expression of this gene in the control group was stable with increasing dusting time,while the expression in the coal dust and quartz groups showed an overall increasing trend.In the quartz group,the expression started to increase at week 8 and reached a peak at week16(P <0.05).In the coal dust group,the expression started to increase at week 4 and reached a peak at week 16(P<0.05).(2)miRNA-29a-3p: The expression of this gene increased in the control,coal dust and quartz groups with the increase of dusting time,and reached the highest expression in all groups at week 16(P<0.05).(3)miRNA-26a-5p: The expression of this gene was stable in the control group,quartz group and coal dust group in the early stage with the increase of dusting time,and showed a significant decreasing trend in the 8th week(P<0.05).(4)miRNA-34a-5p: with the increase of dusting time,the expression of this gene was stable in the control group and the coal dust group in the early stage and decreased at week 16(P<0.05);while the expression of this gene decreased in the quartz group from week 8 and was the lowest at week 16(P<0.05).Conclusion:1.Dust exposure causes changes in the expression profile of exosomal miRNA in bronchoalveolar lavage fluid of rats.2.From 2 to 16 weeks after dust exposure,the expressions of exosomal miRNA-21-5p and miRNA-29a-3p in the bronchoalveolar lavage fluid of rats showed an upward trend,while the expressions of exosomal miRNA-26a-5p and miRNA-34a-5p showed a downward trend in the dust exposure group. |
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