Establishment Of Pneumoconiosis Mouse Model And Analysis Of Transcriptome

Pneumoconiosis is an occupational disease caused by the accumulation of dust particles in the lungs.The main symptoms of pneumoconiosis are chest tightness,cough,fatigue and other symptoms.The pathogenesis of pneumoconiosis is still remain elusive.High-throughput sequencing technology has becoming an important auxiliary means to search for key pathogenic genes.The differential expressed genes and alternative splicing events in the pathogenesis of pneumoconiosis can be screened by using RNA-seq,the miRNAs which regulate target gene expression can also be identified by miRNA sequencing.It is helpful to explore the pathogenesis of pneumoconiosis by establishing regulatory network based on transcriptome high-throughput sequencing and bioinformatics analysis.The mouse pneumoconiosis model was established through unexposed trachea and movable dust.Based on mRNA and miRNA high-throughput sequencing,the genes with significant changes in expression and alternative splicing events with different pattern were identified,the miRNA-target gene network diagram composed of miRNA and its target genes was explored,for understanding the pathogenesis of pneumoconiosis from the perspective of gene expression regulation.The specific experimental results of this paper are as follows:1.The mouse model of pneumoconiosis was established by non-exposure tracheal method.Micron-sized silicon dioxide particles with concentrations of 20 mg/ml and 60mg/ml were injected into the lung tissue.On the 28 th and 56 th days,mice were killed,and lung tissue,serum were taken out.The changes of inflammatory response,oxidative stress response and collagen accumulation were detected after pulmonary fibrosis.HE staining showed that the lung tissue morphology changed obviously,and obvious silicon nodules appeared after 56 days of stress.Compared with normal saline control group,the contents of inflammation related factors in the experimental group increased.The levels of MDA and ROS in oxidative stress factors increased significantly,while superoxide dismutase decreased,the level of collagen-related genes and the expression of HYP were significantly increased.These experimental results confirmed that the lung of the animal model constructed by non-exposure trachea produced significant fibrosis.2.Establishment of pneumoconiosis model in mice by dynamic exposure to dust.Mice exposed to dust simulated each stage of pulmonary fibrosis.They were stressed with silicon dioxide for 7 days,14 days,28 days and 56 days,and stressed for 2h,4h and 8h every day,respectively,in order to simulate the various stages of pulmonary fibrosis HE staining section demonstrated that with the increase of dust exposure time,the degree of pulmonary fibrosis became more and more obvious,and the content of inflammatory factors was positively correlated with stress time.Masson staining and HYP content test confirmed that collagen accumulation in lung tissue increased.Combined with these experimental results,it can be deduced that lung tissue showed obvious fibrosis after long-term exposure to dust in mice.3.mRNA sequence analysis of genes.(1)The experimental group exposed to dynamic dust for 4h every day was selected for high-throughput sequencing,and the differentially expressed genes and differential alternative splicing events were analyzed at different stages.Venn map showed that there were 14 differentially expressed genes in each experimental group.GO enrichment analysis showed that differentially expressed genes were mainly involved in cell cycle regulation and immune response regulation.KEGG enrichment analysis showed that differentially expressed genes were mainly involved in inflammatory reaction,pneumoconiosis pulmonary complications and fibrosis formation.(2)Screening the differentially expressed alternative splicing events,it was found that each experimental group had the most different splicing events of exon skipping pattern.GO enrichment analysis showed that the event of differential alternative splicing was mainly involved in RNA splicing regulation,nervous system regulation and metabolic regulation.KEGG enrichment analysis showed that differential alternative splicing events mainly occurred in phosphatidylinositol,metabolism,osteoclast differentiation,MAPK and TNF signaling pathways.4.miRNA sequence analysis of genes.The control group,the 7-day,and 28-day/4h dynamic dust exposure group were selected for sequencing and post-analysis,and the results showed that there is one significantly differentially expressed miRNA in the 7-day stress group and 14 significantly differentially expressed miRNAs in the 28-day stress group.Enrichment analysis of genes regulated by differential miRNA revealed that the significantly enriched GO term was mainly related to neural regulation and biological regulation.The KEGG enrichment signaling pathway is mainly related to metabolism,signaling pathway and biosynthesis.In conclusion,stable mouse models were established by non-tracheal exposure and dynamic dust exposure,respectively,and the stability of the animal model was tested by detecting hydroxyproline and the changes of various factors.Besides,transcriptome sequencing was performed on the lung tissue to screen out the key genes and miRNA,and enrichment analysis was performed on the differentially expressed genes,laying a foundation for subsequent exploration of the molecular mechanism of pneumoconiosis.