Screening And Identification Of Pneumoconiosis-Associated Genes In The TGF-β/Smad Signaling Pathway

Pneumoconiosis is one of the most frequently occurring and dangerous occupational diseases in China.Due to the lack of specific methods for early diagnosis and precise cure for pneumoconiosis,patients bear great economic pressure and the society also carry a huge burden.According to the report of the National Health and Family Planning Commission,the number of pneumoconiosis patients accounts for a large proportion in the total occupational diseases in recent years.The lungs of workers exposed in industrial dust environment with a high concentration for a long time will be repeatedly damaged,which will eventually lead to pulmonary fibrosis and loss of physiological function.Therefore,identifying the pathogenic factors,drug targets and bio-molecular markers in the process of pulmonary fibrosis is essential for the cure for pneumoconiosis.In this study,NIH-3T3 fibroblast model induced by TGF-β1,the mouse model of natural Si O₂ inhalation and the mouse model of pulmonary fibrosis by non-tracheal exposure was established.And then,the relevant metabolic pathways that play an important regulatory role in the process of pulmonary fibrosis were screened via transcriptome sequencing,bioinformatics analysis and experimental validation.The several key genes with significant expression changes in the pathways were found by functional enrichment analysis and literature mining.The screened genes were further excavated,which may provide an important reference for the prevention and cure of pneumoconiosis in the future.The results of the study are as follows.1.A single cytokine TGF-β-treated mouse NIH-3T3 fibroblast model was established.NIH-3T3 cells in logarithmic growth phase were starved and induced by TGF-β1（10 ng/m L）for 12 h.The expression level of fibrosis marker genes and proteins was identified.The results showed that compared with that in the control group,cell debris increased and fibrous contact angle was more obvious in the experimental group.And the expression level of five fibrosis marker genes（Collagen1α1,Collagen1α2,α-SMA,TGF-β1,TGF-β3）were significantly up-regulated（P<0.01）.There was no significant change in Smad3 protein,and the expressions of phosphorylation of Smad3（P-Smad3）and marker proteinsα-SMA were significantly up-regulated（P<0.01）.It indicates that the addition of TGF-βfactor can activate the downstream Smad signaling pathway,and the phosphorylation level of Smad3protein was significantly increased,which may further significantly up-regulate the expression ofα-SMA protein.The expression of collagen factors such as Collagen1α1,Collagen1α2,α-SMA and pro-fibrotic factors TGF-β1 and TGF-β3 in this pathway also showed a significant up-regulation trend.The experimental results showed that the cell model was successfully established and could be used to perform the subsequent transcriptome sequencing.2.Based on the cell model,cells were collected,total RNA was extracted and transcriptome sequencing was performed.Bioinformatics analysis revealed that a total of1284 genes were significantly differentially expressed,of which 495 genes were up-regulated and 789 genes were down-regulated.GO and KEGG enrichment analysis revealed that the differentially expressed genes were enriched in many pathways,including ECM pathway,TNF pathway,TGF pathway,MAPK pathway and cytokine-cytokine receptor pathway when P_adjust<0.05.25 key genes were screened from these pathways for experimental validation.The results showed that the expression of Itga5,Itga2,Itgb1,Itgb3,Thbs1,Smad7,Fbn1,TGF-β1,TGF-β3,Cx3cl1,IL-6,Fgf2,Fdgfc,Gadd45b and Ngf genes were significantly up-regulated（P<0.01）.Thbs2,Dcn,Smad3,Cxcl5,Cxcl1,Angpt2,Vegfd,Pdgfra,IL-33,IL-7 genes expression were significantly down-regulated（P<0.01）.The above expression trends of the 25 key genes screened by real-time PCR method in the TGF-β-induced NIH-3T3 cell model were consistent with the RNA-sequencing results.3.The mouse model of Si O₂ natural inhalation was established to verify gene and protein expression.The mice were randomly divided into control group,natural Si O₂inhalation exposure group for 28 days and natural Si O₂ inhalation exposure group for 56days.A part of lung tissue was processed by Masson staining,and the degree of fibrosis was determined by observing collagen content.In the other part,total RNA and total protein were extracted,the expression of the above five marker genes and the m RNA of seven key genes in the TGF pathway obtained by sequencing were detected by q RT-PCR and the changes in the protein levels of important markers such as Smad3,P-Smad3,andα-SMA were detected by Western blot.Masson staining results showed that the collagen fiber content in lung tissue increased obviously with the increase of natural Si O₂ inhalation days,indicating that pulmonary fibrosis had occurred in mice at this stage.And the expression levels of fibrosis marker genes and proteins in lung tissue increased significantly with the increase of natural Si O2 inhalation time（P<0.01）.Among them,the expression of TGF-β1 gene was up-regulated,and the phosphorylation level of Smad was significantly increased.The expression levels of seven key genes in the TGF pathway were also consistent with those in the cellular model,which further confirmed the results from the RNA-sequencing of cell model.4.A mouse model of pulmonary fibrosis was established by non-tracheal exposure method.After acclimation for 1 week,50μL Si O₂ suspension（200 mg/m L）was directly administered by intratracheal instillation into mice.And the mice were normally raised for two months.After sacrifice,lung tissues were taken to extract primary fibroblast.The cells were cultured for 2-3 generations and the expression of fibrosis marker genes and important marker proteins,as well as key genes in the five pathways obtained by sequencing,were detected by q RT-PCR and Western Blot.The results showed that the expression of the marker genes and proteins were significantly up-regulated（P<0.01）.The key genes and osteopontin in the five pathways screened by sequencing were verified by experiments.The expression trend of genes and proteins was consistent with the results from RNA-sequencing and NIH-3T3 cell model.The above experimental results showed that the reliability of the experimental results was verified at three levels:transcriptome sequencing,the mouse model of natural Si O₂inhalation and the mouse model of pulmonary fibrosis by non-tracheal exposure.The genes and proteins in the five pulmonary fibrosis-related pathways identified by transcriptome sequencing,bioinformatics analysis and experimental validation are involved in the regulation of the pulmonary fibrosis process.Although the precise molecular mechanisms,in which these genes regulate pulmonary fibrosis,need to be further investigated,the identification of these genes has an important reference for understanding the key markers of pathogenic factors and drug targets of TGF-βregulation of pulmonary fibrosis,as well as for molecular marker screening.