ABSTRACT
OBJECTIVE: To analyze transforming growth factor-β1( TGF-β1)-induced differentially expressed genes( DEGs) in human embryonic lung fibroblast( IMR-90) using microarray,and to screen the key genes and signaling pathways related to trans-differentiation of fibroblast.METHODS: The gene chip GSE17518,attained from TGF-β1 stimulated IMR-90 cells,was downloaded from the Gene Expression Omnibus database.The DEGs were screened by GENE-E software.Then,the DEGs were imported into the DAVID online database for Gene Ontology( GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes( KEGG) pathway enrichment analysis.The proteinprotein interaction( PPI) network was constructed and the hub genes were screened using STRING database and Cytoscape software.RESULTS: A total of 394 DEGs related to TGF-β1 stimulation were identified,including 171 down-regulated genes and 223 up-regulated genes.The results of GO analysis showed that the DEGs were widely distributed in cytoplasm,cell membrane,extracellular matrix( ECM) and exosomes,regulating biological functions such as ECM organization,cell migration and adhesion,cell proliferation and apoptosis.The results of the KEGG pathway analysis indicated that most of DEGs were enriched in cell focal adhesion,ECM-receptor interaction and phosphoinositide 3 kinase-Protein kinase B( PI3K-Akt) signaling pathways.The PPI network screened 10 core genes,included nucleolar protein 2( NOP2),succinate dehydrogenase B,glutamyl-prolyl-tRNA synthetase( EPRS),FtsJ homolog 3( FTSJ3),prefoldin subunit 4,Ras-related C3 botulinum toxin substrate 2,signal recognition particle receptor subunit beta,succinate-Co A ligase GDPforming beta subunit,pumilio RNA binding family member 3( KIAA0020),and general vesicular transport factor p115.NOP2,EPRS,FTSJ3,KIAA0020 were mainly distributed in M1 module.The NOP2 is the core gene with the highest number of nodes in M1 module.CONCLUSION: A total of 10 core differential genes and 7 signaling pathways related to TGF-β1 stimulation were screened.Among them,focal adhesion,ECM-receptor interaction,PI3K-Akt and NOP2,EPRS,FTSJ3,KIAA0020 may provide new direction for research of mechanisms of abnormal activation of fibrotic diseases including silicosis in incidence and development of multiple lung fibrotic diseases.
ABSTRACT
OBJECTIVE: To investigate the epithelial-mesenchymal transition(EMT) induced by direct or indirect exposure to free silicon dioxide(SiO_2) and the expression of surface protein marker in rat typeⅡalveolar epithelial cell RLE-6TN.METHODS: i) The alveolar macrophages(AM) were isolated from specific pathogen-free SD rat by bronchoalveolar lavage.AM and RLE-6TN were treated with 0-140 mg/L(final concentration) of SiO_2 suspension and were cultured conventionally for 24,48 and 72 hours. The cell viability was detected by CCK-8 assay. The result of CCK-8 essay was used to choose the SiO_2 concentration for the following study. ii) To establish models of RLE-6TN co-cultured with AM that were seeded in Transwell. The cells were divided into 4 groups: the direct control group(RLE-6TN,no SiO_2 exposed),the direct exposure group(RLE-6TN,treated with 100 mg/L SiO_2),the indirect control group(RLE-6TN and AM were cocultured,no SiO_2 exposed) and the indirect exposure group(RLE-6TN and AM were co-cultured,AM was treated with 100 mg/L SiO_2 directly). Western blotting was used to detect the expression of E-cadherin(E-cad) and α-smooth muscle protein(α-SMA) after cells were cultured for 0,24,48 and 72 hours. RESULTS: i) According to the CCK-8 assay,the final concentration of 100 mg/L SiO_2 was chosen for the following study. ii) The difference of relative expression of E-cad andα-SMA in RLE-6TN was statistically significant in different treatment combination and time(P < 0. 01). The E-cad expression of RLE-6TN at 48 and 72 hours in the direct exposure group and the indirect exposure group was lower than that in direct control group at the same time point(P < 0. 05). The E-cad expression in RLE-6TN at 72 hours in the direct exposure group was lower than that in the 0 and 24 hours(P < 0. 05). The E-cad expression in RLE-6TN at 48 and 72 hours in the indirect exposure group was lower than that in the 0 hour(P < 0. 05). At 48 and 72 hours,the α-SMA expression in the indirect exposure group and the direct exposure group was higher than that in their control groups at the same time point(P < 0. 05). The expression of α-SMA in the indirect exposure group was higher than that in the direct exposure group(P < 0. 05). The expression of α-SMA in both exposure groups increased in a time-effect relationship(P <0. 05). CONCLUSION: Direct or indirect exposure to free SiO_2 can induce EMT in RLE-6TN,and decrease the expression of E-cad and increase the expression of α-SMA in a time-effect relationship. Indirect exposure group is more susceptible to EMT.