ABSTRACT
OBJECTIVE: To investigate the differentially expressed microRNAs(miRNAs) in human embryonic lung fibroblast MRC-5 cells stimulated by transforming growth factor-β1(TGF-β1) using microarray chip, and screen for key genes and signaling pathways of fibroblast trans-differentiation. METHODS: The miRNA expression gene chip dataset GSE43992 on TGF-β1 stimulated MRC-5 cells were downloaded from high-throughput Gene Expression Omnibus(GEO) database of National Center for Biotechnology Information of the United States. The R language Limma package was used to screen the differentially expressed miRNAs. Corresponding target genes were predicted by miRWalk database performed by Gene Ontology(GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) signaling pathway enrichment analysis. The protein-protein interaction(PPI) network was constructed by the search tool for the Retrieval of Interacting Genes database. RESULTS: A total of five differentially expressed miRNAs were identified, including four up-regulated miRNAs and one down-regulated miRNA; and 42 corresponding differentially expressed target genes were predicted. GO analysis indicated that the target genes were significantly enriched in collagen catabolic process, extracellular matrix organization, membrane organization, collagen fibril organization, and cellular response to amino acid stimulus. The results of KEGG pathway analysis showed that the signaling pathways corresponding to miRNAs and target genes were mainly concentrated in 18 signaling pathways, that were mainly related to the age-ethnic signaling pathways and protein digestion and absorption miRNAs in tumors and diabetic complications. The core genes transfected into the myofibroblasts by the three fibroblasts screened by the PPI network were threonine kinase 1, estrogen receptor 1 and β-catenin. CONCLUSION: Five differentially expressed miRNAs, 42 target genes, 18 signaling pathways, and 3 core genes related to TGF-β1-induced MRC-5 cell trans-differentiation were screened. It can provide new reference for the treatment and research of many diseases including pneumoconiosis and pulmonary fibrosis.
ABSTRACT
OBJECTIVE: To investigate the effects of nicotinamide adenine dinucleoside phosphate oxidases 1(NOX1) and nuclear factor-kappa B(NF-κB) in tumor necrosis factor-α(TNF-α)-induced oxidative damage in A549 cells. METHODS: i) TNF-α was used to stimulated A549 cells at the concentrations of 0.0, 10.0, 25.0 and 50.0 μg/L. Cell inhibition rate in each group was tested by CCK-8 assay to select the appropriate concentration. ii) A549 cells in logarithmic growth phase were divided into blank control group, solvent control group, TNF-α group, diphenylene iodine(DPI) group and TNF-α+DPI group for NOX1 inhibitor experiment. Logarithmic growth phase A549 cells were divided into blank control group, TNF-α group, BAY11-7082 group and TNF-α+BAY11-7082 group for NF-κB inhibitor experiment. The relative expression of NOX1 and p65 protein in each group was detected by Western blot method. The relative expression of intracellular reactive oxygen species(ROS) were detected by flow cytometry. RESULTS: i) The inhibition rate of A549 cells increased with the increase of TNF-α dose(P<0.05), and 25.0 μg/L was selected as the stimulation dose of TNF-α in subsequent experiments. ii) The relative expression of NOX1, p65 protein and ROS in the TNF-α group was higher than that in the blank control group, solvent control group and DPI group, respectively(P<0.05). The above indexes in TNF-α+DPI group were lower than that in TNF-α group(P<0.05), but higher than that in DPI group(P<0.05). The relative expression of NOX1, p65 protein and ROS in the TNF-α group were higher than that in the blank control group and the BAY11-7082 group(P<0.05), while the above indicators in the TNF-α+BAY11-7082 group were lower than that in the TNF-α group(P<0.05). CONCLUSION: Inhibition of NOX1 or NF-κB can alleviate the oxidative damage induce by TNF-α in A549 cells.