Screening And Early Detection Analysis Of Pneumoconiosis Biomarkers Based On ITRAQ Technology

Objectives To screen the differentially expressed proteins in lung tissue of silicosis animal models and verify their expression in animal and human samples.Then to explore the mechanism of differential protein PTPN2 inhibiting pneumoconiosis fibrosis.To explore the diagnostic effect of differential proteins as biomarkers for early diagnosis of pneumoconiosis.Methods iTRAQ proteomics technology was used to analyze the differential proteins in the lung tissues between 24 weeks silicosis rats and control rats.The functional annotation of the identified differential proteins was carried out by Gene Ontology analysis.KEGG was used to identify the metabolic and signal transduction pathways involved in the differential proteins.The lung tissue proteins of 4 weeks,12 weeks and 24 weeks rats in the animal model and the control rats were extracted.The expression of the screened differential proteins in the lung tissue of rats was verified by western blot.The expression of the screened differential proteins in the SiO₂ stimulated mouse alveolar type II cells was also verified by western blot.MLE-12 cells were transfected with PTPN2 lentivirus gene overexpression and gene silencing plasmids.PTPN2 gene overexpression and gene silencing stable transfection cell lines were constructed.Epithelial mesenchymal transformation model was established by SiO₂ stimulation.The expression levels of PTPN2,p-STAT3,STAT3,COLI,?-SMA,E-cad and Vimentin in PTPN2 overexpression and silencing groups were detected by western blot and immunofluorescence staining.From April to December of 2017,191 pneumoconiosis patients and 12 observational cases were recruited from Beidaihe sanatorium of Chinese coal miners.Meanwhile,we recruited 200 healthy miners in Dongpang mine and Gequan coal mine of Jizhong Energy Group Co.,Ltd.Western blot was used to detect the expression of differential proteins in bronchoalveolar lavage fluid(BALF)of pneumoconiosis patients and observation cases.ELISA method was used to detect serum differential protein levels in 191 pneumoconiosis patients and 200 normal miners.ROC curve was used to analyze the AUC,sensitivity,specificity,accuracy and yoden index of CHIA,PTPN2,Factor B and VRK1 in serum to evaluate the effect of early diagnosis of pneumoconiosis.Logistic regression was used to detect the effect of two factors and multiple factors on the early diagnosis of pneumoconiosis.Results 1 iTRAQ was used to analyze the differential proteins expression in lung tissue of rat silicosis model.A total of 471 differential proteins were obtained,including 252 upregulated proteins and 219 down-regulated proteins.Among them,there were 38 differentially expressed proteins with more than 1.5 fold differential expression.2 The results of GO analysis on cell components,the differential proteins mainly concentrated in membrane components,extracellular body components,cell plasma membrane components.The molecular function GO analysis proteins mainly concentrated in G protein coupled receptor active protein,olfactory receptor active protein,poly adenine ribonucleotide RNA binding protein.Biological process GO analysis proteins mainly concentrated in G protein coupled receptor signaling pathway,DNA template transcription regulation,olfactory perception process.3 KEGG signaling pathway analysis showed that 471 differentially expressed proteins involved 217 pathways,mainly concentrated in olfactory pathway,neuroactive ligand receptor interaction pathway,cytokine cytokine receptor interaction pathway.4 The expression levels of CHIA,PTPN2,VRK1,Factor B and MOT4 in the lung tissue of 4 weeks,12 weeks and 24 weeks rats of silicosis group were significantly higher than those in control groups.5 The expression levels of PTPN2,VRK1,Factor B and C4 BPB in MLE-12 cells stimulated by SiO₂ were significantly higher than those in normal MLE-12 cells.6 We constructed lentiviral overexpression vector of p HS-AVC-LW1049 and the lentiviral silencing vectors of p HS-ASR-LW265,LW266,and LW267,which was successfully transfected into MLE-12 cells.7 The overexpression of PTPN2 significantly decreased the expression levels of vimentin,p-stat3 and COLI;the expression of E-cad in MLE-12 cells stimulated by SiO₂ was increased.8 The silencing of PTPN2 significantly increased the expression levels of vimentin,p-stat3 and COLI;the expression of E-cad in MLE-12 cells stimulated by SiO₂ was decreased.9 The expression of CHIA,PTPN2,Factor B and VRK1 in pneumoconiosis group was significantly higher than those in healthy group.10 ROC curve analysis showed that the area under the curve of each protein was higher than 0.746,and the area under curve of PTPN2(0.858)was the highest,and its specificity(91.04%)and the accuracy of pneumoconiosis diagnosis(88.3%)were also the highest.When the critical values of CHIA,PTPN2,VRK1 and Factor B was 509,1836,284 and 868 pg/m L,respectively,the specificity of the three proteins was more than 75%.11 The combination of PTPN2 and Factor B had the highest AUC(0.856),and the specificity and diagnostic accuracy of these two proteins were 85.71% and 94.67%,respectively.In the combined diagnosis of multiple factors,the combination of PTPN2,Factor B and VRK1 had the highest AUC(0.872),while the combination of PTPN2,Factor B and CHIA had the highest specificity and accuracy.Conclusions 1 We successfully screened 471 differential proteins in lung tissue of rats with silicosis.2 CHIA,PTPN2,VRK1 and Factor B in lung tissue of silicosis rats,MLE-12 cells stimulated by SiO₂,alveolar lavage fluid and serum of pneumoconiosis patients increased significantly.3 PTPN2 inhibited pneumoconiosis fibrosis through dephosphorylation of STAT3.4 Among the four differential proteins,PTPN2 was the best protein for the early diagnosis of pneumoconiosis.5 The combination of PTPN2,Factor B and CHIA was the best combination for early diagnosis of pneumoconiosis.Figure 42;Table 19;Reference 194...