| Part ? Screening of biomarkers and establishment of the serum proteomic diagnostic model for childhood asthma by i TRAQ quantitative proteomicsBackground: Bronchial asthma is a common chronic respiratory tract diseases characterized by chronic airway inflammation,airway hyperresponsiveness and irreversible airway remodeling.The incidence of the disease grows higher than ever,and about 1/3 childhood asthma will continue and finally develop to adult asthma.Accurate assessment of the condition is the key to effective prevention and control of childhood asthma.The different levels of protein expression in different periods and pathological states of childhood asthma can screen out specific proteins related to childhood asthma,which can be used as biomarkers to predict,diagnose,evaluate and prognosis of childhood asthma.Proteomics technology can realize the analysis of serum protein from the overall level.As a powerful proteomics technology,i TRAQ stable isotope labeling,two-dimensional liquid chromatography nanoelectrospray ionization and high resolution tandem mass spectrometry using the hybrid quadrupole time-of-flight platform(i TRAQ-2DLC-MS/MS)as a powerful proteomics technology can be used for screening and identification of disease-related proteins.Methods: A total of 190 serum samples were collected,including 55 healthy children and 135 childhood asthma.The differentially expressed serum proteins from15 healthy children and 15 childhood asthma were screened by using i TRAQ-2DLC-MS/MS.The gene ontology(GO)was analyzed using the DAVID on the differentially expressed proteins,and the functional classification and functional enrichment analysis of cellular components,molecular functions and biological processes were conducted respectively.The Kyoto Encyclopedia of Genes and Genomes(KEGG)database was used for signal transduction pathway analysis.The Search Tool for the Retrieval of Interacting Genes / Proteins(STRING)software was used for protein-protein interaction network analysis.Enzyme-linked immunosorbent assay was used to verify differential proteins in serum samples from 120 childhood asthma and 40 healthy children.Receiver operating characteristic curve(ROC)andmultivariate logistic regression analysis were used to analyze the sensitivity and specificity of the single protein and combined proteins for the diagnosis of childhood asthma.Biomarker Patterns Software 5.0 software was used to analyze and compare the weight combinations of differentially expressed proteins in childhood asthma.The serum proteomic diagnostic model for childhood asthma was established to determine the normal threshold,positive predictive value(PPV),and Youden index of the model.Sensitivity and specificity of the serum proteomic diagnostic model for childhood asthma were assessed by a 10-fold cross-validation.Results: Using i TRAQ-2DLC-MS/MS to detect protein in the serum samples of childhood asthma and healthy controls,a total of 78 differentially expressed proteins were screened(fold change>1.2 or fold change<0.8).The differentially expressed proteins were mainly involved in 22 important biological processes,with 9 molecular functions and 8 cellular components.It was mainly involved in 53 pathways,including 16 differentially expressed proteins involved in complement and coagulation cascade reaction pathways.ELISA results showed that apolipoprotein C-?(Apo C ?),apolipoprotein E(Apo E),and serum amyloid A(SAA)levels in childhood asthma were significantly higher than those in healthy children(P<0.05,P<0.001,P<0.001).ROC curve analysis was performed on the three serum proteins Apo C?,Apo E and SAA,the area under the ROC curve(AUC)of a single serum protein of Apo C ?,Apo E and SAA was 0.622,0.763 and 0.756,respectively.Multiple logistic regression method calculated the AUC of three serum protein compositions to be 0.850,the sensitivity was 77.5%,and the specificity was 85.0%.A decision tree modeling was further used to construct a serum proteomic diagnostic model for childhood asthma,the sensitivity,specificity,PPV and Youden index of the model were 79.2%,92.5%,96.9% and 0.717,respectively.Conclusions:(1)Serum proteins Apo C?,Apo E and SAA were screened and validated by i TRAQ-2DLC-MS/MS and ELISA,and can be as candidate protein biomarkers for childhood asthma,which can be further verified in clinical and functional aspects.(2)Screening of 78 childhood asthma-related differential proteins were mainly involved in the metabolic process,response to biological regulation andstimulus,mainly involved in complement and coagulation cascade reaction pathways,and there was a close functional connection between the differential proteins.,the results lay a foundation for the further to explore the pathogenic mechanism of childhood asthma.(3)Serum proteomic diagnostic model for childhood asthma composed of Apo C?,Apo E and SAA constructed by decision tree modeling,which has better diagnosis capability and provided a new method for the diagnosis of childhood asthma and can provide core data and technology for the development of protein chip kits for the diagnosis of childhood asthma.Part ? Screening of biomarkers and establishment of the serum micro RNAs diagnostic model for childhood asthmaBackground: mi RNAs play an important role in guiding the differentiation of asthma dendritic cells,regulating the proliferation,survival,differentiation and cytokine production of asthma T lymphocytes,and play an important role in airway inflammation and airway remodeling of asthma,and can be used as a potential new target for the prediction,diagnosis and evaluation of childhood asthma.The emergence of high-throughput sequencing technology makes the analysis of all the childhood asthma-related mi RNAs in the serum possible,with the advantages of high throughput,high accuracy and high stability.Methods: A total of 170 serum samples were collected,including 75 healthy children and 95 childhood asthma.The differentially expressed serum mi RNAs from15 healthy children and 15 childhood asthma patients were screened by using the Illumina sequencing method.Quantitative real time polymerase chain reaction assay(q RT-PCR)was used to validate differentially expressed serum mi RNAs from 60 healthy children and 80 childhood asthma.The receiver operating characteristic(ROC)curve and multivariate logistic regression analysis were used to analyze the sensitivity and specificity of the single mi RNA and combined mi RNAs for the diagnosis of childhood asthma.Biomarker Patterns Software 5.0 software was used to analyze and compare the weight combinations of differentially expressed mi RNAs in childhood asthma.The serum mi RNAs diagnostic model for childhood asthma was established to determine the normal threshold,PPV,and Youden index of the model.Sensitivity and specificity of the serum mi RNAs diagnostic model for childhood asthma were assessed by a 10-fold cross-validation.Web-based programs,namely mi Randa and Target Scan,were employed to predict specific mi RNA target genes,and GO function analysis,signal transduction pathway enrichment analysis and mi RNA-Gene network analysis were performed on specific mi RNAs target genes.Results: Illumina sequencing was used to detect serum mi RNAs in childhood asthma and healthy children.A total of 111 differentially expressed serum mi RNAs were screened(fold change>1.5or fold change<0.5).q RT-PCR results showed that hsa-mi R-17-5p,hsa-mi R-18a-5p,hsa-mi R-106a-5p,hsa-mi R-144-3p and hsa-mi R-375 levels in childhood asthma were significantly higher than those in healthy children(P<0.0001,P<0.0001,P<0.0001,P<0.0001,P<0.0003),and hsa-mi R-19b-3p level in childhood asthma was significantly lower than that in healthy children(P<0.0293).ROC analysis was performed on the six serum mi RNAs of hsa-mi R-17-5p,hsa-mi R-18a-5p,hsa-mi R-19b-3p,hsa-mi R-106a-5p,hsa-mi R-144-3p and hsa-mi R-375,the AUC were 0.787,0.799,0.608,0.774,0.756 and 0.678,respectively.Multivariate logistic regression analysis of a combination of six serum mi RNAs revealed that the AUC was 0.924,the sensitivity was 90.4%,and the specificity was 86.7%.Six childhood asthma-specific serum mi RNAs were mainly involved in 25 important biological processes,mainly with 15 molecular functions and 8 cellular structure,mainly involved in 203 signal pathways,regulating a total of13,190 target genes,of which 152 target genes may be related to the pathogenesis of asthma,two target genes of MAP3K1 and FRS2 were co-regulated by five mi RNAs of hsa-mi R-19b-3p,hsa-mi R-144-3p,hsa-mi R-18a-5p,hsa-mi R-106a-5p and hsa-mi R-17-5p.Conclusions:(1)Six serum mi RNAs of hsa-mi R-17-5p,hsa-mi R-18a-5p,hsa-mi R-19b-3p,hsa-mi R-106a-5p,hsa-mi R-144-3p and hsa-mi R-375 were screened and validated by Illumina sequencing method and q RT-PCR,and can be used as candidate mi RNA biomarkers of childhood asthma,which can be further verified inclinical and functional aspects.(2)Serum mi RNAs diagnostic model for childhood asthma composed of four mi RNAs of has-mi R-18a-5p,has-mi R-106a-5p,has-mi R-144-3p and has-mi R-375 constructed by decision tree modeling,which had better diagnosis capability and provided a new method for the diagnosis of childhood asthma.(3)Screening of six childhood asthma-specific serum mi RNAs related main target genes were mainly involved in gene expression,protein phosphorylation and signal transduction,mainly involved in Wnt signaling pathway,vascular smooth muscle contraction,T cell receptor signaling pathway,and encoded proteins were mainly involved in inflammation,immune and transcription efficiency,etc.The results can lay the foundation for further exploring the pathogenesis of mi RNAs in children with asthma. |
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