| Background and objectiveIdiopathic pulmonary fibrosis(IPF)is a fatal lung disease characterized by progressive and irreversible lung fibrosis of unknown etiology.In recent years,the incidence of IPF has been increasing globally.This disease has affected more than 3 million people worldwide.The prognosis of IPF is extremely poor,with a median transplant-free survival of only 3-5 years after diagnosis,and only 3-4 months’survival time after an acute exacerbation.However,therapies for IPF are very limited currently.Up to now,only pirfenidone and nintedanib are effective drugs for IPF patients.Therefore,exploring the possible pathogenesis and finding effective and safe therapeutic targets of IPF are the research hotspots and difficulties needed to be solved urgently at home and abroad.Proteomics is a science that studies the composition and change rules of cells,tissues or organisms at the overall level.Proteomics is considered to be the best way to discover new molecular markers,explain the mechanism of phenotype,and discover new mechanisms of drug action.Bioinformatics is an emerging discipline in recent years,which combines life science and computer science,and is more and more widely used in medical research.With the development of these technologies,medical research has entered a new era in which computer science and traditional basic medicine are combined,The medical research model that combines omics technology,bioinformatics analysis,and basic medical experiments has become more and more popular.The key genetic information obtained from these also continuously provides new clues for the treatment of diseases.This project aims to explore the role and mechanism of LTBP2 in the occurrence and development of IPF through proteomics technology,bioinformatics analysis,and basic medical experiments,and lay the foundation for LTBP2 as a biomarker for the diagnosis of IPF and a potential molecular therapeutic target.Science experiment technology.The research of this subject is mainly divided into four parts.The first part:the use of proteomic sequencing to analyze the differentially expressed proteins in bleomycin-induced pulmonary fibrosis in mice.The second part:GSE17978,GSE53845,and GSE122960 datasets were screened from the gene expression omnibus(Gene expression omnibus,GEO)database,and key genes involved in the occurrence and development of IPF were identified;then,the above-mentioned genes were intersected with the genes encoding differentially expressed proteins in mouse pulmonary fibrosis tissue obtained in the first part.Finally,the LTBP2 gene was selected as the follow-up research target from the screened key genes.The third part:the regulation of down-regulation of LTBP2 on the activation,secretion,proliferation,migration and invasion of human lung fibroblasts.The fourth part:the mechanism of LTBP2 in the occurrence and development of IPF.Part I Analysis of differential expressed proteins in bleomycin-induced mice pulmonary fibrosis modelsMethods1.Thirty 6-8-week-old male C57BL/6J mice were randomly divided into Bleomycin(BLM)group receiving intratracheal instillation of BLM,and Saline group receiving intratracheal instillation of sterile saline.Each group consisted of 15 mice.BLM and Saline were instilled in the trachea respectively,and the bodyweight of the mice after anesthesia was recorded before installation.After installation,these mice were routinely fed for 28 days to develop pulmonary fibrosis mice models.2.Post-anesthesia body weights of surviving mice were recorded on the 28th day after intratracheal instillation of BLM or saline.Lung tissue samples were collected from surviving mice to perform HE and Masson staining and the detection of hydroxyproline content.Meanwhile,quantitative real-time reverse transcriptionpolymerase chain reaction(qRT-PCR)were performed to detect of Acta2(the gene encoding the protein α-SMA),Collal(the gene encoding the protein Collagenlal)and Fnl(the gene encoding the protein Fibronectin 1)3.Four pairs of lung tissues of mice(BLM Vs Saline)were utilized to perform mass spectrometry-based proteomic sequencing.Then differential expressed proteins were analyzed.Meanwhile,using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)to annotate these genes encoding differentially expressed proteins.4.Building BLM-induced mice lung fibrosis models again with a larger scale of samples(15 pairs of mice,BLM Vs Saline).Using mass spectrometry-based targeted protein verification technology—Parallel reaction monitoring(PRM)to verify 20 proteins from the proteomics sequencing results.Then qRT-PCR was used to verify the genes encoding the proteins which were verified by PRM and conformed to the results of proteomic sequencing.Results1.On the 28th day of intratracheal instillation of BLM or Saline,9 mice survived in the BLM group and 15 mice survived in the Saline group.2.The weight of the mice in the BLM group generally decreased,while the weight of the mice in the Saline group increased.The weight of the mice in the BLM group was significantly lower than that in the Saline group(p<0.001).HE staining showed that the lung tissue of the mice in the BLM group was characterized with obvious manifestations of alveolitis,widening of alveolar septa,destruction of the alveolar structure,and a large number of fibroblasts in the interstitium.Masson staining showed that the collagen fibers in the lung tissue of the mice in the BLM group were significantly increased,the interstitium contains a large number of collagen fibers.The content of hydroxyproline in lung tissue of mice in BLM group was significantly higher than that in Saline group(p<0.001).The results of qRT-PCR showed that the transcription levels of Acta2,Col1a1 and Fn1 in the BLM group were significantly increased than that in Saline group,with a significant statistical difference.3.The results of mass spectrometry-based proteomic sequencing(4 pairs of mice,BLM Vs Saline)showed that 1351 proteins were significantly differentially expressed between the two groups(foldchange>1.5,p<0.05),of which 633 proteins were significantly up-regulated and 718 were significantly down-regulated.The results of GO and KEGG annotation of genes encoding these 1351 differentially expressed proteins were as follows.The main biological process(BP)enrichment included:positive regulation of fibroblast proliferation,extracellular matrix organization,etc.The main cellular component(Cellular Component,CC)enrichment included:platelet alpha granule,adherens junction,etc.The main molecular function(Molecular Function,MF)enrichment included:alpha-actin binding,collagen binding,extracellular matrix binding,etc.The KEGG enrichment mainly included:ECM-receptor interaction,cell adhesion molecules,etc.4.The results of PRM showed that the relative expression levels of Tgm1,Tnc,Cfd,Fmod,Mmp2,Ltbp2,Fbln2,Lgmn,Serpinf2,Ahsg,Vtn,Mrc2,and Lgals3 in the BLM group were significantly higher than those in the Saline group(p<0.05).The relative expression levels of Dcn,Sod2,Cav1,Gsta3,Emp2,Cyp1a1 were significantly lower than those in the Saline group(p<0.05).While the relative expression levels of Thbsl had no significant difference compared with the Saline group.The qRT-PCR results were consistent with the PRM results.qRT-PCR showed that in the BLM group,the relative expression levels of Tgm1,Tnc,Cfd,Fmod,Mmp2,Ltbp2,Fbln2,Lgmn,Serpinf2,Ahsg,Vtn,Mrc2,and Lgals3 were significantly higher than those in the Saline group(p<0.05).The relative expression levels of Dcn,Sod2,Cav1,Gsta3,Emp2,and Cyp1a1 were significantly lower than those in the Saline group(p<0.05).Part Ⅱ Analysis of the expression level of LTBP2 in idiopathic pulmonary fibrosis based on bioinformaticsMethods1.Microarray profiling datasets GSE17978 and GSE53845 based on transcriptome sequencing of human IPF lung tissue total RNA were obtained from the Gene Expression Omnibus(GEO)database.Differentially expressed genes(DEGs)were analyzed after normalization by using the GEO2R tool.The thresholds of DEGs were set as follows:|logFC|>1 and adj.p.value<0.05.The obtained DEGs from the above datasets were intersected.GO and KEGG pathway enrichment analysis annotations were performed on these intersected DEGs.Then these intersected DEGs were used for subsequent analysis.2.The intersected DEGs from the GSE17978 and GSE53845 datasets,and the 19 genes(Tgm1、Tnc、Cfd、Fmod、Mmp2、Ltbp2、Fbln2、Lgmn、Serpinf2、Ahsg、Vtn、Mrc2、Lgals3、Dcn、Sod2、Cav1、Gsta3、Emp2、Cyp1a1)encoding differentially expressed proteins that were validated in Part Ⅰ in mice pulmonary fibrosis were intersected.3.Pearson correlation analysis was used to analyze the co-expression relationship of LTBP2 with ACTA2,COL1A1 and FN1 in human IPF lung tissue,respectively.4.The IPF single-cell transcriptomic expression profile dataset GSE122960 was obtained from the GEO database.Then the lung tissues of 4 IPF patients and 8 donor lungs in this dataset were analyzed to explore the expression of LTBP2 in each cell population in IPF lungs situation,looking for cell populations that differentially express LTBP2.5.Using the IPF lung tissue and donor lung tissue obtained in our unit,after paraffin embedding,the immunohistochemical experiment of LTBP2 was performed.Results1.A total of 996 DEGs were identified in the GSE17978 dataset,and 925 DEGs were identified in the GSE53845 dataset.After taking the intersection of the DEGs in the two datasets,155 DEGs were jointly up-regulated or down-regulated.The results of GO functional enrichment of these 155 DEGs were as follows.The main BP enrichment included:extracellular matrix organization,extracellular structure organization,etc.The main CC enrichment included:collagen trimer,extracellular matrix component,etc.The main MF enrichment included:extracellular matrix structural components,G protein-coupled receptor binding,etc.KEGG enrichment mainly included cytokine-cytokine receptor interaction,chemokine signaling pathway,etc.2.After the intersection of 155 intersected DEGs obtained from GSE17978 and GSE53845 datasets and 19 genes encoding differentially expressed proteins that were validated in Part Ⅰ,the only target gene LTBP2 was obtained.3.In IPF lung tissue,the expression level of LTBP2 was positively correlated with the expression levels of ACTA2,COL1A1 and FN1(p<0.05).4.LTBP2 is mainly expressed in the fibroblast population.In the fibroblast population,the expression of LTBP2 was significantly increased in fibroblasts from IPF compared to fibroblasts from healthy donors(p<0.001).5.LTBP2 was strongly expressed in fibroblasts/myofibroblasts within fibroblast foci of IPF lung tissue.Part Ⅲ The role of LTBP2 regulating activation,secretion,proliferation,migration and invasion in human lung fibroblast and regulating pulmonary fibrotic lesions in miceMethods1.Human lung fibroblasts(MRC-5 and CCC-HPF-1)were cultured in vitro.After starving in serum-free DMEM medium for 12 h,MRC-5 and CCC-HPF-1 cells were stimulated with serum-free DMEM medium at a concentration of 1Ong/ml TGF-β1 for 12h,24h,and 48h,respectively,to activate fibroblasts and construct fibrotic phenotype of fibroblasts.Next,qRT-PCR was used to detect the human lung fibroblast activation markers(ACTA2 and DESMIN),the expression levels of COL1A1 and FN1 genes encoding the main components of extracellular matrix(ECM),and the expression level of LTBP2.2.Synthesis of RNA-interfering lentiviruses of three LTBP2 genes based on human LTBP2 mRNA sequences(sh-LTBP2(1),sh-LTBP2(2)and sh-LTBP2(3)).Synthesis of 1 negative control gene RNA interference lentivirus(sh-NC).MRC-5 cells were infected with them,and the infection efficiency and the silencing level of LTBP2 were detected 48h after infection.3.The expressions of ACTA2,DESMIN,COL1A1 and FN1 were detected by qRT-PCR and WB experiments,and the secretion of COL1A1 and FN1 was detected by ELISA experiment to explore the effect of down-regulation of LTBP2 on the activation and secretion capacity of human lung fibroblasts under TGF-β1-driven and TGF-β1-independent driving conditions.The groups were set as follows:(一)Blank control group without TGF-β1 induction(Blank#group),blank cell group induced by TGF-β1 for 48h(TGF-β1+Blank group),group of unrelated sequences induced by TGF-β1 for 48h(TGF-β1+sh-NC group),after lentivirus-mediated shRNA-LTBP2 infection,the infection group induced by TGF-β1 for 48h(TGF-β1+sh-LTBP2 group);(二)Blank control group not infected with lentivirus(Blank group),unrelated sequence group(sh-NC group),infection with lentivirus-mediated shRNA-LTBP2 group(sh-LTBP2 group).4.CCK8 assay and flow cytometry were used to detect the effect of down-regulation of LTBP2 on the proliferation and cell cycle of human lung fibroblasts under TGF-β1-driven and TGF-β1-independent driving conditions.Set the grouping as above.5.Scratch assay and Transwell assay were used to detect the effect of down-regulation of LTBP2 on the migration and invasion of human lung fibroblasts under TGF-β1-driven and TGF-β1-independent driving conditions.Set the grouping as above.6.Adeno-associated virus(AAV)-mediated shRNA-LTBP2 and unrelated sequence sh-NC were instilled into the trachea of mice,and the expression of LTBP2 was down-regulated in animals by HE,Masson staining and WB experiments.Improvement in pulmonary fibrotic lesions.Results1.The results of qRT-PCR showed that in MRC-5 cells,the expressions of LTBP2,ACTA2,DESMIN,COL1A1 and FN1 all increased with the increase of TGF-β1 stimulation time,and the expression reached the highest at 48h.In CCC-HPF-1 cells,the expression of LTBP2,ACTA2,DESMIN,COL1A1 and FN1 also increased with the increase of TGF-β1 stimulation time,and the expression reached the highest at 48h.When stimulated by TGF-β1,the expression level of LTBP2 in MRC-5 cells was higher than that in CCC-HPF-1 cells.Therefore,the conditions of TGF-β1 stimulation for 48h were selected,and the MRC-5 cell line was used for subsequent experiments.2.The efficiency of lentivirus infection in each group was above 70%.The target sequences sh-LTBP2(1),sh-LTBP2(2)and sh-LTBP2(3)could silence LTBP2 expression in MRC-5 cells.While LTBP2-RNAi(3)had the best effect of silencing LTBP2 expression.Therefore,in the subsequent experiments,the target sequence sh-LTBP2(3)was selected for the experiment.3.Down-regulation of LTBP2 can inhibit the synthesis of ACTA2,DESMIN,COL1A1 and FN1 and the secretion of COL1A1 and FN1 in human lung fibroblasts under TGF-β1-driven and TGF-β1-independent driving conditions.4.Down-regulation of LTBP2 can inhibit proliferation of human lung fibroblasts under TGF-β1-driven and TGF-β1-independent driving conditions.Block occured in G2/M phase.5.Down-regulation of LTBP2 can inhibit rates of healing and invasion in human lung fibroblasts under TGF-β1-driven and TGF-β1-independent driving conditions.6.The results of WB experiment showed that down-regulation of Ltbp2 could reduce the expression of α-SMA,Collal,and Fnl in mice’ pulmonary fibrosis tissues.HE and Masson staining showed that down-regulation of Ltbp2 could reduce inflammation and fibrotic lesions in mice’ pulmonary fibrotic tissues.Part Ⅳ The role of LTBP2 in the development of idiopathic pulmonary fibrosisMethods1.Transcription factor of target gene,possible binding sites for transcription factors and gene promoters were predicted utilizing the following tools:NCBI’s Gene database,the genome browser tool on the online website of The University of California Santa Cruz(UCSC),and the JASPAR database.2.The IPF single-cell transcriptomic expression profile dataset GSE122960 was obtained from the GEO database.Then the lung tissues of 4 IPF patients and 8 donor lungs in this dataset were analyzed.Analyzing expression level of TCF4 in fibroblast populations within IPF lung tissue and healthy donor lung tissue.Analyzing the expression correlation of TCF4 and LTBP2 in fibroblast populations.3.MRC-5 cells were cultured in vitro and stimulated with TGF-β1 for 48 h,then ChIP-PCR was used to detect whether TCF4 was a transcription factor of LTBP2.4.The dual-luciferase reporter assay detected whether TCF4 was a transcription factor of LTBP2.5.Searching for differentially expressed molecules downstream of LTBP2 using mass spectrometry-based proteomic sequencing.The experimental conditions were as follows:knockdown of LTBP2 in human lung fibroblasts(MRC-5)under TGF-β1-driven and TGF-β1-independent driving conditions.Then using bioinformatics analysis to find potential functional molecules for follow-up studies.Setting four groups,the group information was as follows:unrelated sequence group(sh-NC group),infection group using lentivirus-mediated sh-LTBP2(sh-LTBP2 group),group of unrelated sequences induced by TGF-β1 for 48h(TGF-β1+sh-NC group),After lentivirus-mediated sh-LTBP2 infection,the infection group induced by TGF-β1 for 48h(TGF-β1+sh-LTBP2 group).6.The relative expression levels of ITGA3,PI3K,p-PI3K,AKT,and p-AKT were detected by WB EXPERIMENT after LTBP2 was knocked down under TGF-β1-driven and TGF-β1-independent driving conditions.Set the grouping as above.Results1.The results of transcription factor prediction showed that TCF4 might be a transcription factor of LTBP2.2.TCF4 is expressed in multiple cell populations such as fibroblasts,endothelial cells,macrophages,dendritic cells,etc.In the fibroblast population,the expression level of TCF4 was significantly higher in fibroblasts from IPF than in fibroblasts from healthy donor lungs(p<0.001).In the fibroblast population,the expression level of TCF4 and LTBP2 was positively correlated,and the correlation was significantly higher in the fibroblast population in IPF lungs than in healthy donor lungs(p<0.05).3.The enrichment of TCF4 to the promoter sequence of LTBP2 was observed in human lung fibroblasts under TGF-β1driven conditions.4.TCF4 could interact with the LTBP2 promoter region.5.A total of 2253 differentially expressed proteins were obtained,among which ITGA3 may be a functional downstream molecule of LTBP2 under TGF-β1-driven and TGF-β1-independent driving conditions.6.Knockdown of LTBP2 could inhibit the relative protein expression levels of ITGA3,p-PI3K and p-AKT under TGF-β1-driven and TGF-β1-independent driving conditions.Conclusions1.In the BLM-induced mice pulmonary fibrosis model,there are many differentially expressed genes,among which the LTBP2 gene is highly expressed in the mouse lung fibrosis tissue.2.LTBP2 is highly expressed in fibroblast populations in human IPF lung tissue.3.Down-regulation of LTBP2 can inhibit the activation,secretion,proliferation,migration and invasion of human lung fibroblasts under TGF-β1-driven and TGF-β1-independent conditions.Meanwhile,down-regulation of Ltbp2 could ameliorate BLM-induced pulmonary fibrosis in mice.4.TCF4 can regulate the transcription of LTBP2 in human lung fibroblasts,and down-regulation of LTBP2 suppresses the fibrotic phenotype of human lung fibroblasts under TGF-β1-driven and TGF-β1-independent driving conditions through the ITGA3/PI3K/AKT signaling axis. |
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