Associative Analysis Of Multi-omics Indicates That Acetylation Modification Is Involved In Cigarette Smoke-induced COPD

Objective:To study the role of acetylation modification in the chronic obstructive pulmonary disease(COPD)and find potential biomarkers by measuring and associating the transcriptomics,proteomics and acetylomics data of lung tissue from a mouse COPD model.Methods:(1)Established the mouse COPD model.Six male specific pathogen-free(SPF)grade C57BL/6J mice were randomly and equally divided into the control group(Control)and the cigarette smoke treatment group(CS).Mice in the CS group were exposed to cigarette smoke,twice a day and 30 min one time,at an interval of 4 h,5 days/week for 24 weeks,while the mice in the control group were free to breathe-fresh air.(2)Verified the effect of modeling.After the exposure,mice were anesthetized by intraperitoneal injection with 2%pentobarbital sodium(0.1ml/100g),and then the tracheae of the mice were cut and connected to the small animal ventilator.Set the parameters,measured and recorded the airway resistance(RN),tissue damping(G),tissue elasticity(H),peak expiratory flow(PEF),forced expiratory volume in 100 ms(FEV0.1),forced vital capacity(FVC),and FEV0.1/FVC ratio.After the lung function measurement,the mice were euthanized by intraperitoneal injection with 2%pentobarbital sodium.The left lower lung tissue of mice were collected and embedded in paraffin for the H&E staining.The stained sections were observed,evaluated,and photographed under the optical microscope.The Image-Pro Plus software was used to analyze the images and measure the mean linear intercept(MLI).(3)Measured the transcriptomics,proteomics and acetylomics data.The transcriptomics data of the left upper lung tissue of mice were determined using the high-throughput sequencing(HTS)technology,and the proteomics and acetylomics data of the right lower lung tissue were determined using the liquid chromatography-tandem mass spectrometry(LC-MS/MS)technology.(4)Performed associative analysis on these omics data.Firstly,the transcriptomics and proteomics were associated.Screened the genes that were significantly differentially expressed in at least one omics data,and grouped them according to their different expression trend types in the transcriptome and proteome.Performed the GO functional enrichment through the gene ontology(GO)database separately with the differentially expressed genes in various groups,and identified the enriched pathways for different groups through the Kyoto Encyclopedia of Genes and Genomes(KEGG)database,all the functional enrichments and pathways that were significant(p<0.05)in at least one regulatory relationship group were screened and clustered.Next,performed the bioinformatics analysis of the acetylomics data,all the proteins corresponding to the differentially expressed acetylation modification sites were annotated and functionally enriched with GO and KEGG,the InterPro domain database was used to annotate the protein domains,then enrichment-based clustering was performed.Finally,the proteomics and acetylomics data were associated.All the differentially expressed proteins and proteins corresponding to the differentially expressed acetylation sites were collected.These proteins were searched against the STRING database for protein-protein interactions and performed clustering and functional enrichment of the densely connected regions.In addition,genes differentially expressed at all the three levels of transcription,protein,and acetylation modification simultaneously were screened.(5)Explored potential biomarkers of COPD.The single-cell RNA sequencing(scRNA-seq)data(GSE136831)of whole lungs dissociated from Homo sapiens were downloaded from the National Center of Biotechnology Information(NCBI)Gene Expression Omnibus(GEO),selected eight samples of patients with COPD as the COPD group and eight samples of healthy humans as the control group.After quality control,the data were standardized and scaled using the Seurat R package.The cell subpopulations in lung tissue were identified based on the expression of classical correlative biomarkers.Analyzed the expression of the genes differentially expressed in all the three omics data in each cell subpopulation,selected genes with the consistent differential expression trend in the scRNA-seq data as potential biomarkers of COPD.(6)Conducted in vivo verification on the selected potential biomarkers of COPD.Validated the differential expression and the level of acetylation modification of the selected proteins in the lung tissue of COPD mice through quantitative real-time PCR(qRT-PCR),Western blotting(WB),immunofluorescence(IF)and immunoprecipitation(IP)with the right upper lung tissue of mice and the paraffin blocks mentioned above.Results:(1)The lung function of mice treated with long-term cigarette smoke treatment was impaired.The results of the pulmonary function test showed that the FEV0.1,FVC,FEV0.1/FVC ratio,tissue damping(G),and tissue elastance(H)decreased significantly in the CS group(p<0.05),while the differences of the PEF and airway resistance(Rn)were not significant(p>0.05),indicating airflow restriction and impairment of lung tissue elasticity in the CS group.(2)Long-term cigarette smoke treatment caused histopathological damage in lung tissue of mice.H&E staining showed that in the CS group,alveolar cavities were larger,part of the alveolar septa were broken,alveolar cavities were fused,emphysema was formed,and the MLI was significantly increased compared with the control group(p<0.05).(3)The transcriptomics and proteomics associative analysis indicated that 2479 genes and 564 proteins were significantly differentially expressed in the lung tissue of mice exposed to cigarette smoke at the transcriptome and proteome levels,respectively.Among these,only 162 genes were differentially expressed with consistent trends at both the transcriptome and proteome levels(135 down-down,27 up-up),and the two omics data were moderately correlated(R=0.53).The GO functional enrichment of various groups was mainly concentrated in terms such as mitochondria,respiratory electron transfer chain(ETC)and lipid metabolism,and the KEGG pathway enrichment were mainly concentrated in pathways related to inflammation,immunity,mitochondrial respiratory electronic chain,fatty acid and amino acid metabolism.The acetylomics identified 444 upregulated sites of 255 proteins and 89 downregulated sites of 62 proteins in the CS group compared with the control group,and the subcellular structure localization of proteins corresponding to differentially expressed acetylation modification sites was mainly the cytoplasm,mitochondria,nucleus,and extracellular.The GO functional enrichment of proteins corresponding to differentially expressed acetylation modification sites was mainly concentrated in terms such as extracellular space,extracellular matrix(ECM),fatty acid metabolism,cell respiration,and enzymatic activity of acyl-CoA dehydrogenase.The KEGG pathway enrichment was mainly concentrated in pathways such as citrate cycle(TCA cycle),fatty acid metabolism,amino acid metabolism,ECM-receptor interaction,focal adhesion,and tight junction.In addition,the protein domain enrichment of proteins corresponding to differential acetylation sites was mainly focused on laminin,epidermal growth factor(EGF),myosin,and acyl-CoA.The proteomics and acetylomics associative analysis indicated that the most closely interacting proteins were mainly enriched in four pathways such as oxidative phosphorylation(OXPHOS),fatty acid degradation,complement and coagulation cascade,and hypertrophic cardiomyopathy(HCM).In addition,19 genes were differentially expressed at all the three levels of transcription,protein,and acetylation modification simultaneously.In all,nine genes,namely,ENO3,PFKM,ALDOA,ACTN2,FGG,MYH3,MYH8,MYL1,and TTN,were involved in these four pathways,while the other 10 genes,namely,ACTA1,ATP2A1,CKM,COROIA,EEF1A2,AKR1B8,MB,MYH1,MYLPF,and STAT1,were not involved in either of the pathways.(4)A total of 14 cell subpopulations were identified from GSE136831.Among the 19 genes mentioned above,only ALDOA and CORO 1A were differentially expressed in some cell subpopulations of COPD patients’ lung tissue,which was consistent with the trend of the difference(downregulated)of our transcriptome data.In addition,both genes were downregulated in proteomics and upregulated in acetylomics.ALDOA was widely expressed in various cell subpopulations and downregulated in alveolar type 2 cells(AT2),goblet cells,B lymphocytes(B cell),natural killer cells(NK cells),and fibroblasts(Fibs),while CORO1 A was only downregulated in T lymphocytes(T cell),B cells and NK cells.(5)In vivo experiments indicated that ALDOA in lung tissue of mice in CS group was downregulated.The results of qRT-PCR showed that ALODA mRNA expression in the CS group was significantly downregulated(p<0.05).WB showed that the protein expression level of ALODA was significantly decreased in the CS group(p<0.05).IF showed that fluorescence staining of ALDOA in the CS group was lower than that in the control group(p<0.05).IP showed that the overal l acetylation level of ALDOA in the lung tissue of COPD mice was increased,but not significant(p>0.05).Conclusion:Long-term cigarette smoke exposure can cause damage to lung function and structure of lung tissue of mice,and eventually lead to the formation of emphysema.Differential expression of protein acetylation modification may participate in the pathogenesis of cigarette smoke-induced COPD through mitochondrial and energy metabolism-related pathways.The downregulation and hyperacetylation of ALDOA may be breakthrough points in the study of COPD.