| Background Pemphigus is a kind of rare but serious autoimmune disease（AID）and it is also a group of organ-specific autoimmune mucocutaneous disease that characterized by intraepithelial blisters and erosions of the skin and mucous membranes.Pemphigus can be divided into several different types: pemphigus vulgaris（PV）,pemphigus foliaceus（PF）,pemphigus erythematosus（PE）and paraneoplastic pemphigus（PNP）,et al.PV is the major clinical types of this disorder,followed by PF and PE.Several studies found that the recurrence rate of PE was frequent,but it was similar to PV in terms of disease remission,duration and iatrogenic complications.This study mainly focused on PV and PE.The cause of pemhigus is complex and still uncertain,genetic factors and environment factors may both affect pemhigus.In recent years,with the arrival of the "postgenomics",various omics technology appeared,such as transcriptomics,proteomics,metabolomics,etc,and had become an important tool of scientific research.Gene expression can be obtained through the analysis of the transcriptomics study of RNA level,and transcriptomics study can be used to explore gene expression,biological physiological and pathological conditions of the inner link between the phenomena of the life.Through proteomics analysis,we can analyze the composition and expression levels of proteins in cells and tissues,and to explore the expression and function patterns of all proteins.Metabolomics is the downstream of transcriptome and proteomics,and changes in the metabolic components are the body’s final response to heredity,environment or disease.Biological responses are usually not caused by a single factor,but by a combination of genes,m RNA,protein groups,and metabolites.Therefore,it is necessary to integrate the omics data of different platforms.To analyze the expression data of the combined transcriptome and proteome is one way.The conjoint analysis between them can reveal the gene expression of transcription regulation,achieve the complementary and integration between them,so the joint analysis of transcription and protein group has become the inevitable developing trend of medical research.The biological response of pemphigus is complicated,is the result of joint action of many factors.However,there are some limitations by uses a certain omics study,and there is no multi-omics studies about pemphigus.In the previous research of our group,we used multi-omics to explore the pathogenesis of systemic lupus erythematosus（SLE）,and we found the expression of multiple genes m RNA,protein and metabolites in SLE patients were abnormal,and these molecules were involved in a variety of biological responses and signal transduction pathways..In this study,by performing transcriptome,proteome and metabolome experiments,we set out to find the expression levels of gene m RNA,protein and metabolite in pemphigus patients and healthy controls,identify different expression molecules between the two groups,and further carry out bioinformatic analysis.In addition,we integrated data on the expression levels of molecules in transcriptome and proteome experiments to investigate the associations between gene m RNA and protein expression level.Furthermore,we integrated data on KEGG enrichment analysis in transcriptome,proteome and metabolome to find the key pathway in the pathogenesis of pemphigusPart 1 The study of transcriptome,proteome and metabolome in the patients with pemphigusObjective To identify the different expressed gene mRNAs,proteins and metabolites in the patients with pemphigus and to find the biological reactions and signal transduction pathways in which these molecules are involved.Methods In our study,a total of 17 cases of pemphigus were collected,including 12 PV and5 PE patients.Healthy controls were matched by gender and age.Blood samples were collected from pemphigus patients and healthy controls.The transcriptome assays were performed by RNA sequencing（RNA-seq）.The proteome assays were performed by isobaric tags for relative and absolute quantitation（i TRAQ）.The metabolome assays were performed by high liquid chromatography tandem mass spectrometry（LC-MS）.For the transcriptome and proteome experiment,within each group（pemphigus patients and healthy controls）,equal amounts of sample from 2 or 3 different individuals were randomly pooled.Therefore,three biological replicate samples of pemphigus patient and healthy control were generated.For the transcriptome experiment,FDR ≤ 0.001 and an absolute value of log2 Ratio ≥ 2.00 were used as the threshold to judge the significance of gene expression difference.For the proteome experiment,the differentially expressed proteins have been considered as significant if they met the following criteria: fold change of ratio ≥ 1.2 or ≤ 0.833 and P < 0.05.For the metabolome experiment,we used the partial least-squares discriminant analysis（PLSDA）and volcano figure to identify differentially expressed metabolites.The differentially expressed metabolites have been considered as significant if they met the following criteria: variable importance in the projection（VIP）≥ 1 in PLS-DA,fold change ≥1.2 or ≤ 0.833 and Q value <0.05 in volcano figure.GO enrichment analysis was used to find the biological functions in which the differentially expressed molecules are involved.KEGG enrichment analysis was used to find the signal transduction pathways in which the differentially expressed molecules are involved.Results（1）Among the quantified gene m RNAs in transcriptome experiment,871 gene mRNAs showed significant change in expression pattern with 43 being up-regulated and 828 down-regulated in PV patients when compared with healthy controls,and 478 gene m RNAs showed significant change in expression pattern with 34 being upregulated and 444 down-regulated in PE patients when compared with healthy controls.GO and KEGG enrichment analysis of these differentially expressed gene m RNAs revealed molecular function such as potassium ion antiporter activity and pathways such as RNA degradation,transcriptional misregulation in cancer and ribosome,etc in PV patients.And these differentially expressed gene m RNAs revealed cellular component such as cytosolic ribosome,cytosolic large ribosomal subunit and nuclear cap binding complex,and revealed biological processes such as nuclear-transcribed m RNA catabolic process,nonsense-mediated decay,SRP-dependent cotranslational protein targeting to membrane and cotranslational protein targeting to membrane etc.And revealed pathways such as RNA degradation,m RNA surveillance pathway and cytosolic DNAsensing pathway,etc in PE patients.Among the quantified gene lnc RNAs in transcriptome experiment,1 924 gene lnc RNAs showed significant change in expression pattern with 65 being up-regulated and 1 859 down-regulated in PV patients when compared with healthy controls.We then identified the inferred functions that were enriched among the lnc RNAs target genes differentially expressed between the pemphigus patients and HC group by GO and KEGG analyses.In total,in PV patients,2 in molecular function were significantly enriched.These two GO terms in molecular function were nucleic acid transmembrane transporter activity and RNA transmembrane transporter activity.In PE patients,1 in molecular function were significantly enriched,the GO term was haptoglobin binding.There were eight pathways significantly enriched in PV patients,including RNA degradation,spliceosome and lysine degradation pathways,etc.There were eight pathways significantly enriched in PE patients,including RNA degradation,spliceosome and basal cell carcinoma pathways,etc.（2）Among the quantified proteins in proteome experiment,288 proteins had increased expression and 53 proteins had decreased expression in PV patients,and 122 proteins had increased expression and 76 proteins had decreased expression in PE patients.GO enrichment analysis indicated the participation of differentially expressed proteins in a diverse array of biological process in PV patients,including regulation of cellular ketone metabolic process,regulation of G1/S transition of mitotic cell cycle,etc.In PE patients,GO terms in molecular function were serine-type peptidase activity,serine hydrolase activity and serine-type endopeptidase activity,etc,and GO terms in biological processes were transcription from RNA polymerase Ⅱ promoter,regulation of peptide secretion and regulation of insulin secretion,etc.KEGG enrichment analysis identified proteasome,peroxisome,pentose and glucuronate interconversions and glioma as the most relevant pathway in PV patients,and complement and coagulation cascades,propanoate metabolism and Ras signaling pathway as the most relevant pathway in PE patients.（3）In metabolome experiment,in positive ion mode,a total of 88 significantly altered metabolites were found in PV patients,and 2 significantly altered metabolites were found in PE patients.In negative ion mode,a total of 144 significantly altered metabolites were found in PV patients,and 5 significantly altered metabolites were found in PE patients.Using KEGG,metabolic pathways that were altered in PV and PE patients were identified,in positive ion mode,there were 27 pathways significantly enriched in PV patients,including metabolic pathways,sphingolipid metabolism,glycerophospholipid metabolism,etc,and there were 2 pathways significantly enriched in PE patients,including metabolic pathways and primary bile acid biosynthesis pathway.In negative ion mode,there were 29 pathways significantly enriched in PV patients,including metabolic pathways,bile secretion,etc,and there were no pathways significantly enriched in PE patients.Conclusion The dysregulation of a variety of gene m RNA,protein and metabolite was found in pemphigus patients.These molecules participate in many biological functions and signal transduction pathways.Part 2 Identification of novel markers for pemphigus using integrative transcriptome and proteome and to explore the Pathogenesis of pemphigusObjective Using the combined analysis of transcriptome and proteomics to find the different molecules in gene and protein levels of pemphigus patient,and using bioinformatics methods to understand their biological characteristics and functions.The correlation between them and pemphigus was discussed by analyzing the plasma concentration of differential protein.To provide the basis of the pathogenesis of pemphigus.Methods We investigated the correlations between transcriptome and proteome expression profiles.First,we compared the protein expression profile which was obtained based on the reference gene with the transcriptome expression profile.When a certain protein was expressed at the transcriptome level,this molecule was considered to be related.Then,we used the pearson correlation coefficient to reflect the correlations of these related molecules between transcriptome and proteome expression profiles.Finally,we clustered these related molecules into different groups based on the pattern of changes at the gene m RNA and protein levels.By using the ELISA method,the proteins in the association were verified by the expression trend,and the related proteins were analyzed by GO and pathway annotation.The plasma protein concentration was tested with k-s to verify whether it was in normal distribution for PV,PE patients and healthy control group.If the variables were normal distribution,we used （?）±S to describe,if the variables were skewed distribution,we used M(P25,P75)to describe.was expressed in Two independent sample t tests or two independent sample nonparametric tests were used to test the candidate plasma protein concentration.Statistical analysis was performed using SPSS 23.00 software,and the test level was 0.05.Results（1）The significant difference protein was correlated with the significant difference gene,and the correlation coefficient was calculated,PV: the correlation coefficient was-0.1091,PE: the correlation coefficient was 0.1079.（2）PE: the correlation coefficient was 0.4000,there were five molecules expressing the same trend,including RBFA,PZP,CERU,S100A9 and HEP2;PV: the correlation coefficient was 0.6108,there were eight molecules expressing the same trend,including DEF1,HBB,TAGL2,HBA,ANTXR2HUMAN,CD14,TBB5 and AHNK2.（3）According to the correlation analysis results,13 molecules（proteins）with common expression differences（up-regulated or down-regulated）were identified for ELISA verification.The level of CD14 and ANTXR2 in HC group were 64.26（60.10,70.90）μg/ml and 476.03（429.22,913.37）pg/ml,was higher than the PV patients,there was significant difference between the two groups.The level of DEF1、TAGLN2 and TBB5 were 128.40（100.13,237.79）ng/L,1 335.25（1 109.65,15 80.89）pg/ml and4 624.04（3 422.90,6 081.51）pg/ml,were higher than HC group.The expression levels of CD14,TBB5,DEF1 and TAGLN2 were consistent with the results of correlation analysis.（4）The proteins were mainly significantly enriched in eleven cellular component terms,seven molecular function terms and nineteen biological process terms in PV patients.The proteins were mainly significantly enriched in eight cellular component terms,four molecular function terms and fourteen biological process terms in PE patients.In total,there were 17 pathways significant enriched in PV patients,the NODlike receptor signaling pathway,NF-kappa B signaling pathway,Toll-like receptor signaling pathway and Hematopoietic cell lineage were related to immune system.There were 3 pathways significant enriched in PE patients,complement and coagulation cascades and IL-17 signaling pathway were related to immune system.Conclusion PV and the PE group has multiple associated proteins,ELISA experiment found DEF1,TAGL2 and CD14 results consistent with the results of sequencing,associated proteins are mainly involved in the immune related on sample the NOD-like receptor signaling pathway,NF-kappa B signaling pathway and Toll-like receptor signaling pathway,help to clarify the pathogenesis of pemphigus.Combined application of new technology and new method can provide a new direction for the diagnosis and treatment of pemphigus... |
PDF Full Text Request