Microarray Analysis Of Long Non-coding RNA And MRNA Expression In Peripheral Blood Mononuclear Cells From Patients With Active Tuberculosis

Background and Objective:Tuberculosis(TB)is a chronic infectious disease caused by Mycobacterium Tuberculosis(M.tb).It is still one of the main causes of death worldwide and seriously threatens human health.It is one of the top ten "killers" that cause human death and seriously threatens human health.Long non-coding RNA(lncRNA)is a type of non-coding RNA longer than 200 nucleotides.It participates in the regulation of important life processes such as material metabolism,cell differentiation,and individual development on multiple levels.And is closely related to human disease.One of the more researched and mature mechanisms is the competitive endogenous RNA(ceRNA)gene expression regulation mechanism.This mechanism believes that lncRNA can bind with miRNA,thereby regulating the expression of miRNA target genes,and then affecting the occurrence and development of diseases.Therefore,this study aims to provide clues for the research and diagnosis of diseases by constructing a molecular regulatory network with lncRNA as the core in PBMCs of active tuberculosis patients.Materials and methods:The microarray data for lncRNAs and mRNAs were obtained from the Gene Ontology(GEO)datasets of the National Center for Biotechnology Information(NCBI).The datasets were for PBMC samples from patients with active TB and healthy donors control individuals.Multiple mRNA expression profile are merged and batch normalizationed,which was implemented using R(Combat bag).We then used the Limma bag that contains linear model and empirical Bayes statistics to filter out nonspecific expression data.Bioinformatics prediction of target miRNAs with differentially expressed long non-coding RNAs using the miRcode database.The miRDB,miRTarBases and Targetscan databases were also searched for regulatory interactions between miRNAs and their target genes.Based on the results of the above bioinformatics prediction,a lncRNA-miRNA-mRNA ceRNA regulatory network was constructed.Functional annotation of genes(GO-Analysis)and gene pathway enrichment analysis(Pathway-Analysis)were performed on important differential genes in the ceRNA regulatory network.The STRING online analysis tool was used to visualize the differential genomic protein-protein interaction(PPI)network in the regulatory network through Cytoscape(version 3.4.0)software.Based on the results of the above-mentioned biological letter analysis,chip data analysis was used to verify the relationship between mRNA,lncRNA,and mRNA expression levels and related miRNA expression levels.QRT-PCR was used to verify the expression level of key genes and analyze whether they were consistent with the trend of the with the GEO data analysis results.Results:In this study,four original gene sets of gene expression profiles were collected and 32 differentially expressed lncRNAS and 747 differentially expressed mRNAs were screened using the limma bag.The global regulatory networks of lncRNA-miRNA-mRNA of 26 differentially expressed lncRNAs,59 differentially expressed mRNAs and 32 predicted miRNAs were constructed in turn,and analyzed by PPI network.The global regulatory networks of lncRNAmiRNA-mRNA of 26 differentially expressed lncRNAs,59 differentially expressed mRNAs and 32 predicted miRNAs were constructed in turn,and analyzed by PPI network.Functional annotation of 59 differentially expressed genes in the regulatory network showed that these genes were involved in 20(P < 0.001)GO biological process(BP),signal pathway enrichment analysis revealed that these genes are involved in 6(P <0.05)KEGG signal pathways.PPI network analysis was performed on 59 differentially expressed genes in the ceRNA regulatory network.Microarray data verification was used to verify genes in the PPI netwrk and the KEGG signal pathway of interest.The expressions of CCD2,E2F5,JAK2,MYC,PTGS2,RRAS2,and SOD2 were verified.Microarray data analysis of the relationship between lncRAN and mRNA expression showed that FAM157 B and EGR2,RP11-290F20.3.1 and IRF1,RP11-213H15.3.1 and PTGS2,RNU12 and RRAS2 all showed positive correlation trends.The results of microarray verification of miRNA expression showed that hsa-miR-137 and hsa-miR-301b-3p showed high expression levels.The mRNA expression levels of genes EGR2,IRF1,PTGS2 and RRAS2 in PBMC of patients with active tuberculosis were detected by qRT-PCR.Among them,the expression level of IRF1 was upregulated and the expression level of RRAS2 was down-regulated.The results were consistent with the results of multi-chip combined analysis.Conclusion:(1)The differential expression profiles of lncRNA and mRNA in peripheral blood mononuclear cells of tuberculosis patients were screened,and the lncRNA-miRNA-mRNA ceRNA regulatory network was constructed based on this.(2)A PPI network of differentially expressed genes in peripheral blood mononuclear cells of tuberculosis patients was established.(3)The expression levels of the molecules EGR2,IRF1,PTGS2 and RRAS2 in the regulatory network were experimentally verified.