Construction And Analysis Of Gene Regulatory Network Of THP-1 Derived Macrophages Infected By Mycobacterium Tuberculosis H37Rv

Background and purpose:In 2016,approximately 1.7 million people died of tuberculosis(TB)worldwide,more than the number of patients who died of HIV infection,and Mycobacterium tuberculosis(M.tb)was the TB pathogen.It has become a major infectious disease that causes human death worldwide.In recent years,due to the emergence and rapid spread of drug-resistant M.tb and even multidrug-resistant M.tb,coupled with the limited protection of the current BCG vaccine,it is difficult to effectively control the TB epidemic.The gene chip technology can simultaneously detect the expression of tens of thousands of genes and draw a genome-wide expression profile.This technology has expanded the biological research methods,and has greatly promoted the elucidation of gene function and the study of gene interactions.This study was based on the microarray of human acute monocytic leukemia cell line(THP-1)infected by M.tb H37Rv.The bioinformatics analysis method was used to construct the gene regulatory network of macrophage-derived macrophages infected by M.tb H37Rv,excavating functional modules related to M.tb infection process,and screening out key genes,which are important for understanding the pathogenesis and host defense mechanisms of tuberculosis,and provides a new perspective for effective diagnosis and treatment of tuberculosis.Materials and methods:A chip dataset of the THP-1 derived macrophage model of human leukemia monocytic cells infected with M.tb H37Rv was obtained from the database of GEO Datasets of NCBI,and the differentially expressed genes were screened using the Lima package of R language.DAVID was used to analysis the differentiallyexpressed genes for GO-Analysis and Pathway-Analysis,and then with the aid of Transcriptional Regulatory Element Database(TRED)analysis,we found the differential expressions of transcription factor and gene targeting relationship to construct a transcription factor-gene regulatory network of THP-1 derived macrophage by M.tb H37Rv,and further use of DAVID for genes and transcription factors in network regulation maps(Transcription)Factor,TF)performs functional annotation analysis and disease correlation analysis.We constructed an cellular model of infection of THP-1 derived macrophages infected by M.tb H37Rv,the morphological analysis method,confocal laser scanning microscopy and ELISA were used to verify the success of the model,and the expression levels of important node genes(CYP19A1)and transcription factors(NFKB1,STAT1,PPARG,and CEBPB)were verified by qRT-PCR in the network.The analysis of the receiver operating characteristic curve(ROC curve)of genes NFKB1,CEBPB,and CYP19A1 were used to distinguish the specificity and sensitivity of infected and uninfected cells.Results:In this study,two original gene sets of gene expression profiles were collected and 625 differentially expressed genes were screened using the limma package,of which 427 genes were up-regulated and 198 genes were down-regulated.GO analysis of differentially expressed genes resulted in 179,61,and 85 gene entries in the Biological Process(BP),Cellular Component(CC),and Molecular Function(MF)categories,respectively.Pathway analysis found that the differential genes were mainly enriched in 47 entries.Based on the differentially expressed eight transcription factors(NFKB1,STAT1,PPARG,CEBPB,E2F2,STAT4,RELB,NFKB2),the TRED database was integrated and a transcription factor-gene regulatory network containing 58 differential genes was established.The transcription factor NFKB1,STAT1,CEBPB,and PPARG regulate most of thedifferentially expressed genes.The genes that are simultaneously regulated by three or more transcription factors in the network are called Hub Genes,including CYP19A1,IL6,and IRF1,of which CYP19A1 is regulated by multiple transcription factors.Further analysis of the 58 genes in the network was mainly noted in inflammatory response,immune response,extracellular space,cytosol,protein binding,transcription factor activity,sequence-specific DNA binding and other functions,and with cancer,infection,immune,renal and reproduction and other diseases related.In terms of biological validation,we successfully established a cellular model of THP-1 derived macrophages infected with M.tb H37Rv,and used qRT-PCR to detect important node genes(CYP19A1)and transcription factors(NFKB1,STAT1,PPARG,CEBPB)in the network.The expression levels of the gene CYP19A1 and the transcription factors NFKB1,PPARG,and CEBPB were up-regulated in addition to the transcription factor STAT1,which was consistent with the gene expression trend analysis from the chip data.The gene CYP19A1 and transcription factors in the infection group were found.The expression of NFKB1 and CEBPB was significantly different from that of the control group,with statistical significance.The area under the curve(AUC)of NFKB1,CEBPB,and CYP19A1 was 0.832,0.816,and 0.710,respectively,indicating that the genes were able to distinguish between infected and uninfected cells.Conclusion:(1)The TF-mRNA gene regulatory network with NFKB1,STAT1,PPARG,and CEBPB as the main transcription factors was constructed,showing macrophage immune responses to interactions between genes and regulatory relationships during M.tb infection.(2)An experimental model of THP-1 derived macrophages infected by M.tb H37Rv was successfully established.(3)We reported gene CYP19A1 up-regulates macrophage immune responses toM.tb infection and its potential function in negatively regulating macrophage chemotaxis,providing new insights into the interaction of macrophage-M.tb clue.