Mining And Verification Of Key Genes Of Macrophage Immune Response Infected By Mycobacterium Tuberculosis Based On Meta-analysis

Background and ObjectiveTuberculosis(TB)is an infectious disease caused by M.tuberculosis(M.tb)infection.It is one of the top ten causes of death in the world and the leading cause of death from a single pathogen infection,even surpassing AIDS.As the cornerstone of the immune system,macrophages are an important part of innate immunity.They can ingest and degrade foreign substances such as aging cells and microorganisms,coordinate the inflammatory process,and become the first line of defense against M.tb.But recent advances in cell mycobacteriology suggest that M.tb uses sophisticated strategies to disrupt macrophage function to protect against host innate and adaptive immune clearance,thereby achieving immune escape.With the popularity of microarray technology,a variety of public platform provides a variety of gene expression data related to physiological and disease conditions.Meta-analysis can systematically and quantitatively analyze multiple independent research results of the same subject on the basis of strict design,greatly improving the statistical significance and credibility of gene expression data analysis.In this study,meta-analysis technique was used to analyze the expression profile of THP-1 derived macrophage cells infected with Mycobacterium tuberculosis H37 Rv,and high-throughput screening of key genes of macrophage immune response.To provide a reference for further exploring the immune response mechanism of M.tuberculosis infection and lay the foundation for the development of new drugs for tuberculosis.Materials and methodsThe expression data of the THP-1 derived macrophage infected by H37 Rv were collected from the GEO Datasets database to form the original data set.Using GEOquery,affy,MetaQC and MetaDE in R language performs standardization processing,quality control and integration analysis on the chip data,and screens for differentially expressed genes.The protein-protein interaction(PPI)network of differentially expressed genes was constructed using the STRING online tool and visualized by Cytoscape(version 3.6.1)software.Use the CentiScape and MCODE plug-ins in Cytoscape software to further explore the functional modules related to the M.tb infection process and screen out the core differential genes.Functional annotation analysis(GO-Analysis)and signal pathway enrichment analysis(Pathway-Analysis)were performed on the core differential genes.The THP-1 derived macrophage was infected by Mycobacterium tuberculosis standard strain H37 Rv,Mycobacterium tuberculosis attenuated strain H37 Ra and BCG strain,the success of the model was verified by laser confocal microscopy.The expression level of the core differential gene was detected by qRT-PCR,and the specific expression gene in the H37 Rv infected group was excavated.Further,the specific gene was interfered in the macrophage to investigate the effect of the gene on the survival of bacteria in the macrophage during the infection of M.tuberculosis.ResultsA total of 6 expression data chip raw data sets were collected in this study.The MetaQC software package and PCA visual analysis were used for quality control.Finally,4 high-quality data sets were included,including 76 independent samples(30 H37 Rv infected samples and 46 Control samples).The MetaDE software package was used to integrate and analyze high-quality datasets,and 306 shared differentially expressed genes were obtained.The CentiScape and MCODE analysis of the PPI network resulted in 32 core differential genes.The function and signal pathway enrichment analysis of differential genes showed that 32 The characteristic genes involved 39(P<0.01)GO biological processes(BP)and 3(P<0.01)KEGG signaling pathways.Detection of core differential genes ISG15,IRF7,OASL and DDX58 by qRT-PCR in THP-1 derived macrophage infected by H37 Rv,H37Ra and BCG strain.The expression levels were significantly up-regulated,consistent with the chip analysis results.Further studies found that OASL has significant expression specificity in the H37 Rv infection group.Interference with the OASL gene in macrophages showed that the number of H37 Rv in macrophages was significantly reduced.Conclusion:(1)This study constructed a differential gene PPI network of H37 Rv infected THP-1 derived macrophages,and analyzed the core differentially expressed genes;(2)Verify the expression levels of core molecules ISG15,OASL,IRF7 and DDX58 in the regulatory network,and the differential expression is consistent with the chip analysis results;(3)OASL gene plays a negative regulatory role in macrophage anti-tuberculosis infection immunity.