Microarray Analysis Of Long Noncoding RNA And MRNA Expression Profiles In Human Macrophages Infected With Mycobacterium Tuberculosis

Background:Mycobacterium tuberculosis(MTB)was the causative agent of tuberculosis.MTB is able to evade the major antimicrobial mechanisms of macrophages by a variety of bacterial immune subversion mechanisms,allowing the bacteria to survive in cell via the utilization of various intracellular resources.LncRNAs are arbitrarily defined as transcripts longer than 200 nt in length without protein-coding capacity,which have a central role in fundamental biological processes,such as genomic imprinting,chromosome modification,immune response,tumorigenesis,cellular development and metabolism,etc.However,the function of lncRNAs in regulating the immune response of macrophages infected with MTB is just beginning to be understood.ObjectiveIn this study,we investigated expression patterns of IncRNA and mRNA in human macrophages infection with H37Ra or H37Rv,in order to provide novel insight into the mechanisms of the pivotal MTB-macrophage interactions as well as find the novel diagnostic and therapeutic strategies for tuberculosis.We try to investigate the function of IncRNA-ENST00000360485 in macrophage with MTB infection,in order to provide a novle antimicrobial mechanism of macrophages.MethodMicroarray analysis was performed on human macrophages at 72 h post infection with avirulent H37Ra and virulent H37Rv MTB strains.And an Arraystar Human LncRNA Microarray V3.0(Array-Star,Inc.,Rockville,MD,USA)was employed in this study;To validate our microarray data,6 lncRNAs were randomly selected for detection by quantitative real-time PCR(qPCR);Then the functions of mRNAs were evaluated through gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.The expression level of lncRNAs,which were selected based on their significant upregulation in macrophages with MTB infection in vitro,were detected by qPCR in PBMC samples from 32 patients with active pulmonary tuberculosis(PTB patients)and 32 healthy people vaccinated with BCG;siRNA were performed to silence the expression of lncRNA-ENST00000360485;Then colony-forming unit(CFU)assay was used to test the survival of intracellular mycobacteria after inhibition of ENST00000360485;And qRT-PCR were used to detect the expression of TNF-?,IL-1?,IL-6 and the neighbouring genes of ENST00000360485.Western blot was used to measure the protein level of microtubule-associated protein light chain 3(LC3),a marker of autophagy.ResultsThe results of microarray showed that there were 972 lncRNAs and 2138 mRNAs which were differentially expressed in macrophages infected with H37Ra,while 1417 lncRNA and 2478 mRNAs were differentially expressed in macrophages after H37Rv infection.The expression of the lncRNAs and mRNAs were highly consistent with the result of microarray data.GO and KEGG pathway analysis showed the function and pathways of the differentially expressed mRNA sassociated with H37Ra and H37Rv infection respectively.3 lncRNAs(ENST00000360485,MIR3945HG V1 and MIR3945HG V2)were differentially expressed between the healthy controls and tuberculosis patients.The results of ROC curve analysis showed that AUC(the area under the ROC curve)of ENST00000360485,MIR3945HG V1 and MIR3945HG V2 was 0.798,0.925 and 0.956.And the sensitivity was 83.97%,90%and 89.66%respectively,while the specificity was 71.88%,81.25%,90.63%.ENST00000360485 do not affect the macrophage-mediated elimination of MTB,the expression of the neighbouring genes and RNA levels of TNF-?,IL1-? and IL-6,as well as the activation of autophagy pathway in macrophages.ConclusionIn summary,our study revealed a great many of lncRNAs were consistently induced in macrophages infected with H37Ra and H37Rv,respectively,which indicated that IncRNAs might be crucial to regulate the antimicrobial mechanisms of macrophages.GO analysis and KEGG pathway analysis was performed on the differentially expressed mRNAs and the results revealed the broad function and metabolic pathways associated with tuberculosis infection.Our study also found three lncRNAs,ENST00000360485,MIR3945HG V1 and MIR3945HG V2,which have potential as diagnostic markers for tuberculosis.Finally,the preliminary functional study of ENST00000360485 lays the foundation for subsequent in-depth study.