Deregulated lncRNAs in B Cells from Patients with Active Tuberculosis

<div><p>Role of lncRNAs in human adaptive immune response to TB infection is largely unexplored. To address this issue, here we characterized lncRNA expression profile in primary human B cell response to TB infection using microarray assay. Several lncRNAs and mRNAs were chosen for RT-qPCR validation. Bioinformatics prediction was applied to delineate function of the deregulated mRNAs. We found that 844 lncRNAs and 597 mRNAs were differentially expressed between B cell samples from individuals with or without TB. KEGG pathway analysis for the deregulated mRNAs indicated a number of pathways, such as TB, TLR signaling pathway and antigen processing and presentation. Moreover, corresponding to the dysregulation of many lncRNAs, we also found that their adjacent protein-coding genes were also deregulated. Functional annotation for the corresponding mRNAs showed that these lncRNAs were mainly associated with TLR signaling, <a href="http://www.ncbi.nlm.nih.gov/pubmed/27197161" target="_blank">TGF-β signaling</a>. Interestingly, SOCS3, which is a critical negative regulator of cytokine response to TB infection and its nearby lncRNA XLOC_012582, were highly expressed in active TB B cells. Subsequent RT-qPCR results confirmed the changes. Whether upregulated XLOC_012582 causes SOCS3 overexpression and is eventually involved in the context of exacerbations of active TB represents an interesting issue that deserves to be further explored. Taken together, for the first time, we identified a set of deregulated lncRNAs in active TB B cells and their functions were predicted. Such findings provided novel insight into the pathogenesis of TB and further studies should focus on the function and pathogenic mechanisms of the lncRNAs involved in active TB.</p></div

Overexpressed miRNAs in TB sputum.

<p>Column “Name” contained the name of miRNA; Column “TB patients versus control” contained level ratio of TB/control; Column “Chromosome” meant distribution of each miRNA on chromosome.</p><p>Each miRNA spot was replicated for four times on the same slide and two microarray chips were used for each group. After normalization, obtained average values for each miRNA spot were used for statistics. The <i>P</i> values for these miRNAs were less than 0.05 in TB group compared with controls.</p

Expression signatures of deregulated lncRNAs in ATB group versus control group.

<p>(A) Classification of lncRNAs. They were mainly intergenic. (B) Length distribution of lncRNAs. They were mainly between 401 and 800 nt in length. (C) Chromosome distribution of lncRNAs. Chromosome 1 and 2 were the most frequent ones and numbers of the deregulated lncRNAs on the two chromosomes accounted for approximately 11% and 9% of total deregulated ones, which were higher than the expected numbers based on chromosome total lncRNA numbers, respectively.</p

RT-qPCR validation results of the randomly selected lncRNAs and mRNAs between ATB group and control group.

<p>Four lncRNAs RP11-99H8.1, ENST00000507373, RP1-90G24.6, TCONS_00024847 and four mRNAs CH25H, NRG1, IL-32, HOXC4 were chosen for the validation of the microarray data usingRT- qPCR. The height of the columns in the figure represents the lncRNA expression level relative to GAPDH; the bars represent SD. *<i>P</i> <0.05 versus control group.</p

Confirmation miRNA level by RT-qPCR.

<p>RT-qPCR analysis confirmed microarray data. After normalization to U6 RNA, data were presented as mean ± SD (n = 30) and obtained average value for each miRNA was used for statistics. MiR-19b-2* was underexpressed while miR-3179 and miR-147 were overexpressed in TB sputum compared with controls. The experiment was conducted in triplicate. ^#<i>P</i><0.05 versus control.</p

Underexpressed miRNAs in TB sputum.

<p>Column “Name” contained the name of miRNA; Column “TB patients versus control” contained level ratio of TB/control; Column “Chromosome” meant distribution of each miRNA on chromosome.</p><p>Each miRNA spot was replicated for four times on the same slide and two microarray chips were used for each group. After normalization, obtained average values for each miRNA spot were used for statistics. The <i>P</i> values for these miRNAs were less than 0.05 in TB group compared with controls.</p

Deregulated lincRNAs and associated coding gene pairs (distance<300 kb).

<p>Deregulated lincRNAs and associated coding gene pairs (distance<300 kb).</p

Deregulated antisense lncRNAs and their associated coding genes.

<p>Deregulated antisense lncRNAs and their associated coding genes.</p

Deregulated enhancer-like lncRNAs and nearby coding genes (distance<300 kb).

<p>Deregulated enhancer-like lncRNAs and nearby coding genes (distance<300 kb).</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p