Identification of Serum microRNA Biomarkers for Tuberculosis Using RNA-seq

<div><p>Tuberculosis (TB) remains a significant human health issue. More effective biomarkers for use in tuberculosis prevention, diagnosis, and treatment, including markers that can discriminate between healthy individuals and those with latent infection, are urgently needed. To identify a set of such markers, we used Solexa sequencing to examine microRNA expression in the serum of patients with active disease, healthy individuals with latent TB, and those with or without prior BCG inoculation. We identified 24 microRNAs that are up-regulated (2.85–1285.93 fold) and 6 microRNAs that are down-regulated (0.003–0.11 fold) (P<0.05) in patients with active TB relative to the three groups of healthy controls. In addition, 75 microRNAs were up-regulated (2.05–2454.58 fold) and 11 were down-regulated (0.001–0.42 fold) (P<0.05) in latent-TB infected individuals relative to BCG- inoculated individuals. Of interest, 134 microRNAs were differentially-expressed in BCG-inoculated relative to un-inoculated individuals (18 up-regulated 2.9–499.29 fold, 116 down-regulated 0.0002–0.5 fold), providing insights into the effects of BCG inoculation at the microRNA level. Target prediction of differentially-expressed microRNAs by microRNA-Gene Network analysis and analysis of pathways affected suggest that regulation of the host immune system by microRNAs is likely to be one of the main factors in the pathogenesis of tuberculosis. qRT-PCR validation indicated that hsa-miR-196b and hsa-miR-376c have potential as markers for active TB disease. The microRNA differential-expression profiles generated in this study provide a good foundation for the development of markers for TB diagnosis, and for investigations on the role of microRNAs in BCG-inoculated and latent-infected individuals.</p></div

Demographic characteristics of the study population.

<p>LTBI, individuals with latent TB infection.</p

Venn diagram illustrating the distribution of microRNAs which showed significantly altered expression in TB patients compared with three control groups (LTBI, BCG-inoculated, and un-inoculated individuals).

<p>A, microRNAs which were up-regulated in serum from TB patients compared with the control groups; B, microRNAs which were down-regulated in serum from TB patients compared with the control groups. BCG, BCG-inoculated; LTBI, individuals with latent TB infection; Healthy, un-inoculated controls.</p

Effects of miRNA overexpression on NFAT5 mRNA levels (2^−ΔΔCT) in HEK293 cells.

<p>HEK293 cells were transfected respectively with four miRNA mimics (<i>hsa-miR-376c</i>, <i>hsa-miR-516b</i>, <i>hsa-miR-486-5p</i> and <i>cel-miR-67-3p</i>) and incubated at 37°C (5% CO₂) for 24 h. NFAT5 mRNA levels were measured in extracts of total RNA using RT-qPCR. NC: negative control (<i>cel-miR-67-3p</i>). Data presented are mean values from three independent experiments, n = 3. Error bars show the s.e.m. The significance of comparisons was tested using the Student’s <i>t</i>-test; *: P<0.05.</p

microRNA-gene network for hsa-miR-196b, hsa-miR-516b, hsa-miR-376c and hsa-miR-486-5p.

<p>The microRNA-gene network was built using gene expression data and predicted interactions from the TargetScan and PicTar microRNA databases. Red circles represent microRNAs and blue squares represent genes; their relationship is represented by the edges.</p

Up-regulation of I-309, IL-8, and MIG in the serum of active TB patients.

<p>(A-C) quantitative humane cytokine array analysis of peripheral serum samples from health controls (n = 20), individuals with LTBI (n = 20) and patients with active TB (n = 20). Blue, I-309; red, IL-8; yellow, MIG. (D-F) ELISA analysis for the confirmation of I-309, IL-8 and MIG expression in peripheral serum samples from health controls, patients with active TB, and individuals with LTBI or other pulmonary diseases (n = 45 in each group). Mean values ± standard error are shown, * P<0.05; ** P < 0.01 (Student’s <i>t</i>-test).</p

The arrangement of cytokines and antigens on TB-relate cytokine and TB-specific antigen array.

<p>Mouse anti-humane-IgG IgG was used as positive control and BSA were used as negative control.</p

I-309, IL-8 and MIG over-expression is stimulated by Mtb antigens (ESAT-6 and CFP-10) in active TB.

<p>Expression of I-309 (A), IL-8 (B), and MIG (C) induced by PBS or specific TB antigens in whole blood displayed a gradual increase from health controls (n = 80) to individuals with LTBI (n = 45), to those with active TB (n = 80). Mean values ± standard error are shown, *P < 0.05; **P < 0.01 (Student’s <i>t</i>-test).</p

Determination of cut-off values for I-309, IL-8 and MIG in the diagnosis of active TB.

<p>Determination of cut-off values for I-309, IL-8 and MIG in the diagnosis of active TB.</p