Characterization of a novel panel of plasma microRNAs that discriminates between Mycobacterium tuberculosis infection and healthy individuals

Abstract

Cavities are important in clinical diagnosis of pulmonary tuberculosis (TB) infected by Mycobacterium tuberculosis. Although microRNAs (miRNAs) play a vital role in the regulation of inflammation, the relation between plasma miRNA and pulmonary tuberculosis with cavity remains unknown. In this study, plasma samples were derived from 89 cavitary pulmonary tuberculosis (CP-TB) patients, 89 non-cavitary pulmonary tuberculosis (NCP-TB) patients and 95 healthy controls. Groups were matched for age and gender. In the screening phase, Illumina high-throughput sequencing technology was employed to analyze miRNA profiles in plasma samples pooled from CP-TB patients, NCP-TB patients and healthy controls. During the training and verification phases, quantitative RT-PCR (qRT-PCR) was conducted to verify the differential expression of selected miRNAs among groups. Illumina high-throughput sequencing identified 29 differentially expressed plasma miRNAs in TB patients when compared to healthy controls. Furthermore, qRT-PCR analysis validated miR-769-5p, miR-320a and miR-22-3p as miRNAs that were differently present between TB patients and healthy controls. ROC curve analysis revealed that the potential of these 3 miRNAs to distinguish TB patients from healthy controls was high, with the area under the ROC curve (AUC) ranged from 0.692 to 0.970. Moreover, miR-320a levels were decreased in drug-resistant TB patients than pan-susceptible TB patients (AUC = 0.882). In conclusion, we identified miR-769-5p, miR-320a and miR-22-3p as potential blood-based biomarkers for TB. In addition, miR-320a may represent a biomarker for drug-resistant TB.

Fig 1. Flow chart of the experimental design.

Fig 2. MiRNA expression in plasma derived from CP-TB, NCP-TB patients and healthy controls (H).

Fig 3. Detection of TB using 3 plasma miRNAs as a biomarker.

A hydrolysis probe–based qRT-PCR assay was used to measure the relative levels of the 3 miRNAs in 64 CP-TB patients, 64 NCP-TB patients and 64 healthy controls (in both the training and validation set). Each point represents the mean of the results for triplicate. The asterisks indicate significant differences compared to healthy controls. * P<0.05; ** P<0.01; *** P<0.001. (A) miR-769-5p, (B) miR-320a and (C) miR-22-3p.

Fig 4. ROC curves to compare the ability of miRNA to distinguish TB patients from the healthy controls.

(A-D) miR-769-5p, miR-320a, miR-22-3p and the three-miRNA panel for discriminating between NCP-TB patients and healthy controls; (E-H) miR-769-5p, miR-320a, miR-22-3p and the three-miRNA panel for discriminating between CP-TB patients and healthy controls; (I-L) miR-769-5p, miR-320a, miR-22-3p and the three-miRNA panel for discriminating between TB patients and healthy controls.

Fig 5. Downregulation of miR-320a in plasma from drug-resistant TB patients compared with drug-susceptible TB patients.

(A) Relative concentration of miR-320a in plasma derived from drug-resistant and drug-susceptible TB patients. (B) ROC curves to comparing the ability of miR-320a to distinguish between drug-resistant TB and drug-susceptible TB.