Serum Exosomal miRNAs Are Associated with Active Pulmonary Tuberculosis

Abstract

Introduction: Tuberculosis (TB) remains a major threat to human health. Due to the limited accuracy of the current TB diagnostic tests, it is critical to determine novel biomarkers for this disease. Circulating exosomes have been used as diagnostic biomarkers in various diseases.

Objective of the study: In this pilot study, we examined the expression of miRNAs as biomarker candidates for the diagnosis of TB infection.

Methods: Serum-derived exosomes were isolated from TB patients and matched control subjects. The expression of miR-484, miR-425, and miR-96 was examined by RT-PCR methods.

Results: The expression of miR-484, miR-425, and miR-96 were significantly increased in serum of TB patients which correlated with the TB infection level. A receiver operating characteristic (ROC) curve analysis showed the diagnostic potency of each individual serum exosomal miRNA with an area under the curve (AUC) = 0.72 for miR-484 (p < 0.05), 0.66 for miR-425 (p < 0.05), and 0.62 for miR-96 (p < 0.05).

Conclusion: These results demonstrate that exosomal miRNAs have diagnostic potential in active tuberculosis. The diagnostic power may be improved when combined with conventional diagnostic markers.

Figure 1

Characterization of the serum exosomes. (a) Transmission electron microscopy showing that serum exosomes are spherical particles with an average size of 70 nm. The bar represents 100 nm. (b) Detection of exosomal CD81 surface markers using flow cytometry. MFI (mean fluorescence intensity) represents the expression of CD81 on the surface of exosomes. The results are representative of three independent experiments. (c) Exosomal RNA analysis by Agilent Bioanalyzer indicated a population of 18–28 nt without prominent ribosomal RNA peaks. Results are representative of three independent experiments.

Figure 2

The relative expression of exosomal miR-484, miR-425, and miR-96-3-p in TB patients compared to that in control subjects. Real-time PCR of exosomal miR-484, miR-425, and miR-96 indicated upregulation in TB patients compared to control subjects (^∗p < 0.05 and ^∗∗p < 0.01 significantly different compared to control). Data represent mean ± SEM of data from 25 patients in each group. Each sample was analyzed twice in triplicate to give a single value for each subject. The relative value in control subjects is defined by the dotted blue line.

Figure 3

Correlation between the exosomal level of miRNAs and the grade of smear positivity. Real-time PCR showed increased exosomal miRNA expression with increasing infection rates in comparison with healthy controls. Data represent mean ± SEM from 25 patients of 2 independent analyses each repeated in triplicate for each subject. ^∗p < 0.05 and ^∗∗p < 0.01 significantly different compared to control. The relative value in control subjects is defined by the dotted blue line.

Figure 4

Diagnostic power of exosomal miR-484 (a), miR-425 (b), and miR-96 (c) determined by ROC curve analysis. The results show the area under the ROC curve (AUC) for the sensitivity and specificity of each miRNA. The improved AUC (95% CI) for the combination of all miRNAs is shown in (d).