Circulating microRNAs in patients with active pulmonary tuberculosis

Abstract

Emerging evidence shows that microRNAs (miRNAs) play an important role in pathogen-host interactions. Circulating miRNAs have been repeatedly and stably detected in blood and hold promise to serve as molecular markers for diverse physiological and pathological conditions. To date, the relationship between circulating miRNAs and active pulmonary tuberculosis (TB) has not been reported. Using microarray-based expression profiling followed by real-time quantitative PCR validation, the levels of circulating miRNAs were compared between patients with active pulmonary tuberculosis and matched healthy controls. The receiver operating characteristic curve was used to evaluate the diagnostic effect of selected miRNA. Bioinformatic analysis was used to explore the potential roles of these circulating miRNAs in active pulmonary tuberculosis infection. Among 92 miRNAs significantly detected, 59 miRNAs were downregulated and 33 miRNAs were upregulated in the TB serum compared to their levels in the control serum. Interestingly, only two differentially expressed miRNAs were increased not only in the serum but also in the sputum of patients with active pulmonary tuberculosis compared to the levels for the healthy controls. Upregulated miR-29a could discriminate TB patients from healthy controls with reasonable sensitivity and specificity. A number of significantly enriched pathways regulated by these circulating miRNAs were predicted, and most of them were involved in acute-phase response, inflammatory response, and the regulation of the cytoskeleton. In all, for the first time our results revealed that a number of miRNAs were differentially expressed during active pulmonary tuberculosis infection, and circulating miR-29a has great potential to serve as a marker for the detection of active pulmonary tuberculosis infection.

Fig. 1.

Hierarchical clustering of miRNA in serum. Samples were clustered according to the expression profile of 92 differentially expressed miRNAs. Data from each miRNA were median centered. Samples are in columns and miRNAs are in rows. Red indicates high relative expression, and green indicates low relative expression. The P values for these miRNAs were less than 0.05 for active TB compared to controls.

Fig. 2.

Confirmation miRNA expression by quantitative real-time PCR. Quantitative real-time PCR analysis confirmed microarray data. After normalization to U6 RNA in each group, data were represented as the means ± SD (n = 30), and obtained average values for each miRNA were used for statistics of the active TB group compared to healthy controls. Serum miR-3125 was decreased while miR-93* and miR-29a were increased in active TB patients compared to levels in healthy controls. At the same time, sputum miR-29a also was present at higher abundance while there was no significant difference for sputum miR-93* in active TB patients compared to that in healthy controls. The experiments were conducted in triplicate.

Fig. 3.

Increased serum level of miR-29a for patients with active pulmonary TB versus healthy controls, with sensitivity of 83% and specificity of 80%, respectively.