Figure 1.
Unsupervised hierarchical clustering of the expression of 451 miRNAs.
The tree was generated by unsupervised hierarchical clustering of PBMC miRNA profiles for patients with active TB, subjects with LTBI, and healthy controls. MiRNAs with 2-fold up- or down-expression compared with the median intensity across all samples and differential expression values greater than 10% of all samples were selected for unsupervised analysis (n = 415 miRNAs). Sample clusters can be compared with the clinical parameters displayed in blocks underneath each profile. A key is provided at the bottom of the figure. Subjects' clinical status is indicated as follows: patients with active TB are indicated by red rectangles; subjects with LTBI by blue rectangles; and healthy controls by black rectangles. Age is indicated as: subjects between 15–24 years old are indicated by olive green rectangles; subjects 25–34 years old by brown rectangles; subjects 35–44 years old by purple rectangles; and subjects 45–54 years old by khaki rectangles. Females are indicated by hoar rectangles and males by darkish rectangles.
Table 1.
Characteristics of study participants with active tuberculosis (TB), latent TB infection (LTBI) and healthy controls.
Figure 2.
Multi-dimensional scaling of the relationships and unsupervised hierarchical clustering based on the 17 miRNAs expression pattern.
A. Multi-dimensional scaling generated by BRB Arraytools of the relationships between the 15 samples based on the 17 miRNAs expression pattern. Red dot, active TB; Blue dot, latent TB; Black dot, healthy controls. B. The tree generated by unsupervised hierarchical clustering (Pearson's correlation, average linkage) of 15 samples (columns) and 17 miRNAs differentially expressed among the 3 groups predicted by using the SVM method. Sample clusters can be compared with the clinical parameters displayed in blocks underneath each profile. A key is provided at the bottom of the figure. Subjects' clinical status is indicated as follows: patients with active TB are indicated by red rectangles; subjects with LTBI by blue rectangles; and healthy controls by black rectangles. Age is indicated as: subjects between 15–24 years old are indicated by olive green rectangles; subjects 25–34 years old by brown rectangles; subjects 35–44 years old by purple rectangles; and subjects 45–54 years old by khaki rectangles. Females are indicated by hoar rectangles and males by darkish rectangles.
Figure 3.
Quantitative PCR (qPCR) validation of miRNA expression levels in samples from the active tuberculosis (TB) group versus the latent tuberculosis infection (LTBI) groups.
We confirmed the gene expression levels of miRNAs using real-time qPCR. We analyzed the expression of 7 miRNAs (hsa-miR-223, hsa-miR-365 hsa-miR-424, and hsa-miR-451, hsa-miR-144, hsa-miR-500 and hsa-miR-21*) selected from the microarray data by qPCR. The 2−ΔΔCT method was used to normalize the relative gene expression data in the qPCR assay. U6 snRNA was set as the reference gene. The miRNA expression value in one subject with LTBI was normalized to 1. Statistical analysis was performed using the unpaired t-test. ** P<0.01, * P<0.05, NS: not significant.
Figure 4.
MiRNA-Gene-Network for the transition from latent to active tuberculosis.
The MiRNA-Gene-Network was built using the gene expression data and the predicted interactions in the TargetScan miRNA database. The mRNA profile was obtained from previously published studies [10]. miRNA–mRNA modules obtained via population-based probabilistic learning was used to determine the interactions in the network (Materials and Methods). Circles represent genes and squares represent miRNAs; their relationship is represented by one edge. The center of the network represents the degree (i.e., the interaction of one miRNA with the genes around and the interaction of one gene with the miRNAs around. The key miRNAs and genes in the network always have the highest degrees.
Table 2.
Differently expressed miRNAs in the samples from study participants with active tuberculosis (TB) versus latent tuberculosis infection (LTBI).