Plasma-derived exosome characterization reveals a distinct microRNA signature in long duration Type 1 diabetes

Abstract

Type 1 diabetes mellitus (T1DM) results from an autoimmune attack against the insulin-producing ß cells which leads to chronic hyperglycemia. Exosomes are lipid vesicles derived from cellular multivesicular bodies that are enriched in specific miRNAs, potentially providing a disease-specific diagnostic signature. To assess the value of exosome miRNAs as biomarkers for T1DM, miRNA expression in plasma-derived exosomes was measured. Nanoparticle tracking analysis and transmission electron microscopy confirmed the presence of plasma-derived exosomes (EXOs) isolated by differential centrifugation. Total RNA extracted from plasma-derived EXOs of 12 T1DM and 12 control subjects was hybridized onto Nanostring human v2 miRNA microarray array and expression data were analyzed on nSolver analysis software. We found 7 different miRNAs (1 up-regulated and 6 down-regulated), that were differentially expressed in T1DM. The selected candidate miRNAs were validated by qRT-PCR analysis of cohorts of 24 T1DM and 24 control subjects. Most of the deregulated miRNAs are involved in progression of T1DM. These findings highlight the potential of EXOs miRNA profiling in the diagnosis as well as new insights into the molecular mechanisms involved in T1DM.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

Work flow of study design and sample processing. Plasma from T1DM (n = 36) and control subjects (n = 36) was collected. Exosomes were isolated by ultracentrifugation and characterized by TEM and NTA analysis. Total exosome RNA was isolated and use for the miRNA microarray analysis (discovery set) or for the qRT-PCR validation (validation set) (A). Plasma exosomes were analyzed under electron microscopy which displayed the same morphology in T1DM and control subjects (B,C). Particle and Size distribution of exosomes analyzed by the Nanoparticle tracking analysis of T1DM and control subjects (D,E). Exosomal RNAs determined by the Agilent RNA Pico Chip. Exosomal RNA samples contained no detectable 18S and 28S rRNAs (G). Validation of selected exosome protein expression by flow cytometry (control read peak) (F). Small RNA Densitometry traces profiles were used to quantify and compare the relative abundance of various small RNAs in T1DM (H) and Control subjects (I).

Figure 2

Differential miRNA content in T1DM and control subjects. Heatmap of the per-row normalized expression levels of selected miRNAs differentially expressed in T1DM and control subjects plasma-derived exosomes (A). Scatter plot of proteins enriched in Control or T1D subjects, n = 12 per group, using P < 0.05 (multiple t-test) and array threshold as cutoffs. Noted in the legend are Control subjects overexpressed miRNAs (green), T1D overexpressed miRNAs (red) (B). Correlation plot of exosome miRNAs, indicating in red and green those miRNAs expressed differentially T1DM and control subject’s exosomes (C).

Figure 3

Differential microarray miRNA expression in T1DM and control subjects. Individual changes in the miRNA plasma exosome levels of the one up-regulated and six down-regulated miRNAs in T1DM patients (n = 12) and control subjects (n = 12). The signal intensities were normalized to the total signal intensity of the microarray. The horizontal lines indicate the mean normalized signal intensity for each group. Statistically significant differences were determined by Student’s t-test.

Figure 4

Box-and-whisker plots of the expression levels of the seven selected miRNAs in an independent set of T1DM (n = 24) and control subjects (n = 24). The comparative cycle threshold (Ct) method was used to quantify the levels of exosome miRNAs in T1DM and control subjects. The relative ratio was calculated using the ΔCt method. The Ct value of miR-631 was used as an internal standard. (*p-value 

Figure 5

Effect of plasma-derived exosomes from T1D and controls on insulin secretion of perifused islets. Groups of 350 IEQ islets each, were stimulated every minute as shown in (A) for low (3 mM) → high (11 mM) → low (3 mM) glucose challenge followed by a KCl step. Pancreatic islet perifusion, normalized iInsulin secretion data to islet volume (IEQ). Data are expressed as ng insulin/islet equivalents (IEQs) per minute (A,B) or area under the curve (AUC) for each secretagogue (C–E). Statistical analysis was performed by Student’s t-test.