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.
Researchers from the University of Miami measured miRNA expression in plasma-derived exosomes to assess the value of exosome miRNAs as biomarkers for T1DM. 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. The researchers 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.
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).