Circulating microRNAs (miRNAs) are useful biomarkers of hemolysis. Since blood cells are the main origins of circulating miRNAs, we evaluated blood cell-related pre-analytical modification of the miRNA signatures during blood drawing and serum processing. The levels of miRNA before and after ex vivo blood drawing were analyzed with the reverse transcriptase-based polymerase chain reaction method. Furthermore, the changes of miRNA signatures caused by different time-lag between blood drawing and serum preparation by 24 h were evaluated. Finally, we compared the miRNA levels between leftover samples and samples of hemolytic diseases. Blood drawing procedure induced increments of red blood cell (RBC)-related miRNAs (miR-451a, miR-486) about 2-fold. One hour standing of blood samples before serum separation induced almost the same increases in RBC-related miRNAs. To test the clinical usefulness of miR-451a as a biomarker of hemolytic diseases, we analyzed miRNAs of samples from 10 normal subjects, 30 leftover samples in the clinical laboratory, and 20 samples from patients with hemolytic diseases. Serum miR-451a significantly increased in patients with hemolytic anemia more than the levels of pre-analytical modification. In conclusion, the pre-analytical modification of serum miRNAs did not disturb the usefulness of RBC-derived miRNAs as biomarkers of hemolytic diseases.

Type 2 Diabetes mellitus (T2DM) is one of the most common diseases in the world and its prevalence ratio is still increasing. Patients with T2DM have diverse pathophysiological changes like as macrovascular, microvascular diseases, cancers as well as abnormal glucose metabolism. Thus, there are urgent needs to develop relevant biomarkers for the broad range of pathophysiology in patients with T2DM. We analyzed the signatures of serum miRNAs with the miRNA array analysis and reverse-transcription based quantitative polymerase chain reaction (RT-qPCR) in 50 patients with type 2 DM (T2DM) and 15 normal subjects. Array analysis showed that 19 miRNAs were up-regulated more than 2-fold and 71 miRNAs were down-regulated less than 0.5 in T2DM in comparison with normal subjects. Top 5 of up-regulated miRNAs were miR-3619-3p, miR-557, miR-6850-5p, miR-3648, miR-4730, and 5 of most down-regulated miRNAs were miR-5100, miR-4454, miR-1260b, miR-7975, miR-6131. We selected 4 miRNAs for validation analysis with RT-qPCR based on the abundance enough for reliable analyses and disease-specificities reported in previous reports. Serum miR-126-3p was down-regulated (3.21-fold, p<0.05) in T2DM, and miR-10a up-regulated (1.94-fold, p<0.05). However, none of single miRNA had significant correlation with clinical data and state. Data of the paired miRNAs: miR-10a and miR-200c, or miR-126 and miR-10a, clearly differentiated T2DM patients from normal subjects (p<0.05). Our study showed the paired-miRNA analyses as the more effective diagnostics for T2DM than the single miRNA analysis.

Extracellular vesicles (EVs) are nano-sized particles released from cells and transferring molecules (proteins, lipids and nucleic acids such as mRNA, tRNA and miRNA) to recipient cells. Surface antigens and components are important for the functions as cell-to-cell communication of EVs. Thus, EVs are useful biomarkers for various diseases including leukemias and other types of malignancies. We evaluated whether miRNAs in EVs released from chronic myelogenous leukemia (CML) cells could be used for diagnosis. Microarray analysis of miRNAs in EVs obtained from the culture supernatants of two CML cell lines showed that miR-494 and miR-373-5p were significantly decreased by tyrosine kinase inhibitor for BCR-ABL1. Validation analysis with Taqman-based qRT-PCR of whole serum obtained patients with CML in the chronic phase (n = 5) did not show a significant difference in miR-494 levels compared to the CML accelerated phase and blast crisis patients (n = 5). However, the levels of miR-494 were 2.9-fold higher in the accelerated phase or blast crisis than in the chronic phase (p < 0.05). These results indicate that it is important to measure miR-494 using only EVs rather than whole serum. Our data suggest that EV-miR-494 is a useful biomarker of CML progression and evaluation of response to tyrosine kinase inhibitors.