P67

Abstract

Differently sized microparticles, including exosomes (30–100 nm), prostasomes (50–500 nm), oncosomes (50–500 nm) and other microparticles (100–1000 nm) were found in blood and urine. Exosomes from prostate cancer (PCa) patients can potentially contain cancer-specific nucleic acids, and thus can represent a valuable source of diagnostic material. In this study, we have investigated microvesicles and miRNA from urine of healthy donors and PCa patients. To isolate miRNAs from urine and microparticles, novel methods for miRNA isolation were elaborated (Rus. patent application No 2014137763, priority date 17.09.2014). The study population included 14 patients with PCa (63–82 years, T2-3NxMx1) and control group of 20 healthy volunteers with no previous history of prostate disease (48–73 years). Urine was clarified by two serial centrifugations at 400g, 20 °C, 20 min and at 17000g, 20 °C, 20 min. Microparticles were precipitated from the resulting supernatant by high-speed centrifugation at 100000g, 18 °C, 90 min, the pellet was resuspended and pelleted by centrifugation under the same conditions. To isolate exosomes, total microparticles were filtered through 0.1 μm pore filters and reprecipitated. The resulting pellets were resuspended and exosome samples were investigated by transmission electron microscopy (TEM). MiRNAs were isolated by one-step single-phase protocol and purified using “BioSilica” spin- columns (Zaporozhchenko et al., Anal. Biochem, upcoming, doi: 10.1016/j.ab.2015.03.028) and by recently developed method based on precipitation of excess biopolymers, allowing to isolate miRNAs with better efficiency than commercially available kits. The size and quantity assessment of extracellular RNA were performed using capillary electrophoresis system on Agilent 2100 Bioanalyzer. Concentrations of miRNAs (miR19b, miR25, miR205, miR125b, miR126) were measured by qRT-PCR and normalized to miR-16 using dCq method.

TEM demonstrated the presence of 20–300 nm microparticles in urine of healthy donors and PCa patients. Approximately 50–70% of all urine microparticles are represented by 30–100 nm exosomes and residual 30–50% by particles larger than 100 nm. (The pool of urine microvesicles consists of 50–70% exosomes and 30–50% particles larger than 100 nm.).

The major part of extracellular RNA found both in exosomes and total microparticles fraction of healthy donors and patients with PCa, is 25–200 nt long and can include tRNA (73–93 n.), 5.8 rRNA (∼150 n.), snoRNK (10–20 n.), snRNA (60–300 n.) piRNAs (29–30 n.), miRNAs (20–25 n.), siRNA (21–25 n.). Concentration of extracellular urine RNA in exosomes and total microparticles of healthy donors and patients with PCa amounts to100 pg/ml of urine on average.Receiver Operating Characteristic (ROC) curve analysis of all miRNAs in samples isolated from total urine did not demonstrate any diagnostic significance. In contrast, in training cohort concentration of miR-19b in exosomes and total microparticles fraction provide 95%/75% and 93%/100% sensitivity and specificity, respectively.

Thus, microparticles, isolated from urine of PCa patients represent a valuable source of diagnostically significant miRNAs. To investigate miR-19b, a large testing cohort is required. Search for other potential miRNA-markers by microarray profiling of cell-free miRNA isolated from urine of PCa patients is also required to reveal a sold set of markers for confident PCa diagnostics.