title: Lectin-induced agglutination method of urinary exosomes isolation followed by mi-RNA analysis: Application for prostate cancer diagnostic.
abstract: BACKGROUND
  Prostate cancer is the most common cancer in men. Prostate-specific antigen has, however, insufficient diagnostic specificity. Novel complementary diagnostic approaches are greatly needed. MiRNAs are small regulatory RNAs which play an important role in tumorogenesis and are being investigated as a cancer biomarker. In addition to their intracellular regulatory functions, miRNAs are secreted into the extracellular space and can be found in various body fluids, including urine. The stability of extracellular miRNAs is defined by association with proteins, lipoprotein particles, and membrane vesicles. Among the known forms of miRNA packaging, tumour-derived exosome-enclosed miRNAs is thought to reflect the vital activity of cancer cells. The assessment of the exosomal fraction of urinary miRNA may present a new and highly specific method for prostate cancer diagnostics; however, this is challenged by the absence of reliable and inexpensive methods for isolation of exosomes.
  
  
  METHODS
  Prostate cancer (PC) cell lines and urine samples collected from 35 PC patients and 35 healthy donors were used in the study. Lectins, phytohemagglutinin, and concanavalin A were used to induce agglutination of exosomes. The efficiency of isolation process was evaluated by AFM and DLS assays. The protein content of isolated exosomes was analysed by western blotting. Exosomal RNA was assayed by automated electrophoresis and expression level of selected miRNAs was evaluated by RT-qPCR. The diagnostic potency of the urinary exosomal miRNA assessment was estimated by the ROC method.
  
  
  RESULTS
  The formation of multi-vesicular agglutinates in urine can be induced by incubation with lectin at a final concentration of 2 mg/ml. These agglutinates contain urinary exosomes and may be pelleted by centrifugation with a relatively low G-force. The analysis of PC-related miRNA in urinary exosomes revealed significant up-regulation of miR-574-3p, miR-141-5p, and miR-21-5p associated with PC.
  
  
  CONCLUSIONS
  Lectin-induced aggregation is a low-cost and easily performed method for isolation of exosomes from urine. Isolated exosomes can be further analysed in terms of miRNA content. The miRNA profile of urinary exosomes reflects development of prostate cancer and may present a promising diagnostic tool.
authors.affiliation: Laboratory of Oncoendocrinology, N.N. Petrov Institute of Oncology, Pesochny, Saint-Petersburg, Russia.|Laboratory of Genetic Engineering, Russian Research Centre for Radiology and Surgical Technologies, St. Petersburg, Russia.
authors.forename: Roman
authors.initials: R
authors.lastname: Samsonov
authors.affiliation: Division of Molecular and Radiation Biophysics, SFBI Petersburg Nuclear Physics Institute, Gatchina, Saint-Petersburg, Russia.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Shtam
authors.affiliation: Division of Molecular and Radiation Biophysics, SFBI Petersburg Nuclear Physics Institute, Gatchina, Saint-Petersburg, Russia.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Burdakov
authors.affiliation: Department of Genetics and Biotechnology, Saint Petersburg State University, Saint-Petersburg, Russia.
authors.forename: Andrey
authors.initials: A
authors.lastname: Glotov
authors.affiliation: Laboratory of Oncoendocrinology, N.N. Petrov Institute of Oncology, Pesochny, Saint-Petersburg, Russia.
authors.forename: Evgenia
authors.initials: E
authors.lastname: Tsyrlina
authors.affiliation: Laboratory of Oncoendocrinology, N.N. Petrov Institute of Oncology, Pesochny, Saint-Petersburg, Russia.
authors.forename: Lev
authors.initials: L
authors.lastname: Berstein
authors.affiliation: Department of Urology, N.N. Petrov Institute of Oncology, Pesochny, Saint-Petersburg, Russia.
authors.forename: Alexander
authors.initials: A
authors.lastname: Nosov
authors.affiliation: Laboratory of Genetic Engineering, Russian Research Centre for Radiology and Surgical Technologies, St. Petersburg, Russia.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Evtushenko
authors.affiliation: Division of Molecular and Radiation Biophysics, SFBI Petersburg Nuclear Physics Institute, Gatchina, Saint-Petersburg, Russia.
authors.forename: Michael
authors.initials: M
authors.lastname: Filatov
authors.affiliation: Laboratory of Oncoendocrinology, N.N. Petrov Institute of Oncology, Pesochny, Saint-Petersburg, Russia.|Division of Molecular and Radiation Biophysics, SFBI Petersburg Nuclear Physics Institute, Gatchina, Saint-Petersburg, Russia.|Laboratory of Cell Migration and Invasion, Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Malek
mesh_terms: D000328:Adult; D000368:Aged; D000372:Agglutination Tests / Q000379:methods; D015415:Biomarkers / Q000652:urine; D055354:Exosomes / Q000378:metabolism*; D006801:Humans; D037102:Lectins / Q000494:pharmacology; D008297:Male; D035683:MicroRNAs / Q000652:urine*; D008875:Middle Aged; D060787:Neoplasm Grading; D009367:Neoplasm Staging; D011471:Prostatic Neoplasms / Q000175:diagnosis / Q000473:pathology / Q000652:urine; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
chemical_list: D015415:Biomarkers; D037102:Lectins; D035683:MicroRNAs
keywords: diagnostic; exosomes; lectin; miRNA; prostate cancer