Research Article: Data analysis algorithm for the development of extracellular miRNA-based diagnostic systems for prostate cancer

Date Published: April 10, 2019

Publisher: Public Library of Science

Author(s): O. E. Bryzgunova, I. A. Zaporozhchenko, E. A. Lekchnov, E. V. Amelina, M. Yu. Konoshenko, S. V. Yarmoschuk, O. A. Pashkovskaya, A. A. Zheravin, S. V. Pak, E. Yu. Rykova, P. P. Laktionov, MOHAMMAD Saleem.


Urine of prostate cancer (PCa) carries miRNAs originated from prostate cancer cells as a part of both nucleoprotein complexes and cell-secreted extracellular vesicles. The analysis of such miRNA-markers in urine can be a convenient option for PCa screening. The aims of this study were to reveal miRNA–markers of PCa in urine and design a robust and precise diagnostic test, based on miRNA expression analysis. The expression analysis of the 84 miRNAs in paired urine extracellular vesicles (EVs) and cell free urine supernatant samples from healthy donors, patients with benign and malignant prostate tumours was done using miRCURY LNA miRNA qPCR Panels (Exiqon, Denmark). Sets of miRNAs differentially expressed between the donor groups were found in urine EVs and urine supernatant. Diagnostically significant miRNAs were selected and algorithm of data analysis, based on expression data on 24-miRNA in urine and obtained using 17 analytical systems, was designed. The developed algorithm of data analysis describes a series of steps necessary to define cut-off values and sequentially analyze miRNA expression data according to the cut-offs to facilitate classification of subjects in case/control groups and allows to detect PCa patients with 97.5% accuracy.

Partial Text

Timely detection of neoplasms is the key to successful tumor therapy. Thus, the urgent task of modern diagnostic medicine is the detection of cancer at the early stages of development (pre-cancer and early cancer), monitoring of the effects of anticancer therapy and detection of metastases and recurrences [1, 2].

Urine samples were obtained from 10 healthy men (HD, age range 48–65 years, mean age– 57.4 years), 10 patients with benign prostatic hyperplasia (BPH, age range 52–80 years, mean age– 57.4 years) and 10 previously untreated PCa patients (PCa, age range 56–82 years, mean age– 70.7). Diagnosis was confirmed based on clinical, morphological, radiological examination and the results of surgical intervention. Criteria for the PCa group were stage T2-3N0M0, PSA blood levels above 10 ng/ml and the absence of previous oncological diseases of the prostate or other localizations in the anamnesis. Criteria for the BPH group were clinical confirmation of the diagnosis and the absence of oncological diseases of the prostate or other localizations in the anamnesis. Men with a normal blood PSA level (below 2.8 ng/ml), no clinical history of oncological diseases and complaints from the urogenital system were included in the HD group. Samples were obtained from “E. Meshalkin National medical research center” of the Ministry of Health of the Russian Federation, (Novosibirsk, Russia). The work was conducted in compliance with the principles of voluntariness and confidentiality in accordance with the “Fundamentals of Legislation on Health Care”. The study was approved by the ethics committees of ICBFM SB RAS, “E. Meshalkin National medical research center” of the Ministry of Health of the Russian Federation (Novosibirsk, Russia) and Novosibirsk Regional Oncology Center (Novosibirsk, Russia) and written informed consent was provided by all participants.

To study the distribution and representation of PCa-specific miRNAs in urine, a comparative analysis of 84 miRNA expression was performed in paired samples of EVs and cell-free urine supernatant from healthy men, patients with BPH and PCa using miRCURY LNA miRNA qPCR Panels (Exiqon Ltd, Denmark). On average, at least 70 of the 84 miRNAs were detected in each sample, with 72 miRNAs expressed in more than 70% of the samples. For the great majority of miRNAs the difference in expression between EVs and urine supernatant was quantitative, but a few miRNAs were predominantly present in only one of the fractions. The most obvious examples are miR-143-3p and miR-451a, which were present in cell-free urine supernatant and absent almost in all EVs samples. This can be due to the secretion or efflux of miR-451a as a part of protein and/or lipoprotein complexes from blood cells where it is highly expressed, followed by infiltration into the urine [23, 24]. However, during ultracentrifugation–based isolation of EVs the concentration of circulating miRNA complexes in the samples is reduced by more than twenty times.

Prostate cancer is the second most diagnosed cancer among males worldwide [25]. The highest incidence rates are in the U.S. where it kills over 27000 men annually [26]. According to GLOBOCAN 2012 mortality rate in less developed countries is even higher. Despite a slight decrease in mortality from PCa since 1992, due to the widespread PSA testing of men’s blood, the incidence of PCa continues to increase every year [27]. An overwhelming majority (99%) of all prostate cancers occur in men over 50 and are mostly slow-growing. In contrast, in younger men the development of aggressive PCa disease resulting in patient’s death is significantly more likely [27].

The expression and distribution of 84 miRNAs in two urine fractions (EVs and cell-free supernatant) was studied in healthy donors, patients with BPH and PCa. An approach to the development of a miRNA-based diagnostic system was proposed and resulting panels used to classify PCa and BPH patients and HD with 100% specificity and 97.5% accuracy. The diagnostic system based on the expression of 5 miRNA pairs (miR-30a: miR-125b; miR-425: miR-331; miR-29b: miR-21; miR-191: miR-200a; miR-331: miR-106b), can be potentially used to identify PCa and aid in therapy optimization.




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