Малоинвазивная диагностика рака легкого на основе анализа внеклеточной микроРНК крови

Introduction. The high mortality rate in patients with lung cancer (LC) is due to the lack of highly sensitive diagnostic markers of this disease. Genetic and epigenetic alterations in tumor cells, for example, aberrant microRNA expression, can be proposed. It is known that extracellular/circulating microRNA of biological fluids, in complexes with proteins, or packaged in extracellular vesicles is of interest for the diagnosis of tumor diseases.Aim. To perform a comparative analysis of miRNA expression in plasma and plasma extracellular vesicles of LC patients and healthy donors. Based on the obtained results, to propose a diagnostic panel to identify patients with LC.Materials and methods. Blood plasma was obtained from blood samples of healthy donors and LC patients by sequential centrifugation.  Then, a fraction of extracellular vesicles (40–150 nm in size) was isolated from a part of the obtained plasma supernatant by the method of aggregation-precipitation with polyethylene glycol/blue dextran. MicroRNAs were isolated from both blood plasma fractions of patients and healthy donors using guanidine isothiocyanate and octanoic acid. Expression of 17 miRNAs most characteristic for the development of LC according to our and literature data in the above-mentioned blood plasma fractions was analyzed by stem-loop reverse transcription polymerase chain reaction.Results. 29 and 10 miRNA pairs were differentially expressed in plasma extracellular vesicles and plasma of lung cancer patients and donors. Thus, plasma extracellular vesicles are characterized by greater potential as a source for miRNA based lung cancer diagnostic panels in comparison with blood plasma. Diagnostic algorithm based on aberrant miRNA expression of 8 different miRNAs (miRNA-30e, -1, -125b, -133, -222, -374, -425, -660) composed in 6 pairs was designed. This algorithm allows to diagnose 100 % of patients with lung cancer stages II–IV.Conclusion. Extracellular plasma vesicles represent a promising source of diagnostically significant microRNAs compared to plasma microRNAs. For the diagnosis of patients with non-small cell lung cancer with 100 % sensitivity and specificity, a panel of 8 microRNAs (6 miRNA pairs) was proposed.Введение. Одной из причин высокой смертности больных раком легкого (РЛ) является нехватка высокочувствительных диагностических маркеров этого заболевания. в качестве таковых могут быть предложены маркеры генетических и эпигенетических процессов, характерных для опухолевых клеток, например микроРНК. известно, что внеклеточная/циркулирующая  микроРНК биологических жидкостей в комплексах с белками или упакованная во внеклеточные везикулы представляет интерес для диагностики опухолевых заболеваний.Цель исследования – выполнить сравнительный анализ экспрессии микроРНК в составе внеклеточных везикул и супернатанта плазмы крови больных РЛ и доноров и предложить на основании полученных результатов диагностическую панель для выявления пациентов с данной патологией.Материалы и методы. из образцов крови доноров и больных РЛ методом последовательного центрифугирования была получена плазма крови. Затем из части супернатанта плазмы методом агрегации – осаждения полиэтиленгликолем/синим декстраном выделена фракция внеклеточных везикул (размером 40–150 нм). из обеих собранных фракций плазмы крови больных РЛ и доноров с использованием гуанидина изотиоцианата и октановой кислоты получены микроРНК. экспрессия 17 микроРНК, участвующих в механизмах развития РЛ, по нашим данным и данным литературы, в вышеупомянутых фракциях плазмы крови была проанализирована методом петлевой полимеразной цепной реакции с обратной транскрипцией.Результаты. В ходе исследования во фракциях внеклеточных везикул и супернатанта плазмы крови обнаружены 29 и 10 пар микроРНК соответственно, экспрессия которых достоверно различалась между больными немелкоклеточным РЛ и донорами. Таким образом, внеклеточные везикулы плазмы обладают большим потенциалом с точки зрения диагностики РЛ на основе оценки относительной экспрессии микроРНК по сравнению с плазмой крови. Разработан диагностический алгоритм, основанный на исследовании аберрантной экспрессии 8 различных микроРНК (miRNA-30e, -1, -125b, -133, -222, -374, -425, -660) в составе 6 пар, позволяющий выявить немелкоклеточный РЛ II–IV стадии в 100 % случаев.Заключение. внеклеточные везикулы являются более перспективными, диагностически значимыми микроРНК по сравнению с микроРНК плазмы крови. для диагностики больных немелкоклеточным РЛ предложена панель из 8 микроРНК, характеризующаяся 100 % чувствительностью и специфичностью

Isolation of Extracellular Vesicles: General Methodologies and Latest Trends

Background. Extracellular vesicles (EVs) play an essential role in the communication between cells and transport of diagnostically significant molecules. A wide diversity of approaches utilizing different biochemical properties of EVs and a lack of accepted protocols make data interpretation very challenging. Scope of Review. This review consolidates the data on the classical and state-of-the-art methods for isolation of EVs, including exosomes, highlighting the advantages and disadvantages of each method. Various characteristics of individual methods, including isolation efficiency, EV yield, properties of isolated EVs, and labor consumption are compared. Major Conclusions. A mixed population of vesicles is obtained in most studies of EVs for all used isolation methods. The properties of an analyzed sample should be taken into account when planning an experiment aimed at studying and using these vesicles. The problem of adequate EVs isolation methods still remains; it might not be possible to develop a universal EV isolation method but the available protocols can be used towards solving particular types of problems. General Significance. With the wide use of EVs for diagnosis and therapy of various diseases the evaluation of existing methods for EV isolation is one of the key problems in modern biology and medicine

The Influence of Radical Prostatectomy on the Expression of Cell-Free MiRNA

MiRNAs of blood and urine have been shown to represent a convenient source of biomarkers for prostate cancer (PCa) diagnosis and assessment of the therapy effectiveness due to their high stability and representation and the low invasiveness of sample collection. Here, we studied the influence of radical prostatectomy (RP) on the expression of 12 cell-free miRNAs previously shown as potential markers of PCa (i.e., miR-19b, miR-22, miR-92a, miR-378, miR-425, miR-30e, miR-31, miR-125b, miR-200b, miR-205, miR-375 and miR-660). The relative expression of the miRNAs combined into 31 paired ratios was evaluated in the urine extracellular vesicles (EVs), clarified urine (CU) and blood plasma of healthy donors, pre- and post-RP samples of PCa patients. Nineteen miRNA ratios based on combinations of ten of the miRNAs (miR-19b, miR-30e, miR-31, miR-125b, miR-200b, miR-205, miR-375, miR-378, miR-425, and miR-660) were altered by RP. The comparative expression analysis of the cell-free miRNA ratios between healthy donors and PCa patients revealed miR-125b/miR-30e and miR-375/miR-30e as potential markers for evaluating therapeutic efficacy. MiR-378/miR-19b, miR-425/miR-19b, miR-200/miR-30e, miR-660/miR-30e, and miR-205/miR-30e had minor prognostic value but could be used to increase the steadiness of the diagnostic system. The urine EVs had the highest potential as a source of markers

Searching for the Novel Specific Predictors of Prostate Cancer in Urine: The Analysis of 84 miRNA Expression

The aim of this study was to investigate miRNA profiles of clarified urine supernatant and combined urine vesicle fractions of healthy donors and patients with benign prostatic hyperplasia and prostate cancer (PCa). The comparative analysis of miRNA expression was conducted with a custom miRCURY LNA miRNA qPCR panel. Significant combinations of miRNA pairs were selected by the RandomForest-based feature selection algorithm Boruta; the difference of the medians between the groups and a 95% confidence interval was built using the bootstrap approach. The Asymptotic Wilcoxon-Mann-Whitney Test was performed for miRNA combinations to compare different groups of donors. Benjamini-Hochberg correction was used to adjust the statistical significance for multiple comparisons. The most diagnostically significant miRNAs pairs were miR-107-miR-26b.5p and miR-375.3p-miR-26b.5p in the urine supernatant fraction that discriminated the group of healthy patients and PCa patients, as well as miR-31.5p-miR-16.5p, miR-31.5p-miR-200b, miR-31.5p-miR-30e.3p and miR-31.5p-miR-660.5p in the fraction extracellular vesicles that were different between healthy men and benign prostate hyperplasia patients. Such statistical criteria as the occurrence of individual significant miRNA pairs in the total number of comparisons, median ΔCt difference, and confidence interval can be useful tools for determining reliable markers of PCa