Advances in Molecular OncologyAdvances in Molecular OncologyУспехи молекулярной онкологии2313-805X2413-3787Publishing House ABV Press54310.17650/2313-805X-2023-10-2-78-89EXPERIMENTAL REPORTЭКСПЕРИМЕНТАЛЬНАЯ СТАТЬЯCell-free plasma miRNAs analysis for low invasive lung cancer diagnosticsМалоинвазивная диагностика рака легкого на основе анализа внеклеточной микроРНК кровиhttps://orcid.org/0000-0003-2925-9350KonoshenkoM. Yu.КоношенкоМ. Ю.
Russian Federation

Maria Yu. Konoshenko.

8 Akademika Lavrentieva Prospekt, Novosibirsk 630090; 15 Rechkunovskaya St., Novosibirsk 630055

Коношенко Мария Юрьевна.

630090 Новосибирск, проспект Академика Лаврентьева, 8; 630055 Новосибирск, ул. Речкуновская, 15

msol@ngs.ruhttps://orcid.org/0000-0002-0866-0252LaktionovP. P.ЛактионовП. П.
Russian Federation

8 Akademika Lavrentieva Prospekt, Novosibirsk 630090; 15 Rechkunovskaya St., Novosibirsk 630055

630090 Новосибирск, проспект Академика Лаврентьева, 8; 630055 Новосибирск, ул. Речкуновская, 15

https://orcid.org/0009-0004-4174-7300LancuhajYu. A.ЛанцухайЮ. А.
Russian Federation

15 Rechkunovskaya St., Novosibirsk 630055

630055 Новосибирск, ул. Речкуновская, 15

https://orcid.org/0009-0009-6221-2758PakS. V.ПакС. В.
Russian Federation

15 Rechkunovskaya St., Novosibirsk 630055

630055 Новосибирск, ул. Речкуновская, 15

https://orcid.org/0000-0003-0687-0894KrasilnikovS. E.КрасильниковС. Э.
Russian Federation

15 Rechkunovskaya St., Novosibirsk 630055

630055 Новосибирск, ул. Речкуновская, 15

https://orcid.org/0000-0003-3433-7261BryzgunovaO. E.БрызгуноваО. Е.
Russian Federation

8 Akademika Lavrentieva Prospekt, Novosibirsk 630090; 15 Rechkunovskaya St., Novosibirsk 630055

630090 Новосибирск, проспект Академика Лаврентьева, 8; 630055 Новосибирск, ул. Речкуновская, 15

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of SciencesФГБУ «Институт химической биологии и фундаментальной медицины» Сибирского отделения Российской академии наукMeshalkin Siberian Federal Biomedical Research Center, Ministry of Health of RussiaФГБУ «Национальный медицинский исследовательский центр им. акад. Е.Н. Мешалкина» Минздрава России1505202310278891509202210072023Copyright ©; 2023, Konoshenko M.Y., Laktionov P.P., Lancuhaj Y.A., Pak S.V., Krasilnikov S.E., Bryzgunova O.E.Copyright ©; 2023, Коношенко М.Ю., Лактионов П.П., Ланцухай Ю.А., Пак С.В., Красильников С.Э., Брызгунова О.Е.2023Konoshenko M.Y., Laktionov P.P., Lancuhaj Y.A., Pak S.V., Krasilnikov S.E., Bryzgunova O.E.Коношенко М.Ю., Лактионов П.П., Ланцухай Ю.А., Пак С.В., Красильников С.Э., Брызгунова О.Е.https://creativecommons.org/licenses/by/4.0https://umo.abvpress.ru/jour/article/view/543

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.

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