e15597 Background: Oncolytic virotherapy is developing intensively in modern oncology. Viruses demonstrate the ability to the direct oncolysis and to the stimulation of antitumor immune activity; this experiment was aimed at solving the question of the prevalence of one of them. Glial tumors are the most common brain tumors; oncolytic viruses show certain prospects in their treatment due to the ability to penetrate the blood-brain barrier. The aim of the study was to determine the possible oncolytic effect of new unclassified group K rotaviruses (RVK) on T98G and U87MG glioblastoma cells in vitro. Methods: T98G and U87MG cell cultures were received from Russian banks of cell lines of human and animal tissues. Standard culturing was performed with attenuated apatogenic RVK strains No. 100 and No. 228 at a concentration of 108, 107, 106 and 105 particles/mL. The cytotoxic effect was determined with MTT and Annexin V assays, cell morphology was evaluated by the light-optical method. Results: Both RVK strains demonstrated a dose-dependent cytotoxic activity; the maximal effect was observed in strain No.100 at a dose of 108 particles/mL on U87MG cells (predominantly apoptosis). Studies of cell morphology showed a pronounced effect of RVK on the cell culture: significant degenerative changes in cells, a tendency to a decrease in cluster size, a change in their shape and granularity. Cluster formation in culturing in the serum-free medium is considered in the literature as a property of cancer stem cells responsible in vivo for tumor recurrence and its chemo- and radio-resistance. T98G cells demonstrated morphological changes: nuclear segmentation, diffused cytoplasm, indistinct cell borders with signs of syncytium formation. Conclusions: The established oncolytic effect of RVK strain No. 100 in vitro on glioma cells, presumably with tumor stem cells, indicates a significant potential for the use of these rotaviruses in treatment of glial tumors.
e13506 Background: Gliomas are the most common type of primary brain tumors, with a high level of recurrence and mortality. The purpose of the study was to determine the expression profile of micro-RNA targeting components of the Hedgehog, Notch, Wnt, EGFR, TGFβ, HIF1α signaling pathways according to MirTarBase, miRDB, TargetScan. Methods: The study included 30 patients (15 women and 15 men) aged 27 to 76 years with histologically verified glial brain tumors. The RT2-qPCR method in operational biopsy specimens determined the relative expression of 12 micro-RNAs: hsa-miR-17-3p, hsa-miR-20a-3p, hsa-miR-326, hsa-miR-330-3p, hsa-miR- 107, hsa-miR-143-3p, hsa-let-7a-5p, hsa-miR-146a-5p, hsa-miR-29a-3p, hsa-miR-92a-1-5p, hsa-miR-26b-3p, hsa-miR-96-5p. Hsa-miR-7-5p and hsa-miR-126-3p were used as reference micro-RNA. Results: A statistically significant increase in the expression of hsa-miR-143-3p, hsa-miR-146a-5p and hsa-miR-92a-1-5p by 2, 2.2 and 2.9 times, respectively, was found, as well as a decrease in the expression of hsa-miR-330- 3p by 4.1 times in tumor tissue of the brain relative to normal tissue (p < 0.05). Reduced expression of micro-RNA hsa-miR-330-3p may increase the activity of the VEGFA gene, which leads to intensive vascularization of the tumor. In addition, hsa-miR-330-3p is a validated negative regulator of the E2F1 gene known as a regulator of the cell cycle and tumor suppressor proteins. The TRAF6 gene is the direct validated target of hsa-miR-146a-5p, and its overexpression is associated with the patient's chemoresistance to temozolomide. Increased hsa-miR-143-3p micro-RNA expression can reduce the activity of the EGLN1 gene that also mediates the adaptation of tumor cells to hypoxic conditions. Hsa-miR-92a-1-5p micro-RNA is a negative regulator of PTEN gene expression. Conclusions: A significant decrease in hsa-miR-330-3p expression and an increase in hsa-miR-146a-5p, hsa-miR-143-3p, and hsa-miR-92a-1-5p, which was found in gliomas, can potentially be used to develop prognostic markers and therapeutic targets for targeted therapy.
Objective. The aim of this work was to obtain the primary cell lines of brain malignant tumors using the explant method. Materials and methods. Thirteen patients of both sexes, aged 22 to 66, were recruited. The tumor material of the patients was fragmented and placed in flasks with complete nutrient medium for glial tumor cells. Subsequently, the material was photographed at various stages of cultivation, the cell morphology was determined, and the rate of monolayer formation at the zero and first passages was assessed. Results. As a result, thirteen primary human cell lines of glial tumors were obtained: six glioblastoma lines, two glioblastoma lines with anaplastic astrocytoma, one anaplastic oligodendroglioma line, one diffuse astrocytoma line, one oligoastrocytoma line and one diffuse protoplasmic astrocytoma line, one anaplastic astrocytoma line. In the culture of diffuse astrocytoma, there were observed the cells forming a network at the bottom of the flask. In the culture of anaplastic astrocytoma at a confluence of 3050 %, fibroblast-like cells were presented, and at a confluence of 100 %, a monolayer was formed with cells intimately adjacent to each other. In the culture of oligoastrocytoma, both fibroblast-like cells and islets of closely intertwined fusiform cells were observed. The same was typical for the cells of diffuse protoplasmic astrocytoma. Anaplastic oligodendroglioma during the first week of cultivation was represented mainly by round cells with a contrast agent, which subsequently attached and actively proliferated. At a confluence of 3080 %, fibroblast-like cells were observed, and at 100 %, spindle-shaped cells closely adjacent to each other. In cultures of glioblastomas, no specific character of cell growth was revealed: spindle-shaped, fibroblast-like cells and cells with long processes forming a network were encountered. Glioblastoma cultures against the background of anaplastic astrocytoma were represented by a network of cells with long processes. At the zero passage, the rate of formation of a 100 % confluence monolayer ranged from 22 to 85 days. At the first passage, the cells reached a full monolayer within 4 to 25 days. At the zero passage, the longest time among all the samples to form the monolayer with a 100 % confluence needed glioblastoma lines on average 59 days. The shortest time to reach a 100 % confluence was required for cells of diffuse astrocytoma, anaplastic oligodendroglioma and glioblastoma against the background of anaplastic astrocytoma 2224 days. Conclusions. In our work, it was shown that the explant method ensures the production of viable cells of glial tumors and the possibility of their further cultivation.
Глиомы -наиболее распространенные опухоли головного мозга. Частота встречаемости глиальных опухолей составляет около 5 случаев в год на 100 000 человек. Максимально возможная циторедукция улучшает выживаемость пациентов, однако вопрос дальнейшей терапии глиом остается актуальным. Одним из перспективных подходов может стать разработка эффективных терапевтических схем на основании молекулярно-ориентированных воздействий на опухоль. Целью настоящей работы было исследование относительной экспрессии 15 генов сигнальных путей, ассоциированных с онкотрансформацией клеток, в 54 образцах опухолей в сравнении со здоровой тканью мозга, генотипированных на наличие соматических мутаций в генах IDH1/2 и ATRX. Случаев мутаций в генах IDH2 и ATRX не выявлено. Мутация R132H гена IDH1 была идентифицирована в 18,5% опухолей, достоверно отличавшихся от опухолей IDH1 дикого типа по транскрипционной активности гена SMAD7 (p=0,012). Аберрантный паттерн относительной экспрессии был характерен преимущественно для глиобластом G IV. По результатам исследования можно выделить группу молекулярных маркеров (SMAD7, SMO, EGLN1/3, HIF1A, KDM1A), с высокой частотой изменяющих транскрипционную активность в глиальных опухолях и перспективных для использования в таргетной терапии и дифференциальной диагностике. Ключевые слова: глиомы, экспрессия генов, IDH1/2, ATRX.
; 2 ФГАОУ ВО Южный федеральный университет, Ростов-на-Дону Глиомы высокой степени злокачественности представляют собой агрессивный тип первичных опухолей центральной нервной системы. В связи с этим чрезвычайно актуальными являются поиск эффективных методов ранней диагностики, а также осуществление мониторинга течения заболевания. МикроРНК представляют собой класс малых некодирующих РНК, которые могут быть использованы в качестве потенциальных биомаркеров, поскольку сохраняют высокую стабильность в плазме крови. Целью настоящей работы стало исследование дифференциальной экспрессии онкоассоциированных микроРНК и возможности их применения в качестве маркеров для диагностики глиом высокой степени злокачественности. Методом RT-qPCR была проведена оценка экспрессии микроРНК hsa-miR-22-3p, hsa-miR-122-5p, hsa-miR-107, hsa-miR-324-5p, hsa-miR-34a-5p, hsa-miR-155-5p, hsa-miR-21-5p, hsa-miR-330-3p, hsa-miR-146a-5p и hsa-miR-92a-1-5p в плазме крови 20 пациентов со злокачественными глиомами, 10 пациентов, страдающих метастатическими опухолями головного мозга, и 10 потенциально здоровых волонтеров. Была выявлены сигнатура микроРНК hsa-miR-107 и hsa-miR-122-5p (AUC=0,885), позволяющая определить наличие глиом высокой степени злокачественности. Диагностическая чувствительность и специфичность составили 100% и 71,2% соответственно. Циркулирующие микроРНК hsa-miR-107 и hsa-miR-122-5p потенциально могут быть использованы в качестве неинвазивных биомаркеров для диагностики глиом высокой степени злокачественности. Ключевые слова: циркулирующие микроРНК, глиома, диагностика, hsa-miR-107, hsa-miR-122-5p.
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