The efficient functioning of cells in an organism is carried out using intracellular signaling pathways, the activation of which leads to a change in the profile of genome expression. Differential activation and repression of genes can cause a change in the cell radiosensitivity. Based on published data and the results that we obtained in the experiment, the effect of radiation on the state of some signaling pathways, as well as microRNA, is studied. The possibility of a change in the cell radiosensitivity during the effect on the signaling pathways triggering the programs of cell death or proliferation is analyzed. The approaches of directed influence on the P53-dependent system of maintaining the genome stability for increasing post-radiation survival, as well as the significance of this system in the formation of long-term consequences of radiation exposure, are considered.

The effect of low-dose radiation was studied on the growth dynamics of transfused Lewis carcinoma cells in female C57Bl/6 mice. Fourfold total fractionated irradiation was conducted at a dose of 75 mGy (at a dose rate of 0.154 Gy/min and a voltage of 200 kV), starting from day 10 after the transplantation with a four-day interval. On days 14 and 22, a group of mice was euthanized to study the expression levels of genes and noncoding RNAs (microRNAs and long noncoding RNAs) in tumor cells, as well as in normal tissues (bone marrow, liver, spleen, thymus). The total RNA was obtained, and complementary DNA was synthesized according to the manufacturer's protocol. After that, a real-time PCR reaction was performed using the SYBR Green I dye (Thermo Scientific, United States) or a TaqMan probe and specific primers. Genes and noncoding RNAs were divided into groups of oncogenes and oncosuppressors according to scientific research. In the group of irradiated mice, a decrease in the tumor growth rate was observed, mainly from day 20, accompanied by a change in the activity of the studied genes. The ratio of the activity of oncosuppressors to oncogenes was calculated for the obtained tissues. These fractions were 0.5 for the tumor, 10.7 for the bone marrow, and 2.4 for the spleen and thymus. Thus, it can be stated that in the tumor there was a particularly pronounced activation of oncogenes compared to the activity of oncosuppressors, while the opposite effect was observed in normal tissues.

The activity of genes and noncoding RNAs was studied in 183 hybrid mice (CBA × C57Bl) F1 after a prolonged low-power irradiation (within 21 h at a dose 12.6 Gy with a dose rate 10 mGy/min). The analysis of the studied parameters was carried out in the bone marrow of mice in the eighth and tenth months after irradiation. After ten month, radiation-induced malignant lymphomas in the liver, abdominal cavity, and subcutaneously were found in 14 out of 94 animals. The studied molecular genetic parameters were divided into two groups: oncogenes and tumor suppressors, the activation of which was compared at eighth and tenth months after irradiation. It was detected that the activity of tumor suppressors (PTEN gene and lncp21 long noncoding RNA) in the groups of mice with tumors was lower in comparison with the group of mice without cancer, while the activity of oncogenes (NFkB(p65), IkBα, iNOS, TAL1, CTCF, NEAT1 lncRNAs, and miR-125b) increased. The studied parameters can be considered as potential biomarkers of radiation-induced cancer.

The processes of detection and quantification by real-time PCR in blood of mature microRNAs, including miR-21, should be standardized and validated. Our studies show that the specificity of PCR method for miR-21 in the blood of healthy donors can be demonstrated by the formation of a 67 bp amplicon product, precision under conditions of convergence and reproducibility, expressed using the coefficient of variation, is less than 2% and 3%, respectively, and the linearity of PCR method for miR-21 is confirmed by the correlation coefficient r≥0.99.

Процессы обнаружения и количественного определения методом ПЦР в реальном времени в крови пациентов зрелых микроРНК, в том числе и miR-21, должны быть стандартизированы и валидированы. Проведенные нами исследования, показывают, что специфичность метода ПЦР для miR-21 в крови здоровых доноров может быть демонстрирована образованием продукта-ампликона размером 67 нуклеотидов, прецизионность в условиях сходимости и воспроизводимости, выраженная в виде коэффициента вариации, составляет менее 2% и 3 % соответственно, а линейность метода подтверждается коэффицентом корреляции r≥0,99. The processes of detection and quantification by real-time PCR in blood of mature microRNAs, including miR-21, should be standardized and validated. Our studies show that the specificity of PCR method for miR-21 in the blood of healthy donors can be demonstrated by the formation of a 67 bp amplicon product, precision under conditions of convergence and reproducibility, expressed using the coefficient of variation, is less than 2% and 3%, respectively, and the linearity of PCR method for miR-21 is confirmed by the correlation coefficient r≥0.99.

This review is devoted to various aspects of studying functions of long RNAs (lncRNAs) under radiation exposure. A hypothesis on the lncRNA origin, their functions in cis and trans, and the interaction with other RNAs and cell structures are described. The expression of several lncRNAs (including MALAT1, HOTAIR, etc.) in the radioresponse and their possible contribution to some human pathologies are considered, which can be used both in disease prognosis and as a therapeutical target. Our own data allow us to compare and identify differences in the expression levels of lncRNAs (GAS5, RoR, MALAT1, NEAT1, and HOTAIR) in the formation of an adaptive response in human lymphocytes and lymphoid cells (Jurkat cells) irradiated at low (0.1 Gy) and high (5 Gy) doses.

We used 183 F1 CBA×C57Bl hybrid mice to study the delayed effects of low-power long-term γ-irradiation at a dose of 12.6 Gy (10 mGy/min) 8 and 10 months after the treatment. Eight months after the treatment we found the increased expression of the transcription factor NFκB and its target genes iNOS and G-SCF in the bone marrow (BM). Ten months after the treatment malignant lymphomas were revealed in 14 of 94 mice in the liver, abdominal cavity, and subcutaneously. In the BM of these mice, the transcription of the PTEN, NFκB, and iNOS genes was inhibited and the contents of long non-coding RNAs (lncRNAs) lnc p21, NEAT1, and microRNA miR-125b were decreased. The expression of the NFκB(p65) gene and miR-125b was inhibited in the BM of irradiated mice without tumors ten months after the treatment. These data show the deregulation of the P53 system supporting the genome stability in the BM of irradiated mice. These indices will be studied as potential markers of risk of development of irradiation-induced tumors.

The effect of X-ray radiation (0.1 Gy) on the expression of a number of genes and regulatory RNA (miRNA and long noncoding RNA) in human lymphocytes and T-lymphoblastic cells (Jurkat cells) was studied. One hour after cell irradiation with a low dose of radiation, lymphocytes displayed p53 expression and a decreased level of mature miR-27a and miR-181a having mRNA of gene p53 as target. These cells also had inhibited NFkB activity, which was found from reduced mRNA content of RhoAcdc42 and IL6 genes. By 4 h, their expression was normalized. Unlike in the case of normal cells, increased content of mRNA of NFkB gene (p65) and mRNA of its target IL6 gene was observed in Jurkat cells during this period. Repeated irradiation of cells with 5 Gy carried out after 4 h showed the radiation adaptive response (AO) according to the criterion of lymphocyte survival and its absence in Jurkat cells. The difference between groups of 5 Gy and 0.1 + 5 Gy in lymphocytes that survived after 20 h revealed common AO features (mRNA of p53 gene, NEAT1, miR-181a, miR-107). The results indicate the activation of various intracellular systems after the stress with low doses of radiation on lymphocytes and Jurkat cells. This approach can be used to optimize the efficacy of radiation therapy when preirradiation with a small dose of radiation increases the radioresistance of normal tissues surrounding a tumor.

Abstract—Various authors have shown that the numerous instances of spontaneous DNA damage (DNA breaks, change in bases) that occur daily in a cell as a result of the metabolism of the human body are the basis of mutational selection. Mutant cells have certain advantages in the microenvironment, which changes due to age characteristics. This contributes to the formation of cancer clones and their dissemination. A significant role in this process belongs to hereditary factors; at the same time, there are a number of hereditary diseases (Xeroderma pigmentosum, etc.) that in most cases lead to the occurrence of cancer. Herein, we describe a number of associated genes that control cellular homeostasis and, at the same time, are suppressor genes; changes in their activity or mutations in their structure contribute to carcinogenesis. The role of epigenetic factors, including noncoding regulatory RNAs, the organization of the chromatin structure, and methylation in the carcinogenesis processes is considered. Modulation of the functional activity of genes is carried out by noncoding RNAs; each RNA can have several target genes and can, in turn, be affected by different genes. Regulatory RNAs include microRNAs and long noncoding RNA proteins, changes in the activity of which can serve as biomarkers of disease onset, have a predictive value, and can be used as a target for therapy. New data on the role of noncoding RNAs, methylation, and chromatin organization in carcinogenesis are presented. Our data on the use of the expression level of some genes and noncoding RNAs as prognostic indicators suitable for the evaluation of treatment efficacy for a number of tumors are presented. Special attention is paid to the structural organization of chromatin and methylation in carcinogenesis, as well as to the interaction of cellular structures that affect the activity of genes and their regulation in carcinogenesis. A brief assessment of the carcinogenic effect of exogenous factors such as natural (cosmic factors and some natural compounds) and anthropogenic ones (radiation, chemicals, and cancer viruses) is given.

Цель. Определение прогностической значимости и роли экспрессии некодирующих РНК (длинные РНК и микроРНК), и белка кодирующих генов в патогенезе рака гортани. Методика. Исследован биопсийный материал и периферическая кровь 35 пациентов с диагнозом плоскоклеточный рак гортани (ПРГ) с классификацией от T1N0M0 до T4N1M0. Контролем служили образцы близлежащей гистологически неизмененной ткани гортани тех же больных. Для оценки экспрессии генов исследовали кровь 27 здоровых доноров. Содержание мРНК генов ( р53, CCND1, ORAOV1, hPTEN ), длинных некодирующих РНК (днРНК): NEAT1, MALAT1, ROR , а также зрелых микроРНК (miR-21, miR-27a, miR-34a, miR-101, miR-124, miR-125b, miR-181а) в опухолевой ткани и крови определяли методом ПЦР в реальном времени (ПЦР-РВ). Результаты. Выявлено увеличение содержания мРНК генов CCND1, hPTEN , днРНК NEAT1, MALAT1 и miR-21, miR-27a в крови у пациентов с ПРГ. Установлено, что уровень мРНК генов CCND1, ORAOV1 был значимо выше при исследовании биоптатов у больных 3-й - 4-й стадии, чем у больных 1-й - 2-й стадии заболевания. Такая же закономерность выявлена для днРНК NEAT1, MALAT1 и для miR-101. Экспрессия miR-27a и miR-124 на более поздних стадиях болезни была ниже, чем у пациентов 1-2 стадии. Заключение. Выявлена возможность использования исследованных днРНК, микроРНК и мРНК белоккодирующих генов для индивидуального прогноза заболевания при создании панели биомаркеров. Aim. To study the role of non-coding RNA (long RNAs and microRNAs) expression and protein-coding genes in the pathogenesis of laryngeal cancer to determine their prognostic significance for oncotransformation. Methods. The expression of long non-coding RNAs, microRNAs and protein-coding genes was examined in biopsy samples (fresh frozen tissue) and peripheral blood samples from 35 patients with laryngeal squamous cell cancer (LSCC) at T1N0M0 - T4N1M0 stages. Samples of surrounding, histologically unchanged tissues collected from the same patients were used as control. Gene expression was evaluated in blood samples from 27 healthy donors. Contents of gene mRNAs ( p53, CCND1, ORAOV1, hPTEN ), long non-coding RNAs (IncRNA) ( NEAT1, MALAT1, ROR ), and mature miRNAs (miR-21, miR-27a, miR-34a, miR-101, miR-124, miR -125b, miR-181a) were measured in tissue and blood using real-time PCR. Results. Contents of CCND1 and hPTEN gene mRNAs, lncRNAs ( NEAT1, MALAT1), miR-21, and miR-27a were increased in blood of patients with LSCC. Levels of CCND1 and ORAOV1 gene mRNAs were significantly higher in biopsy samples from stage 3-4 patients compared to stage 1-2 patients. A similar expression pattern was observed for lncRNAs NEAT1 and MALAT1 and miR-101. On the other hand, expression of miR-27a and miR-124 was lower at later stages than at stages 1-2. Conclusion. The studied lncRNAs, microRNAs and protein-coding genes can be used in development of a biomarker panel for individual prognosis of the disease.

В работах различных авторов было показано, что многочисленные спонтанные повреждения ДНК (разрывы ДНК, изменение оснований), ежедневно возникающие в клетке в результате метаболизма организма человека, являются основой мутационной селекции. Мутантные клетки имеют определенные преимущества в микроокружающей среде, изменяющейся вследствие возрастных особенностей, что способствует образованию онкоклонов и диссеминации их. Значительная роль в этом процессе принадлежит наследственным факторам, при этом существует ряд наследственных болезней (пигментная ксеродерма и др.), которые в большинстве случаев приводят к возникновению рака. Описан ряд сопряженных генов, контролирующих клеточный гомеостаз и при этом являющихся генами-супрессорами, изменение активности которых или мутации в их структуре способствуют канцерогенезу. Рассмотрена роль эпигенетических факторов, к которым относятся некодирующие регуляторные РНК, организация структуры хроматина, метилирование в процессах канцерогенеза. Модуляция функциональной активности генов осуществляется некодирующими РНК, каждая из которых может иметь несколько генов-мишеней и, в свою очередь, быть подвержена влиянию различных генов. К регуляторным РНК относятся микроРНК и длинные некодирующие белки РНК, изменения активности которых могут служить биомаркерами начала заболевания, иметь прогностическую ценность и использоваться как мишень для терапии. Представлены новые данные о роли некодирующих РНК, метилирования и организации хроматина при канцерогенезе. Приведены данные авторов по использованию уровня экспрессии ряда генов и некодирующих РНК как прогностических показателей, пригодных для оценки эффективности терапии ряда опухолей. Специальное внимание уделено структурной организации хроматина и метилированию в канцерогенезе, а также взаимодействию клеточных структур, влияющих не активность генов и их регуляцию в канцерогенезе. Дана краткая оценка канцерогенного действия экзогенных факторов: природных (космические факторы, некоторые природные соединения) и антропогенных (радиация, химические вещества, онкогенные вирусы).

This review presents analysis of the literature and our own research with respect to the role of miRNAs in the regulation of activity (expression) of genes controlling cellular homeostasis in human cells when exposed to ionizing radiation. Human cells, on one hand, can have increased resistance to radiation, which hinders the effectiveness of tumor treatment in radiotherapy. On the other hand, increased sensitivity to radiation may be accompanied by the development of several pathologies, including tumorigenesis. This paper examines the role of specific miRNAs in the formation of radioresistance and radiosensitivity of human cells and their impact on the respective target genes. Separate sections are devoted to the role of different miRNAs in radiation therapy of tumors of different localization, as well as their role in the bystander effect. A special section highlights features of gene activity and its regulators, miRNAs, in radiosensitive cells in patients with Down syndrome. The final section provides information about new approaches to change miRNA expression and, accordingly, their target genes by the action of plant and synthetic drugs (crown compounds) which reduce damaging effects of mutagens. It is assumed that antimutagens affecting the expression levels of miRNAs and structural genes may be used to correct the increase and decrease in cellular radioresponse, reducing the risk of development of pathological processes, including tumorigenesis.