Regulatory network of competitively interacting RNAs and effectiveness of rectal tumors radiotherapy.

BACKGROUND: Currently, rectal tumors radiotherapy effectiveness reaches an acceptable level only in a small number of patients (they have a complete clinical response), which is associated with the formation of malignant cells radioresistance. A comprehensive study that integrates various epigenetic parameters would explain a number of molecular mechanisms of rectal tumor cells radioresistance and identify new bio-markers. In the last decade, using high-through-put sequencing, the competitively interacting RNAs regulatory network (long non-coding RNAs, miRNAs and mRNAs) has been shown. PURPOSE: The aim of the study was to analyze the features of competitively interacting RNAs regulatory network functioning in patients with rectal cancer who are radioresistant and sensitive to radiotherapy. The study was performed on 500 patients with dia-gnosed rectal cancer. Radiotherapy was performed on a Novalis TX linear particle accelerator according to the standard protocol (single focal dose 2.4 Gy, total focal dose 54.0 Gy). Total RNA preparations were isolated from paired bio-psy fragments of tumor and non-tumor tissues of the rectum (obtained by video-colonoscopy). The relative abundance of mRNA, miRNA and lncRNA transcripts was assessed by the RT-qPCR method. Using bio-informatic analysis, the probability of potential interactions between the investigated mRNA, miRNA and lncRNA was determined. It has been shown that the effectiveness of radiotherapy depends on the level of miRNA (miRNA-195-5p; miRNA-4257; miRNA-5187-5p; miRNA-149-5p; miRNA-138 -1-3p; miRNA-6798-5p; miRNA-6819-5p; miRNA-4728-5p; miRNA-1249-5p; miRNA-557; miRNA-1273h-5p; miRNA-6737-5p; miRNA-6808-5p; miRNA-3202; miRNA-5195-3p; miRNA-130b-3p) and lncRNA (XIST, HELLPAR, NEAT1, AC008124. 1, LINC01089, LINC01547, and VASH1-AS1) expression, which regulate the DNA repair system (H2AX, RBBP8) and apoptosis (BCL2). CONCLUSION: A comprehensive study of competitively interacting RNAs regulatory network and radiotherapy effectiveness of rectal tumors made it possible to establish the mechanisms of radioresistance formation and its bio-markers.

Features of the Copy Number Variation of Certain Genes in Tumor Cells in Patients with Serous Ovarian Adenocarcinoma.

We analyzed the peculiarities of the copy number variation of genes that regulate apoptosis, DNA repair, cell proliferation, metabolism, and estrogen reception in tumor and normal cells of high-grade and low-grade serous adenocarcinoma of the ovaries. Using real-time qPCR method, the relative copy number of 34 genes (BAX, BCL2, TP53, MDM2, CASP9, CASP3, CASP7, CASP8, PRKCI, SOX2, OCT4, PIK3, PTEN, C-MYC, SOX18, AKT1, NOTCH1, BRCA1, BRCA2, EXO1, SCNN1A, KRAS, EGFR, BRAF, CYP1A1, CYP1A2, CYP1B1, CYP19A, ESR1, ESR2, GPER, STS, SULT1A, and SULT1E1) was determined in normal and tumor cells of the ovaries extracted by contactless capture laser microsection from FFPE-blocks of 200 patients. The most typical molecular markers of ovarian serous adenocarcinoma cells were identified: copy number of PIK3CA, BCL2, BAX, CASP3, and CASP8 genes. Based on the differences in the gene copy number variation, two molecular subtypes of serous adenocarcinoma were identified, corresponding to two histological subtypes: high-grade (MDM2, SOX2, ESR1, CYP1B1, SULT1E1, TP53, BRCA2) and low-grade (PIK3CA, PTEN, BCL2, BAX, and CASP3). Each of these subtypes was also characterized by molecular heterogeneity and can be subdivided into several subgroups: 3 subgroups for high-grade and 4 subgroups for low-grade serous adenocarcinoma. These findings extend our understanding of the molecular mechanisms of carcinogenesis in the ovarian tissue, confirm molecular difference between the two histological subtypes of serous adenocarcinoma probably underlying their different clinical course.

[Regulation of Gene Expression of Cancer/Testis Antigens in Colorectal Cancer Patients].

The transcriptional activity of genes encoding cancer/testis antigens (CTA) and its regulation in colorectal cancer (CRC) is not well understood. The expression of CTA coding genes (CT genes) and possible mechanisms for its regulation, including expression and copy number of DNA methyltransferase genes, copy number of CT genes, microRNA expression, and LINE-1 methylation in CRC were analyzed in this study. The relative expression levels and copy number variation of 19 genes, MAGE-A1, -A2, -A3, -A4, -B1, -B2, GAGE-1, -3, -4, MAGEC1, BAGE, XAGE3, NY-ESO1, SSX2, SCP1, PRAME1, DNMT1, DNMT3A, and DNMT3B, were determined using real-time quantitative PCR. Quantitative methylation of LINE-1 CpG sites was evaluated by pyrosequencing, and multiple parallel sequencing was used to determine the level of microRNA expression. It was found that in colon tumor tissue a multidirectional destabilization of the transcriptional activity of DNMT3A and DNMT3B, associated with copy number variation and a change in expression of the CT genes BAGE, SSX2 and PRAME1, is observed. A strong positive correlation was found between copy number and expression of the BAGE, SSX2, and PRAME1 genes. As a result of multiple parallel sequencing, 6 differentially expressed microRNAs (hsa-miR-143-3p, hsa-miR-26a-5p, hsa-miR-25-3p, hsa-miR-92a-3p, hsa-miR-21-5p, and hsa-let-7i-5p), targeting the CT genes GAGE1, SSX2, PRAME, SCP1, and the gene for DNA methyltransferase 3A (DNMT3A), were found. Data on the mechanisms of the transcriptional activity regulation of CT genes in malignant colon tumors are important for the development of CTA-dependent immunotherapeutic approaches for the treatment of this type of tumor.

Copy Number Variation in Tumor Cells and Extracellular DNA in Patients with Lung Adenocarcinoma.

Copy number variation of some gene loci in lung tumor cells extracted by laser capture microdissection and in cell-free DNA in the plasma of patients with lung cancer was analyzed for identification of potential molecular markers. Tissue specimens fixed in formalin and embedded in paraffin blocks (FFPE-blocks) from 90 patients and extracellular DNA from plasma samples of 70 patients and 30 donors were used. Copy number variation was assayed for 30 genes (BAX, BCL2, C-FLAR, P53, MDM2, CASP9, CASP3, CASP8, SOX2, OCT4, PIK3, MKI67, HV2, HIF1A1, XRCC1, MMP1, TERT, CTNNB1, KRAS, EGFR, GRB2, SOS1, MAPK1, STAT1, BRAF, FTO, and mir3678) and reference loci (ACTB, B2M, and GAPDH) by the real-time quantitative PCR. Changed copy numbers were detected for genes responsible for apoptosis regulation (BAX, P53, and CASP3), proliferation (SOX2), DNA reparation (XRCC1), oxidative phosphorylation (HV2), EGFR signaling pathway (GRB2, SOS1, MAPK1, STAT1, and BRAF), and for mir3678 gene in lung tumor cells and extracellular DNA.

Specific Features of Transcription Activity of Cancer-Testis Antigens in Patients with Metastatic and Non-Metastatic Breast Cancer.

Cancer-testis antigens, effective markers of tissue malignant transformation, are characterized by heterogonous transcription depending on the pathological features of breast cancer. We performed screening of transcription profile of cancer-testis antigens specific for breast tumor tissues in female patients with and without regional metastasis. The relative expression of 16 genes (MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEB1, MAGEB2, GAGE1, GAGE3, GAGE4, MAGEC1, BAGE, XAGE3, NY-ESO1, SSX2, SYCP1, and PRAME1) was analyzed by RT-qPCR method in biopsy specimens of the mammary gland tissues obtained during surgery from 25 patients. Differential transcription activity of cancer-testis antigens genes was observed in patients with metastatic (enhanced expression of MAGEA2, MAGEB1, and XAGE3 genes) and non-metastatic (enhanced expression of GAGE3 and PRAME1 genes) breast cancer.

[The activity of proapoptotic genes increases after renal ischemia/reperfusion].

According to the World Health Organization, pathologies associated with ischemia/reperfusion occupy the leading position in the structure of mortality. The efficiency of localized kidney cancer surgery is limited by the damaging effects of prolonged warm ischemia and reperfusion. Ischemia/reperfusion damage to renal tissue may be related to changes in the expression profiles of pro- and antiapoptotic genes. Here, we have presented the longitudinal expression profiles of apoptosis-related genes in tissues of left and right (intact) kidneys of male rats exposed to unilateral ischemia followed by reperfusion. The profiles have been assessed at time points of 1, 3, and 48 h after the ischemic/reperfusion exposure by RT-qPCR quantification of mRNAs encoded by 13 genes, including BAX, p53, AIFM1, APAF1, CASP8, CASP3, CASP9,CASP7, MDM2, BCL2, CIAP1, XIAP, and ICAD, after normalization with respect to a reference gene ACTB. The study revealed a shift in the expression of pro- and antiapoptotic genes toward the predominance of proapoptotic processes, as was evinced by the increase in expression detected for the BAX, p53, AIFM1, APAF1, and CASP8 genes. One hour after the reperfusion, activation of mitochondrial, or intrinsic apoptosis was detected, while р53-dependent and extrinsic, i.e., receptor-driven, apoptosis joined at later time points. Changes in the level of expression of caspase 7 (CASP7)-encoding mRNA have only been detected 48 h after the restoration of blood flow. Changes have been observed in the transcription of pro- and antiapoptotic genes in tissues of both kidneys, which suggests the involvement of the contralateral kidney in systemic pathological process that develops during unilateral ischemia/reperfusion.

[Changes in the number of copies of genetic loci in gastric cancer].

It is assumed that changes in the number of copies that belong to the basic mechanisms that control the expression of genes are important for malignization. Therefore, the characterization of these genes and the precise assessment of the number of copies are important for understanding the molecular basis of tumor emergence and progression in the human organism, as well as for the identification of predictive markers of malignization. In the present study, the relative number of copies of 19 loci (BAX, GSTP1, CASP3, CASP8, HIF1A, OCT4, C-MYC, SOX2, BCL2, CASP8/FADD, NANOG, P53, CASP9, IL-10, NFKB1, HV2, and ACTB) in cancerous and conventionally healthy tissues from 25 residents of southern Russia with a histologically confirmed diagnosis of adenocarcinoma (stages G1-G2 and G3) or signet cell gastric cancer were determined by quantitative real-time PCR. Changes in the number of copies of the gene were shown to be specific to particular histological types of cancer, as well as to depend on the stage of tumor cell differentiation. The data suggest that the number of copies of changes in the BAX, CASP3, CASP8, OCT4, C-MYC, SOX2, BCL2, NANOG, CASP9, NFKB1, HV2, ACTB, MKI67, IL-10, GSTP1, and P53 genes play an important role in the malignization of gastric tissue.

Effect of delta sleep-inducing peptide on the expression of antioxidant enzyme genes in the brain and blood of rats during physiological aging.

Subcutaneous injections of exogenous delta sleep-inducing peptide in a dose of 100 µg/kg (monthly, 5-day courses) to rats of various age groups (2-24 months) were followed by an increase in the expression of genes for SOD 1 (Sod1) and glutathione peroxidase 1 (Gpx1) in the brain and nucleated blood cells. The expression of these genes was shown to decrease during physiological aging of the body.

[Rat tissues antioxidant status correction by peptide delta sleep during physiological aging of the organism].

It is shown that exogenous delta-sleep inducing peptide increases glutathione antioxidant system level in rat tissues at different stages of ontogenesis, by subcutaneous injection to rats 2-24 months postnatal development in a dose of 100 mg/kg animal body weight by courses of 5 consecutive days per month, and this effect is especially marked in non-renewable postmitotic tissues.

[Influence of delta-sleep inducing peptide on the state of lysosomal membranes and intensity of lysosomal proteolysis in different rat tissues during physiological aging of the organism].

It is shown that subcutaneous injection of exogenous delta-sleep inducing peptide (DSIP) to rats aged 2-24 months in a dose of 100 µg/kg animal body weight by courses of 5 consecutive days per month has a stabilizing effect on the state of lysosomal membranes in rat tissues (brain, heart muscle and liver) at different ontogenetic stages, and this effect is accompanied by increasing intensity of lysosomal proteolysis in these tissues.

[Influence of delta-sleep peptide on the enzymes activity of mitochondrial electron transport chain in various rat tissues during aging of the organism].

It is shown that subcutaneous injection of exogenous delta-sleep inducing peptide (DSIP) to rats aged 2-24 months in a dose of 100 µg/kg animal body weight by courses of 5 consecutive days per month has a stabilizing effect on the NADH-dehydrogenase activity in the mitochondrial fractions of various tissues, which together with increasing capacity of the antioxidant system should reduce the production of free radicals and their adverse action on cells macromolecule, herewith the activity of succinate dehydrogenase did not change.

Effect of delta sleep-inducing peptide on oxidative modification of proteins in rat tissues and blood during physiological aging.

Accumulation of oxidized proteins (evaluated by the levels of carbonyl and SH groups) in tissues of 2-24-month-old rats (spleen>myocardium>testicles>liver>skeletal muscles) has been demonstrated. Exogenous delta sleep-inducing peptide injected subcutaneously to rats of different age in a dose of 100 µg/kg by monthly 5-day courses protected proteins of the studied tissues from oxidation; its effect was tissue-specific. Delta sleep-inducing peptide exhibited a hypoglycemic effect: it prevented nonenzymatic glycosylation of hemoglobin and reduced the level of defective protein molecules during aging.