Search

Our work aimed to evaluate and differentiate the role of ten lncRNA genes ( GAS5 , HAND2-AS1 , KCNK15-AS1 , MAGI2-AS3 , MEG3 , SEMA3B-AS1 , SNHG6 , SSTR5-AS1 , ZEB1-AS1 , and ZNF667-AS1 ) in the development and progression of epithelial ovarian cancer (EOC). A representative set of clinical samples was used: 140 primary tumors from patients without and with metastases and 59 peritoneal metastases. Using MS-qPCR, we demonstrated an increase in methylation levels of all ten lncRNA genes in tumors compared to normal tissues ( p < 0.001). Using RT-qPCR, we showed downregulation and an inverse relationship between methylation and expression levels for ten lncRNAs ( r s < -0.5). We further identified lncRNA genes that were specifically hypermethylated in tumors from patients with metastases to lymph nodes ( HAND2-AS1 ), peritoneum ( KCNK15-AS1 , MEG3 , and SEMA3B-AS1 ), and greater omentum ( MEG3 , SEMA3B-AS1 , and ZNF667-AS1 ). The same four lncRNA genes involved in peritoneal spread were associated with clinical stage and tumor extent ( p < 0.001). Interestingly, we found a reversion from increase to decrease in the hypermethylation level of five metastasis-related lncRNA genes ( MEG3 , SEMA3B-AS1 , SSTR5-AS1 , ZEB1-AS1 , and ZNF667-AS1 ) in 59 peritoneal metastases. This reversion may be associated with partial epithelial-mesenchymal transition (EMT) in metastatic cells, as indicated by a decrease in the level of the EMT marker, CDH1 mRNA ( p < 0.01). Furthermore, novel mRNA targets and regulated miRNAs were predicted for a number of the studied lncRNAs using the NCBI GEO datasets and analyzed by RT-qPCR and transfection of SKOV3 and OVCAR3 cells. In addition, hypermethylation of SEMA3B-AS1 , SSTR5-AS1 , and ZNF667-AS1 genes was proposed as a marker for overall survival in patients with EOC.

The transformation protocol based on the dual selection approach (fluorescent protein and herbicide resistance) has been applied here to produce transgenic plants of two cereal species, emmer wheat and bread wheat, with the goal of activating the synthesis of the stress hormone jasmonates by overexpressing ALLENE OXIDE SYNTHASE from Arabidopsis thaliana ( AtAOS ) and bread wheat ( TaAOS ) and OXOPHYTODIENOATE REDUCTASE 3 from A. thaliana ( AtOPR3 ) under the strong constitutive promoter ( ZmUbi1 ), either individually or both genes simultaneously. The delivery of the expression cassette encoding AOS was found to affect morphogenesis in both wheat species negatively. The effect of transgene expression on the accumulation of individual jasmonates in hexaploid and tetraploid wheat was observed. Among the introduced genes, overexpression of TaAOS was the most successful in increasing stress-inducible phytohormone levels in transgenic plants, resulting in higher accumulations of JA and JA-Ile in emmer wheat and 12-OPDA in bread wheat. In general, overexpression of AOS , alone or together with AtOPR3 , negatively affected leaf lamina length and grain numbers per spike in both wheat species. Double ( AtAOS + AtOPR3 ) transgenic wheat plants were characterized by significantly reduced plant height and seed numbers, especially in emmer wheat, where several primary plants failed to produce seeds.

There are three types of metastases in ovarian cancer: lymphogenous, hematogenous, and peritoneal. Dissemination of the tumor in the peritoneum is directly related with the development of ascites and a poor prognosis. The purpose of this study is to determine changes in the methylation level of a group of long non-coding RNA (lncRNA) genes at different stages of ovarian cancer progression. The methylation level of 7 lncRNA genes (LINC00472, LINC00886, MAFG-DT, SNHG1, SNHG6, TP53TG1, and TUG1) was studied by quantitative methyl-specific PCR in 93 samples of ovarian tumors and 75 paired samples of histologically normal tissue, as well as in 29 peritoneal macroscopic metastases. Using the nonparametric Mann-Whitney test, a significant (p<0.001) increase in the level of methylation of the LINC00886, SNHG1, SNHG6, and TUG1 genes in the tumor tissue was shown. For the LINC00472, LINC00886, and SNHG6 genes, a significant relationship was found with the clinical stage (p≤0.001), as well as with the appearance of metastases for the LINC00472 (p<0.001) and SNHG6 (p=0.005) genes. There was a significant increase in the level of methylation of MAFG-DT and TP53TG1 (p<0.001) genes, as well as a decrease in LINC00886 (p=0.003) in peritoneal metastases relative to the primary focus. Methylation of the LINC00472 and SNHG6 genes can be considered as a factor in initiating ovarian cancer metastasis, and methylation of the LINC00886, MAFG-DT, and TP53TG1 genes as a colonization factor for metastases in the peritoneum. Thus, a relationship between methylation of a group of lncRNA genes at different stages of ovarian cancer dissemination was shown, which is important for understanding the mechanisms of these processes and for developing innovative approaches to ovarian cancer therapy., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Breast cancer is the most common type of cancer among women. The study of the mechanisms of metastasis, the main cause of death from breast cancer, as well as the search for new markers for early diagnosis and prognosis of breast cancer, is an extremely topical issue. New perspectives in the diagnosis and treatment of breast cancer are opened by the mechanisms of gene regulation involving non-coding RNAs, in particular, long non-coding RNAs (lncRNAs). In this work, we analyzed the methylation levels of seven lncRNA genes (MEG3, SEMA3B-AS1, HAND2-AS1, KCNK15-AS1, ZNF667-AS1, MAGI2-AS3, and PLUT) by quantitative methyl-specific PCR on a set of 79 paired (tumor/normal) samples of breast cancer. Hypermethylation of all seven lncRNA genes was revealed, and hypermethylation of HAND2-AS1, KCNK15-AS1, MAGI2-AS3, and PLUT was detected in breast cancer for the first time. It was found that the level of meth ylation of the studied lncRNA genes correlated statistically significantly with the stage of the tumor process, the size of the tumor, and the presence of metastases in the lymph nodes. Thus, methylation of the seven studied lncRNA genes is associated with the development and progression of breast cancer, and these genes can be useful as potential markers in the diagnosis and prognosis of breast cancer.

This paper introduces the reader to the field of liquid biopsies and cell-free nucleic acids, focusing on circulating tumor DNA (ctDNA) in breast cancer (BC). BC is the most common type of cancer in women, and progress with regard to treatment has been made in recent years. Despite this, there remain a number of unresolved issues in the treatment of BC; in particular, early detection and diagnosis, reliable markers of response to treatment and for the prediction of recurrence and metastasis, especially for unfavorable subtypes, are needed. It is also important to identify biomarkers for the assessment of drug resistance and for disease monitoring. Our work is devoted to ctDNA, which may be such a marker. Here, we describe its main characteristics and potential applications in clinical oncology. This review considers the results of studies devoted to the analysis of the prognostic and predictive roles of various methods for the determination of ctDNA in BC patients. Currently known epigenetic changes in ctDNA with clinical significance are reviewed. The possibility of using ctDNA as a predictive and prognostic marker for monitoring BC and predicting the recurrence and metastasis of cancer is also discussed, which may become an important part of a precision approach to the treatment of BC.

Long non-coding RNAs (lncRNAs) are crucial players in the pathogenesis of non-small-cell lung cancer (NSCLC). A competing binding of lncRNAs and mRNAs with microRNAs (miRNAs) is one of the most common mechanisms of gene regulation by lncRNAs in NSCLC, which has been extensively researched in the last two decades. However, alternative mechanisms that do not depend on miRNAs have also been reported. Among them, the most intriguing mechanism is mediated by RNA-binding proteins (RBPs) such as IGF2BP1/2/3, YTHDF1, HuR, and FBL, which increase the stability of target mRNAs. IGF2BP2 and YTHDF1 may also be involved in m 6 A modification of lncRNAs or target mRNAs. Some lncRNAs, such as DLGAP1-AS2, MALAT1, MNX1-AS1, and SNHG12, are involved in several mechanisms depending on the target: lncRNA/miRNA/mRNA interactome and through RBP. The target protein sets selected here were then analyzed using the DAVID database to identify the pathways overrepresented by KEGG, Wikipathways, and the Reactome pathway. Using the STRING website, we assessed interactions between the target proteins and built networks. Our analysis revealed that the JAK-STAT and Hippo signaling pathways, cytokine pathways, the VEGFA-VEGFR2 pathway, mechanisms of cell cycle regulation, and neovascularization are the most relevant to the effect of lncRNA on NSCLC.

The role of methylation of 9 miRNA genes in the pathogenesis of metastatic clear cell renal cell carcinoma was determined by quantitative methylation-specific PCR (MS-PCR). For 5 genes (MIR125B-1, MIR137, MIR193A, MIR34B/C, and MIR375), a significant correlation of high methylation level with late (III-IV) stages, large size (T3+T4) of the tumor, and metastasis to lymph nodes and/or distant organs was revealed. For another group of genes (MIR125B-1, MIR1258, MIR193A, MIR34B/C, and MIR375), a statistically significant correlation of high methylation level with loss of differentiation in the tumor (G3-G4) was found, and the opposite pattern was found for MIR203A. A total of 7 microRNA genes (MIR125B-1, MIR1258, MIR137, MIR193A, MIR203A, MIR34B/C, and MIR375) were identified, the methylation of which is associated with the progression of metastatic clear cell renal cell carcinoma. For 6 of them (except MIR34B/C) these data were obtained for the first time. Thus, new factors of the development and progression of clear cell renal cell carcinoma were identified as potential biomarkers for the early diagnosis and prognosis of metastatic clear cell renal cell carcinoma., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

The search for interacting long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs of protein-coding genes through the mechanism of competing endogenous RNAs in tumors of ovarian cancer patients was carried out. The levels of expression of 24 lncRNAs, 20 miRNAs, and 28 mRNAs of protein-coding genes involved in oncogenesis were determined by real-time PCR on a set of representative samples. Correlations between lncRNAs/miRNA and miRNA/mRNA levels in ovarian cancer samples were analyzed. We identified 8 pairs of lncRNAs/miRNA and 17 pairs of miRNA/mRNA, the expression levels of which have a negative correlation. Five triplets of potentially interacting lncRNAs/miRNA/mRNA have been identified, among which the most significant triplet is the OIP5-AS1/miR-203a-3p/ZEB1. The data obtained determine new epigenetic profiles, as well as new potential biomarkers and targets for targeted therapy of ovarian cancer patients., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

We studied changes in the level of methylation of a number of microRNA genes hypermethylated in non-small cell lung cancer and its histological subtypes as well as the relationship of methylation of a group of microRNA genes with clinical and morphological features of the tumor with smoking status. A significantly high level of methylation of 7 genes (MIR124-1/3, MIR125B-1, MIR129-2, MIR137, MIR1258, and MIR339) was revealed in adenocarcinoma and squamous cell lung cancer in comparison with samples of adjacent histologically unchanged lung tissue. In squamous cell lung cancer, a significantly high level of methylation of the MIR124-2 gene in the tumor was also shown. In addition, differences in the methylation profile of adenocarcinoma and squamous cell carcinoma at stages III-IV of the oncological process were revealed. A high level of methylation of the MIR137 and MIR1258 genes was shown for adenocarcinoma and MIR339, MIR129-2, and MIR124-2 for squamous cell carcinoma. Significant differences in the level of methylation of MIR124-2 and MIR375 genes were revealed for smoking patients with squamous cell lung cancer., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

A decrease in the miR-124 expression was observed in various epithelial cancers. Like a classical suppressor, miR-124 can inhibit the translation of multiple oncogenic proteins. Epigenetic mechanisms play a significant role in the regulation of miR-124 expression and involve hypermethylation of the MIR-124-1/-2/-3 genes and the effects of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) according to the model of competing endogenous RNAs (ceRNAs). More than 40 interactomes (lncRNA/miR-124/mRNA) based on competition between lncRNAs and mRNAs for miR-124 binding have been identified in various epithelial cancers. LncRNAs MALAT1, NEAT1, HOXA11-AS, and XIST are the most represented in these axes. Fourteen axes (e.g., SND1-IT1/miR-124/COL4A1) are involved in EMT and/or metastasis. Moreover, eight axes (e.g., OIP5-AS1/miR-124-5p/IDH2) are involved in key pathways, such as Wnt/b-catenin, E2F1, TGF-β, SMAD, ERK/MAPK, HIF-1α, Notch, PI3K/Akt signaling, and cancer cell stemness. Additionally, 15 axes impaired patient survival and three axes reduced chemo- or radiosensitivity. To date, 14 cases of miR-124 regulation by circRNAs have been identified. Half of them involve circHIPK3, which belongs to the exonic ecircRNAs and stimulates cell proliferation, EMT, autophagy, angiogenesis, and multidrug resistance. Thus, miR-124 and its interacting partners may be considered promising targets for cancer therapy.

We analyzed changes in the level of methylation of CpG islands in four long non-coding RNA (lncRNA) genes MEG3, ZNF667-AS1, GAS5, and SEMA3B-AS1 as promising markers of breast cancer. Methylation analysis was performed by quantitative methylation-specific PCR on a set of 38 paired (tumor/normal) breast cancer samples. Significantly (p<0.001) increased methylation was shown for three of the four lncRNA genes: MEG3, ZNF667-AS1, and SEMA3B-AS1. We found significant correlations of the methylation level of all the studied lncRNA genes with the stage of cancer and with lymphogenic metastasis, and for MEG3 and ZNF667-AS1 also with the tumor size. Methylation of ZNF667-AS1, and SEMA3B-AS1 genes in breast cancer was detected for the first time. Based on these findings, new potential markers for the diagnosis and prognosis of breast cancer can be proposed., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

We studied the correlations between the levels of methylation of a group of 21 microRNA genes in 99 primary tumors and 29 macroscopic peritoneal metastases of ovarian cancer. Analysis of the level of methylation by quantitative methylation-specific PCR showed that co-methylation was detected for 13 pairs of microRNA genes in primary tumors and for 22 pairs in metastases. Pairs of microRNA genes that have shown significant co-methylation can be involved in common processes and pathways of gene regulation and interaction and can have common target genes. The results are highly significant and pairs of microRNA genes can be proposed as new potential markers for the diagnosis and prognosis of ovarian cancer metastasis., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Late diagnosis of ovarian cancer is one of the most important problems in its treatment. Long non-coding RNA (lncRNA) are a poorly studied, but promising type of diagnostic biomarkers. We studied the lncRNA interactome to identify biomarkers with potential significance for molecular diagnostics of ovarian cancer. By screening the TCGA database, we identified differentially expressed lncRNA CCAT1 and SNHG14. Based on the indices of complementarity of CCAT1 and SNHG14 to the mRNA sequences, we selected 5 protein-coding genes MAPK1, c-MET, TGFB2, SNAIL1, and WNT4 associated with the epithelial-mesenchymal transition. Real-time PCR on 54 ovarian cancer samples confirmed the high expression levels of CCAT1 and SNHG14 (logFC>1.5, p<0.05). A positive correlation between the expression levels of two lncRNA and mRNA of 5 genes in 6 pairs was established. The activating effect of CCAT1 and SNHG14 on the expression of these genes can be mediated by miR-203 and miR-124., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Ovarian cancer (OC) is one of the most common types of cancer among malignancies of the female reproductive system. This pathology is asymptomatic until advanced stages and has a poor prognosis. Our study aimed to search for lncRNA-miRNA-mRNA competing triplets that promote ovarian tumorigenesis. For this purpose, we analyzed tumor samples from the TCGA database and verified the results experimentally in a set of 46 paired samples of tumor and matched histologically unchanged ovarian tissues from OC patients. The list of RNAs selected in silico for experimental studies included 13 mRNAs, 10 lncRNAs, and 5 miRNAs related to epithelial-mesenchymal transition and angiogenesis. We evaluated the expression of these RNAs by qRT-PCR and assessed the correlation between levels of miRNAs, mRNAs, and lncRNAs. Sixteen significant triplets were revealed, in some of which, e.g., OIP5-AS1-miR-203a-c-MET and OIP5-AS1-miR-203a-ZEB2, both lncRNA and mRNA had sites for miR-203a direct binding. Transfection of the OVCAR-3 and SKOV-3 cell lines with the miR-203a mimic was used to confirm the novel links of miR-203a with ZEB2 and c-MET in OC. These connections suggest that the interactomes have the potential for diagnostics of metastasis at early onset.

Our work aimed to differentiate 20 aberrantly methylated miRNA genes that participate at different stages of development and metastasis of ovarian carcinoma (OvCa) using methylation-specific qPCR in a representative set of clinical samples: 102 primary tumors without and with metastases (to lymph nodes, peritoneum, or distant organs) and 30 peritoneal macroscopic metastases (PMM). Thirteen miRNA genes ( MIR107 , MIR124-2 , MIR124-3 , MIR125B-1 , MIR127 , MIR129-2 , MIR130B , MIR132 , MIR193A , MIR339 , MIR34B/C , MIR9-1 , and MIR9-3 ) were hypermethylated already at the early stages of OvCa, while hypermethylation of MIR1258 , MIR137 , MIR203A , and MIR375 was pronounced in metastatic tumors, and MIR148A showed high methylation levels specifically in PMM. We confirmed the significant relationship between methylation and expression levels for 11 out of 12 miRNAs analyzed by qRT-PCR. Moreover, expression levels of six miRNAs were significantly decreased in metastatic tumors in comparison with nonmetastatic ones, and downregulation of miR-203a-3p was the most significant. We revealed an inverse relationship between expression levels of miR-203a-3p and those of ZEB1 and ZEB2 genes, which are EMT drivers. We also identified three miRNA genes ( MIR148A , MIR9-1 , and MIR193A ) that likely regulate EMT-MET reversion in the colonization of PMM. According to the Kaplan-Meier analysis, hypermethylation of several examined miRNA genes was associated with poorer overall survival of OvCa patients, and high methylation levels of MIR130B and MIR9-1 were related to the greatest relative risk of death.

Background: The analysis of individual microRNAs (miRNAs) as a diagnostic and prognostic tool for the effective treatment of various diseases has aroused particular interest in the scientific community. The determination of circulating miRNAs makes it possible to assess biological changes associated with nutritional processes, the intake of dietary supplements and drugs, etc. The profile of circulating miRNAs reflects the individual adaptation of the organism to the effect of specific environmental conditions., Objective: The objective of this study is to systematize the data and show the importance of circulating miRNAs as new potential biomarkers of the organism's response to the intake of various dietary supplements, drugs, and consider the possibility of their use in doping control., Methods: A systematic analysis of scientific publications (ncbi.nlm.nih.gov) on the miRNA expression profile in response to the intake of dietary supplements and drugs most often used by athletes, and supposed their role as potential markers in modern doping control was carried out., Results: The profile of circulating miRNAs is highly dependent on the intake of a particular drug, and, therefore, may be used as a marker of the effects of biologically active supplements and drugs including the substances from the Prohibited List of the World Anti-Doping Agency (WADA)., Conclusion: Monitoring of circulating miRNAs can serve as a high-precision marker for detecting doping abuse in elite sports. However, it is necessary to conduct additional studies on the effect of complex drugs on the profile of circulating miRNAs and individual circulating miRNAs on a particular biological process., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Changes in the methylation levels of 21 microRNA genes in 91 breast cancer samples in comparison with paired samples of histologically unchanged tissue were studied by quantitative methylation-specific PCR. For 19 microRNA genes, a significant increase in the methylation level in tumors in comparison with normal tissues was shown (Mann-Whitney test). When considering the data for breast cancer samples only from patients with clinical stages I and II (59samples), 17 genes with a significantly increased level of methylation were identified. Increased methylation level for 11 genes (MIR124-1, MIR124-3, MIR125B-1, MIR127, MIR129-2, MIR132, MIR137, MIR193a, MIR34B/C, MIR375, and MIR9-1) compared to the paired norm was highly significant (p<0.001, FDR=0.01). The ROC analysis was used to optimize a set of markers for diagnosing breast cancer at the early stages consisting of 4 microRNA genes: MIR125B1, MIR127, MIR1258, and MIR132; the system is characterized by 100% specificity, 85% sensitivity, and AUC=0.924. Importantly, 100% specificity eliminates false positive results. Detection of methylation of at least one of the 4 genes of this set is sufficient to classify the patient's sample as breast cancer., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Systemic analysis of the relationship between the levels of methylation of 21 microRNA genes and the parameters of breast cancer progression was performed on a representative sample of 91 paired specimens of breast cancer and histologically normal tissues and a system of markers for prediction of metastasis was proposed. A significant association of hypermethylation of 11 genes with late (III-IV) clinical stages was found, and for 6 genes (MIR124-1, MIR127, MIR34B/C, MIR9-3, MIR1258, and MIR339) this association was highly significant (p≤0.001, FDR=0.01). For MIR9-3 and MIR339, an association with tumor size was demonstrated (p<0.001, FDR=0.01). No association of the levels of methylation of the analyzed microRNA genes with the degree of differentiation were found. An association with lymph node metastasis was established for 9 microRNA genes; the most significant association was shown for 6 genes MIR125B-1, MIR127, MIR9-3, MIR339, MIR124-3, and MIR1258 (p<0.005, FDR=0.05). Based on these 6 genes, a marker system for predicting breast cancer metastasis was developed by ROC analysis. This system is characterized by 87% sensitivity and 77% specificity (AUC=0.894). The proposed system may have clinical application in the personalized treatment of breast cancer patients., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

The fundamental novelty in the pathogenesis of renal cell carcinoma (RCC) was discovered as a result of the recent identification of the role of long non-coding RNAs (lncRNAs). Here, we discuss several mechanisms for the dysregulation of the expression of protein-coding genes initiated by lncRNAs in the most common and aggressive type of kidney cancer-clear cell RCC (ccRCC). A model of competitive endogenous RNA (ceRNA) is considered, in which lncRNA acts on genes through the lncRNA/miRNA/mRNA axis. For the most studied oncogenic lncRNAs, such as HOTAIR, MALAT1, and TUG1, several regulatory axes were identified in ccRCC, demonstrating a number of sites for various miRNAs. Interestingly, the LINC00973/miR-7109/Siglec-15 axis represents a novel agent that can suppress the immune response in patients with ccRCC, serving as a valuable target in addition to the PD1/PD-L1 pathway. Other mechanisms of action of lncRNAs in ccRCC, involving direct binding with proteins, mRNAs, and genes/DNA, are also considered. Our review briefly highlights methods by which various mechanisms of action of lncRNAs were verified. We pay special attention to protein targets and signaling pathways with which lncRNAs are associated in ccRCC. Thus, these new data on the different mechanisms of lncRNA functioning provide a novel basis for understanding the pathogenesis of ccRCC and the identification of new prognostic markers and targets for therapy.

We studied the relationship of the levels of microRNA group expression and methylation with clinical and pathomorphological parameters of breast cancer and its immunohistochemical status. Quantitative methylation specific PCR analysis showed a significant (p<0.001) increase in the methylation level of 4 microRNA genes (MIR127, MIR129-2, MIR132, and MIR148A) and a significant (p<0.001) decrease for gene MIR375 relative to paired histologically normal tissue. Real-time PCR analysis revealed a significant (p≤0.001) decrease in the expression of 4 microRNAs (miR-127-5p, miR-129-5p, miR-132-3p, and miR-148a-3p) and a significant (p≤0.001) increase in the expression of miR-375-3p. A significant (rs=-0.6--0.7, p≤0.001) relationship between changes in the expression level of miR-129-5p, miR-132-3p, miR-148a-3p, and miR-375-3p and the levels of methylation of the corresponding genes in breast cancer was showed by using Spearman's rank correlation test. Analysis of the samples with consideration of the pathophysiological characteristics of the tumor revealed two significant markers of tumor progression: MIR129-2/miR-129-5p and MIR375/miR-375-3p. Both factors, the increase in the level of MIR129-2 methylation (p<0.001) and a decrease in the expression level of miR-129-5p (p<0.001), are significantly associated (p<0.001) with stage III/IV and the absence of HER2 expression. For MIR375/miR-375-3p, on the contrary, an association of low methylation level and enhanced expression with increased Ki-67 level (>30%, p<0.05) was revealed. These findings are of interest for understanding the mechanisms of breast cancer development and can provide the basis for the diagnosis and prognosis of the course of this disease. Moreover, the revealed features can be useful for adjusting the course of treatment with consideration of the pathophysiological characteristics of the tumor., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Recently, a wealth of data have been accumulating on the role of long non-coding RNAs (lncRNAs) in the fine-tuning of mRNA expression. Four new lncRNAs, namely, TMEM92-AS1, FAM222A-AS, TXLNB, and lnc-CCL28, were identified as differentially expressed in ovarian tumors using deep machine learning. The levels of lnc-CCL28 transcripts in both tumors and normal tissue samples were sufficient for further analysis by RT-PCR. In addition, the promising ovarian cancer biomarkers, lncRNAs LINC00152, NEAT 1 and SNHG17 were added to RT-PCR analysis. For the first time, an increase in the level of lnc-CCL28 and SNHG 17 lncRNAs was found in ovarian tumors, and the overexpression of LINC00152 and NEAT1 was confirmed. It seems that lnc-CCL28 is involved in carcinogenesis and, in particular, in ovarian cancer progression. Overexpression of LINC00152 and lnc-CCL28 was significantly associated with the later stages and metastasis.

The role of methylation in the regulation of genes of long noncoding RNA (lncRNA) is still poorly understood. We revealed new hypermethylated lncRNA genes in ovarian tumors and their effect on metastasis of ovarian cancer. A multiple and significant (p<0.001) increase in methylation of a group of lncRNA genes (MEG3, SEMA3B-AS1, ZNF667-AS1, and TINCR) was shown by quantitative methylation-specific PCR using the non-parametric Mann-Whitney test. Moreover, methylation of SEMA3B-AS1, ZNF667-AS1, and TINCR genes in ovarian cancer tumors was detected for the first time. Comparative analysis of 19 samples of peritoneal metastases and paired primary tumors showed a significant decrease in the methylation level of the same 4 genes: MEG3 (p=0.004), SEMA3B-AS1 (p=0.002), TINCR (p=0.002), and ZNF667-AS1 (p<0.001). Reduced methylation of suppressor lncRNA genes in peritoneal metastases is probably associated with the involvement of these lncRNA in the regulation of plastic reversion of the epithelial-mesenchymal transition to the mesenchymal-epithelial transition. Thus, the effect of lncRNA and their methylation on the development of tumors and metastases of ovarian cancer was demonstrated, which is important for understanding of the pathogenesis and mechanisms of metastasis of ovarian cancer. New properties of lncRNA can find application in the development of new approaches in the therapy of ovarian cancer., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Long noncoding RNAs (lncRNAs) have been identified as contributors to the development and progression of cancer through various functions and mechanisms. LncRNA GAS5 is downregulated in multiple cancers and acts as a tumor suppressor in breast cancer. GAS5 interacts with various proteins (e.g., E2F1, EZH2, and YAP), DNA (e.g., the insulin receptor promoter), and various microRNAs (miRNAs). In breast cancer, GAS5 binds with miR-21, miR-222, miR-221-3p, miR-196a-5p, and miR-378a-5p that indicates the presence of several elements for miRNA binding (MREs) in GAS5. Mediated by the listed miRNAs, GAS5 is involved in the upregulation of a number of mRNAs of suppressor proteins such as PTEN, PDCD4, DKK2, FOXO1, and SUFU. Furthermore, the aberrant promoter methylation is involved in the regulation of GAS5 gene expression in triple-negative breast cancer and some other carcinomas. GAS5 can stimulate apoptosis in breast cancer via diverse pathways, including cell death receptors and mitochondrial signaling pathways. GAS5 is also a key player in the regulation of some crucial signal pathways in breast cancer, such as PI3K/AKT/mTOR, Wnt/β-catenin, and NF-κB signaling. Through epigenetic and other mechanisms, GAS5 can increase sensitivity to multiple drugs and improve prognosis. GAS5 is thus a promising target in the treatment of breast cancer patients.

Ovarian cancer (OC) is mostly detected at late stages weighed down with metastasis, and the five-year survival rate of patients is only 30%, which dictates the necessity to develop gentler and more selectively targeted drugs that current chemotherapeutic agents. The search for factors that can influence on the activity of the PD-1/PD-L1 immune checkpoint signaling pathway in tumors is relevant, and micro RNAs (miRNAs) play an important role in it. Over the past 5 years, only a few miRNAs (miR-34a, miR-145, and miR-424), which have a regulatory effect on the PD-1/PD-L1 system in OC patients, have been discovered. In present work, the methylation levels of 13 miRNA genes in 26 primary tumors and 19 peritoneal metastases of OC patients were determined and compared with the level of the soluble form of PD-L1 (sPD-L1) in the blood plasma of the same patients. It was shown that the methylation levels of five miRNA genes (MIR124-2, MIR34B/C, MIR9-1, MIR9-3, and MIR339) in tumors are in direct correlation with the sPD-L1 level in the blood plasma. In addition, when analyzing these five genes, a significant association of the methylation level of the MIR9-1 gene with a decrease in the three-year relapse-free survival, and a trend for decrease in the three-year survival rate with the methylation level of the MIR124-2 gene of OC patients were determined. Thus, the first data suggesting the role of inhibitors of the sPD-L1 immune checkpoint for five miRNAs (miR-124, miR-34b, miR-34c, miR-9, miR-339) and the possibility of using hypermethylated MIR9-1 and, presumably, MIR124-2 genes as independent prognostic markers of poor disease-free survival in OC patients were obtained.

Systems of markers for the diagnosis of breast cancer based on DNA methylation of a group of suppressor protein-coding genes, hypermethylated microRNA genes, and their combinations were compiled. On a representative sample of 70 paired breast cancer specimens (tumor/normal), MS-PCR analysis revealed a significant increase in the methylation frequency of 5 protein-coding genes: RASSF1A suppressor and apoptosis genes APAF1, BAX, BIM/BCL2L11, and DAPK1 (34-61% vs. 4-24%) and 6 microRNA genes: MIRG124G1, MIRG125bG1, MIRG129G2, MIRG148a, MIRG34b/c, and MIRG9G3 (36-76% vs. 6-27%). ROC-analysis showed that a combination of 4 genes (APAF1, BAX, BIM/BCL2L11, and DAPK1) and MIRG125bG1 gene constitute a highly efficient 5-marker system with 100% specificity and sensitivity of 94-96% at AUC=0.98-0.97, suitable also for patients with stage I and II breast cancer. Detection of methylation of at least one gene in this system in biopsy or postoperative material is sufficient to refer the sample to breast cancer.

It was found that the proportion of microRNA genes inactivated by methylation of regulatory CpG islands is several times higher than the genes encoding proteins, which increases their attractiveness as promising markers of cancer. The aim of this work is to evaluate the clinical significance of methylation of 13 tumor-associated microRNA genes (MIR-124a-2, MIR-124a-3, MIR-125-B1, MIR-127, MIR-129-2, MIR-132, MIR-137, MIR-203a, MIR-34b/c, MIR-375, MIR-9-1, MIR-9-3, MIR-339) in 26 patients with ovarian cancer. Methylation level was evaluated by the method of methylation-specific PCR in real time. The data obtained in primary tumors (26), histologically unchanged ovarian tissues (15) and peritoneal metastases (19) were compared using a number of statistical programs. For all 13 genes, an increase in the level of methylation was revealed during the transition from unchanged tissue to primary tumors and further from primary tumors to peritoneal metastases; moreover, in the genes MIR-203a, MIR-375 and MIR-339, the level of methylation in metastases increased most significantly (in 2 and more times). A correlation was observed for the first time, showing a consistency between the increase in methylation level in some miRNA pairs, for example, MIR-129-2/MIR-132 (r s > 0,7; p<0,0001), both in primary tumors and in metastases. An analysis of microRNA gene methylation in clinical samples of ovarian cancer showed a correlation between the observed molecular changes both with the initial stages of tumor formation and with the progression and dissemination of ovarian cancer, with the presence of metastases in a large omentum and with the appearance of ascites. The revealed dependencies deepen the understanding of the mechanism of peritoneal metastasis and can be used to select new diagnostic and prognostic markers of ovarian cancer., Competing Interests: The authors declare no conflict of interest.

Groups of microRNA genes, methylation of which is associated with the initial (I-II) stages of breast cancer, are determined, and new markers and marker systems for the disease diagnosis were created on the basis of these data. A total of 14 genes in which methylation was associated with breast cancer were identified with the use of methyl-specific PCR on a representative sample of 70 tumor specimens. Analysis of 46 specimens from patients with clinical stages I and II detected 9 genes (MIR-124-1, MIR-124-3, MIR-125b-1, MIR-129-2, MIR-132, MIR-148a, MIR-193a, MIR-34b/c, and MIR-9-3), in which methylation was associated with the initial stages of the disease. Using ROC analysis, we formed two systems including 6 markers each and detecting breast cancer at stages I-II with high sensitivity (89 and 91%) and specificity (88%) at AUC=0.92-0.93. These sets were validated on the total sample of 70 specimens including all disease stages; they showed 93 and 94% sensitivities, 88% specificity, and AUC=0.95. Highly sensitive systems of markers, based on microRNA gene methylation, were created for the diagnosis of breast cancer at stages I-II.

We identified a group of miRNA genes whose methylation is associated with ovarian cancer metastasis. Based on these data, new markers and the systems of markers predicting tumor dissemination were selected. Using methylation-specific PCR and a representative set of 54 ovarian cancer samples, we identified 10 microRNA genes (MIR-124a-2, MIR-127, MIR-125b-1, MIR-129-2, MIR-137, MIR-193a, MIR-203a, MIR-34b/c, MIR-130b, and MIR-1258) whose methylation is associated with tumor metastasis. The greatest association was established for 4 genes: MIR-137, MIR-193a, MIR-34b/c, and MIR-130b (p<0.01). ROC analysis revealed 3 most optimal marker systems including 4-5 miRNA genes and characterized by high sensitivity (82-94%) and specificity (76-86%) at AUC=0.89-0.92. Methylation of any three genes from these systems is sufficient to predict metastasis with the specified accuracy. Detection of the group of hypermethylated miRNA genes with predictive value for ovarian cancer metastasis is of great importance for personalized treatment of the patients.

miRNA genes play an important role in cancer pathogenesis, while they may be suppressed by hypermethylation. Here, we assess the diagnostic potential of a group of hypermethylated miRNA genes (MIR-124-1, MIR-124-3, MIR-125B-1, MIR-127, MIR-132, MIR-193a, and MIR-34b/c) in a representative set of 70 breast cancer samples and 17 breast tissue samples from deceased donors with no malignancies. For these seven genes, the methylation status is determined using the methylation-specific PCR. Methylation reached 26-76% in tumor specimens, 1-27% in paired considered normal breast tissues, and 0-18% in breast tissue from deceased donors. By quantitative RT-PCR, reduced expression levels of the investigated miRNAs are detected, with a negative correlation of expression levels with gene hypermethylation. Combinations of three or four hypermethylation biomarkers, namely, MIR-124-1, MIR-125B-1, MIR-127, and MIR-34b/c are found suitable for breast cancer diagnostics; with sensitivity (76-93%), specificity (88-100%), and AUC (0.88-0.94). Notably, the MIR-127 gene was hypermethylated only in the tumor samples of patients with metastases, and, therefore, should be tested as a marker of breast cancer dissemination. These findings may lead to improvement in the management of breast cancer.

The study was designed to determine genes of microRNAs hypermethylated in malignant ovarian tumors and to select new diagnostic and prognostic markers of the disease and effective system of markers. Using methyl-specific PCR and a representative sample of 54 ovarian cancer specimens, we determined 5 microRNA genes (MIR-34b/c, MIR-9-1, MIR-124-3, MIR-129-2, and MIR-107) hypermethylated in the majority of tumor samples in comparison with paired samples of histologically unchanged tissue (48-57% vs. 4-19%, p<0.001). Using ROC-analysis, we selected an effective system of 4 markers for diagnosis of ovarian cancer (MIR-9-1, MIR-124-3, MIR-129-2, and MIR-107) characterized by high sensitivity and specificity (up to 87-94% at AUC=0.92) relative to the conventional norm (54 paired samples of histologically unchanged tissue) and absolute norm (18 ovarian tissue samples from subjects who died from non-tumor diseases). It was also shown that methylation of MIR-129-2, MIR-9-1, and MIR-34b/c genes is significantly (p<0.01) correlated with the clinical stage or the presence of metastases. The results indicate that epigenetic modifications of the studied microRNA genes are involved in the pathogenesis and progression of ovarian cancer and attest to their diagnostic and prognostic potential.

It is known that microRNAs (miRNAs) are able to dynamically regulate gene expression. At the same time, methylation can reduce expression of miRNA encoding genes and, therefore, reduce their inhibitory effects on mRNAs of target genes, including those of oncogenes, that promoting the development of tumors of different localization. The role of miRNA hypermethylation in the pathogenesis of ovarian cancer is not completely understood; so we conducted a search for new hypermethylated and potentially suppressor miRNA genes in ovarian tumors. Four new miRNA genes (MIR-107, MIR-130b, MIR-203a, MIR-1258) commonly hypermethylated (28-52%) in tumor tissues vs 4-7% in paired histologically normal tissues, p < 0.01, were identified in a representative set of 54 ovarian cancer samples using methylation-specific PCR. It was shown that hypermethylation of MIR-130b, MIR-203a, and MIR-1258 genes is significantly (p < 0.05) associated with metastasis of ovarian cancer. These results suggest the involvement of four miRNAs (miR-107, miR-130b, miR-203a, and miR-1258) and hypermethylation of their encoding genes in the pathogenesis of ovarian cancer.

Methylation of promoter CpG islands may suppress the function of miRNAs by inhibiting their expression. Our work analyzes the role of promoter methylation in altering the expression of 12 miRNAs associated with epithelial ovarian cancer (EOC): miR-124-3p, -125b-5p, -127-5p, -129-5p, -132-3p, -137, -148a-3p, -191-5p, -193a-5p, -203a, -339-3p, and -375. The role of methylation in the deregulation of these miRNAs has not been previously assessed in a representative set of EOC samples. Using 76 paired (tumor/matched normal) ovarian samples and methylation-specific PCR, we demonstrated significant aberrations in the methylation patterns of 11 miRNA genes and identified 8 novel hypermethylated miRNA genes (MIR-124-1, -124-2, -124-3, -127, -132, -137, -193A, and -339) as well as one hypomethylated miRNA gene (MIR-191). Quantitative PCR on a subset of 29 paired EOC samples allowed us to establish a strong correlation between methylation status and alterations in expression levels for all 12 miRNAs studied. These findings demonstrate the functional role of aberrant methylation of examined miRNA genes in EOC. Moreover, we showed a significant association of hypermethylation of 10 miRNA genes (MIR-124-2, -124-3, -125B-1, -127, -129-2, -137, -193A, -203A, -339, -375) with EOC metastasis to lymph nodes, peritoneum, and distant organs. Interestingly, MIR-203A and MIR-375 were hypermethylated only in disseminated ovarian tumors, implying that non-suppressor miR-203a and miR-375 have anti-metastatic properties. Hypermethylation of 10 miRNA genes in EOC metastases was validated using an additional sample set of 13 primary tumors and matched peritoneal metastases. Together, these results show the impact of aberrant methylation on deregulation of 12 miRNAs in EOC, the involvement of 10 hypermethylated miRNA genes in metastasis (including peritoneal macro-metastases), and suggest novel potential biomarkers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

MicroRNA and methylation are important epigenetic mechanisms in the pathogenesis of cancer. The role of a group of microRNA hypermethylated genes in the pathogenesis of ovarian cancer was studied and their diagnostic and prognostic potential was evaluated. Studies on a representative sample of 54 ovarian cancer specimens with the use of methyl-specific PCR resulted in detection of five microRNA genes (MIR-9-1, MIR-9-3, MIR-107, MIR-1258, and MIR-130b) methylated in the majority of tumor specimens in comparison with paired specimens of histologically intact tissue (37-57% vs. 4-9%, p<0.01). Methylation of three genes (MIR-9-1, MIR-9-3, and MIR-130b) was significantly (p≤0.05) associated with the parameters of ovarian cancer progress (clinical stage, differentiation degree, tumor size, and presence of metastases). These findings attest to oncosuppressive role of the studied microRNA genes (MIR-9-1, MIR-9-3, MIR-107, MIR-1258, and MIR-130b) in the pathogenesis and progress of ovarian cancer and indicated their prognostic potential.

Changes in the levels of expression of proapoptotic genes APAF1 and DAPK1 and antiapoptotic gene BCL2 were studied by real time PCR in specimens of tumors and histologically intact tissue from 28 patients with breast cancer. The expression of APAF1 and DAPK1 was below the normal in the majority of tumor samples (p<0.05), while the level of BCL2 mRNA more often surpassed the normal (p<0.1). Study of the same sample of specimens by methylspecific PCR showed predominance of APAF1 and DAPK1 hypermethylation (p<0.05 and p<0.1, respectively) and more frequent hypomethylation of BCL2. A significant correlation between changes in the levels of expression and methylation (r=0.40-0.49; p<0.05) was detected for all three genes (APAF1, DAPK1, and BCL2). The results suggest that methylation play an important role in the regulation of these apoptosis system genes in breast cancer.

The methylation of promoter CpG islands and the interaction between microRNAs (miRNAs) and messenger RNAs (mRNAs) of target genes are considered two crucial mechanisms for gene and pathway deregulation in malignant tumors. The aim of this study was to analyze the role of promoter methylation in altering the expression of 13 miRNAs that are associated with breast cancer (BC): miR-124, -125b, -127, -132, -137, -148a, -191, -193a, -203, -212, -34b, -375, -9. The role of methylation in the deregulation of these miRNAs has not been previously assessed in the representative set of BC samples. We used a set of 58 paired (tumor/normal) breast tissue samples and methylation-specific PCR to demonstrate significant aberrations in the methylation patterns of 9 miRNA genes. In particular, we observed hypermethylation of MIR-127, -132, and -193a, and hypomethylation of MIR-191 for the first time. Using quantitative PCR, we established a strong correlation between promoter methylation and expression levels for 12 miRNA genes (all except MIR-212); this finding demonstrates the functional importance of altered methylation patterns. We also performed a correlation analysis between expression levels of the 13 miRNAs and 5 cancer-associated genes, namely RASSF1(A), CHL1, APAF1, DAPK1, and BCL2, which were predicted as targets for these miRNAs, to investigate the impact of these miRNAs on these genes with key cellular functions in BC. Significant negative correlation was revealed for the following miRNA-mRNA pairs: miR-127-5p and DAPK1, miR-375 and RASSF1(A), and miR-124-3p and BCL2. Additionally, we also found a strong association between hypermethylation of MIR-127 and MIR-125b-1 and BC progression, particularly metastasis. Thus, our findings provide evidence for the significant role of methylation in the deregulation of 12 miRNA genes in BC, identify putative novel functional miRNA-mRNA pairs, and suggest MIR-127 and MIR-125b-1 hypermethylation to be potential biomarkers of BC metastasis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The clear-cell carcinoma of kidney is characterized by high rate of lethal outcomes. The lethality makes up to 16% in the first year after disease was diagnosed. The absence of efficient diagnostic at early stages (30% of all cases of clear-cell carcinoma of kidney are found out at late stages if there is metastatic disease) indicates the necessity of searching new biomarkers of clear-cell carcinoma of kidney. The disorders in methylation of regulatory genes of micro-RNA are one the causes of development of tumor. The purpose of the present study is to discover hyper-methylated genes of micro-RNA under clear-cell carcinoma of kidney and to evaluate their diagnostic and prognostic characteristics. The establishment of status of methylation of genes of micro-RNA in samples of DNA from tumor and unaltered tissue of 50 patients with clear-cell carcinoma of kidney was implemented using bisulfite conversion of DNA and subsequent methyl-specific polymerase chain reaction. The frequent hyper-methylation of seven genes of micro-RNA (miR-9-1/3, miR-124a-1/2/3, miR-34b/c, miR-129-2) in tumors of clear-cell carcinoma of kidney. Out of 7 analyzed genes of micro-RNA the systems of markers on 2-4 genes in each one were compiled. According ROC-analysis, the sensitivity of 4 markers systems reaches 88%, specificity - 94% (AUC 0.83-0.84). Furthermore, it is demonstrated that hyper-methylation of 5 genes of micro-RNA (miR-9-1/3, miR-124a-1/2/3, miR-34b/c, miR-129-2) is associated with parameters of progression of clear-cell carcinoma of kidney (stage, size of tumor, degree of differentiation, metastasis in lymph nodes on remote organs). Out of genes which hyper-methylation is associated with metastasis disease (miR-9-1/3, miR-124a-1/2/3, miR-34b/c, miR-129-2) 5 prognostic systems of markers were compiled and characterized. The hyper-methylation of gene miR-129-2 is a new efficient marker of prognosis of metastasis disease (sensitivity 75% and specificity 79%, AUC 0.77) that can be combined with markers discovered in other studies.

Methylation of promoter CpG islands and microRNA (miRNA) interactions with mRNAs of target genes are epigenetic mechanisms that play a crucial role in deregulation of gene expression and signaling pathways in tumors. Altered expression of six chromosome 3p genes (RARB(2), SEMA3B, RHOA, GPX1, NKIRAS1, and CHL1) and two miRNA genes (MIR-129-2 and MIR-9-1) was observed in primary clear cell renal cell carcinomas (ccRCCs, 31-48 samples) by RT-PCR and qPCR. Significant downregulation (p < 0.05, Fisher's exact test) was observed for SEMA3B, NKIRAS1, and CHL1; and differential expression, for the other chromosome 3p and miRNA genes. Methylation-specific PCR with primers to RARB(2), SEMA3B, MIR-129-2, and MIR-9-1 showed that their methylation frequency was significantly (p < 0.05, Fisher's exact test) elevated in the ccRCC samples. Significant correlations between promoter methylation and expression were confirmed for SEMA3B and observed for the first time for RARB(2), GPX1, and MIR-129-2 in ccRCC (Spearman's correlation coefficient rs ranging 0.31-0.60, p < 0.05). The MIR-129-2 and RARB(2) methylation frequencies significantly correlated with ccRCC progression. MIR-129-2 methylation correlated with upregulation of RARB(2), RHOA, NKIRAS1, and CHL1 (rs ranging 0.35-0.53, p < 0.05). The findings implicate methylation in regulating RARB(2), SEMA3B, GPX1, and MIR-129-2 and indicate that miR-129-2 and methylation of its gene affect RARB(2), RHOA, NKIRAS1, and CHL1 expression.

MicroRNAs play an important role in the regulation of expression of many genes involved in cancer pathogenesis. One of the causes of miRNA level deregulation in tumors is the methylation of CpG islands in the promoter regions of the genes that encode them. Hypermethylation may lead to the suppression of miRNA gene expression and, as a consequence, to a decrease in their inhibitory effect on target gene mRNAs. A search for new miRNA genes hypermethylated in breast cancer has been carried out in the present study. The methylation of five miRNA genes associated with breast cancer (miR-132, miR-1258, miR-107, miR-130b, miR-137) has been as studied using a representative set of 41 breast cancer samples by methylation-specific PCR. Three new genes, MIR-132, MIR-137 and MIR-1258, with a high frequency of hypermethylation (41, 37 and 34%, respectively) have been identified in breast cancer. The methylation of these genes in the breast tissues of ten donors without cancer pathology in anamnesis was only found in single cases. These results enable the involvement of three miRNAs (miR-132, miR-137, miR-1258) and the methylation of the genes that encode them in the pathogenesis of breast cancer to be suggested.

The purpose. Identifying the capacity of the medicinal leech novel original recombinant thrombolytic preparation Destabilase-Lysozyme-2 to inhibit the blood platelet aggregation., Methods: Gene of destabilase-lysozyme. ds2 (mlDL-Ds2 ), was cloned in E.coli cells. Recombinant protein was isolated in denaturing conditions using metal-chelate chromatography followed by denaturation of the polypeptide by rapid dilution in exact accordance with the procedure described by Kurdyumov A.S. et al. ( 2016, Russian Journal of Bioorganic Chemistry, v.42, s. 42-52). Blood was collected from the jugular vein of 18 horses. The functional status of platelets in the presence of different destabilase-lysozyme concentrations were evaluated for their aggregation in Platelet Rich Plasma ( PRP) and in Washed Platelet suspension (WP) using aggregometers Chrono-Log-700 and Сhrono-Log-560, USA560, США. As used aggregation inducers of ADP, collagen type III and human thrombin., Results: First demonstrated the ability of newly synthesized (Kurdyumov A.S. et al. 2016, Russian Journal of Bioorganic Chemistry, v42, s. 42-52) thrombolytic recombinant enzyme destabilase-lyzosyme to inhibit more than 40% of ADP-stimulated PRP aggregation and ADP- stimulated aggregation of horse blood washed platelets., Conclusion: The ability of destabilase-lyzosyme -2 to inhibit platelets aggregation extends biological properties of recombinant thrombolytic enzyme, pre-clinical trials which resulted in the end of 2015.

The methylation of promoter CpG islands and interactions between microRNAs (miRNAs) and messenger RNAs (mRNAs) of target genes are considered two crucial epigenetic mechanisms for inducing gene and pathway deregulation in tumors. Here, the expression levels of seven cancer-associated 3p genes (RASSF1(isoform A), RARB(isoform 2), SEMA3B, RHOA, GPX1, NKIRAS1, and CHL1) and their predicted regulator miRNAs (miR-129-2, miR-9-1) were analyzed in breast (BC, 40 samples) and ovarian (OC, 14 samples) cancers using RT-PCR and qPCR. We first revealed a negative correlation between the level of the miR-129-2 precursor and RASSF1(A) and GPX1 mRNA levels in BC (Spearman's correlation coefficient (rs) was − 0.26 in both cases). Similar results were observed for the miR-129-2 precursor and the RASSF1(A), GPX1, RARB(2), and CHL1 genes in OC (rs was in the range − 0.48 to − 0.54). Using methylation-specific PCR, a significant correlation was shown between promoter hypermethylation and the down-regulation of the RASSF1(A), GPX1, RARB(2), SEMA3B, MIR-129-2, and MIR-9-1 genes in BC (rs = 0.41 to 0.75) and of the RASSF1(A) gene in OC (rs = 0.67). We first demonstrated a high hypermethylation frequency of MIR-129-2 and SEMA3B (up to 45 to 48%) in both BC (69 samples) and OC (41 samples). Moreover, we observed a positive correlation between the hypermethylation of MIR-129-2 and the up-regulation of the RASSF1(A) and GPX1 genes in BC (rs = 0.38 and 0.42, respectively). QPCR analysis of the expression of RASSF1(A) and mature miR-129-2 in additional BC sample set (24 samples) revealed a negative correlation between them (rs = − 0.41) that strengthened the results obtained during the analysis of miR-129-2 precursor level. In summary, the obtained data indicate the involvement of methylation in the down-regulation of the studied coding and miRNA genes and suggest the involvement of miR-129-2 in the deregulation of RASSF1(A) via a direct interaction or/and mediators in common pathways (according to KEGG, Gene Ontology (FDR < 0.01), and GeneCards data) in the examined gynecological tumors.

The methylation of CpG islands in promoter regions, together with the interaction of miRNAs with the mRNAs of their target genes on the posttranscriptional level, are complex epigenetic mechanisms that perform the delicate and dynamic regulation of genes and signal transduction pathways in the cell. This review summarizes the results obtained by the authors, as well as the literature data, on the roles of methylation in regulating the protein-coding genes of chromosome 3 and a number of miRNA genes in clear-cell renal cell carcinomas. The results are based on the use of genomic NotI-microarrays (which allow the identification of both methylation and deletions in genes containing CpG islands) and on some other approaches. The application of NotI-microarray technology to the analysis of the chromosome-3 short arm, a region of frequent deletions in tumors, gave us the opportunity to identify many novel genes associated with kidney cancer pathogenesis. The relationship between alterations in the expression leyels and methylation of chromosome 3 genes, kidney cancer progression, and metastasis was shown. New microRNAs involved in kidney cancer pathogenesis were identified as well. The functions of microRNA genes methylated in kidney cancer were discussed.

The SEMA3B gene is located in the 3p21.3 LUCA region, which is frequently affected in different types of cancer. The objective of our study was to expand our knowledge of the SEMA3B gene as a tumor suppressor and the mechanisms of its inactivation. In this study, several experimental approaches were used: tumor growth analyses and apoptosis assays in vitro and in SCID mice, expression and methylation assays and other. With the use of the small cell lung cancer cell line U2020 we confirmed the function of SEMA3B as a tumor suppressor, and showed that the suppression can be realized through the induction of apoptosis and, possibly, associated with the inhibition of angiogenesis. In addition, for the first time, high methylation frequencies have been observed in both intronic (32-39%) and promoter (44-52%) CpG-islands in 38 non-small cell lung carcinomas, including 16 squamous cell carcinomas (SCC) and 22 adenocarcinomas (ADC), and in 83 clear cell renal cell carcinomas (ccRCC). Correlations between the methylation frequencies of the promoter and the intronic CpG-islands of SEMA3B with tumor stage and grade have been revealed for SCC, ADC and ccRCC. The association between the decrease of the SEMA3B mRNA level and hypermethylation of the promoter and the intronic CpG-islands has been estimated in renal primary tumors (P < 0.01). Using qPCR, we observed on the average 10- and 14-fold decrease of the SEMA3B mRNA level in SCC and ADC, respectively, and a 4-fold decrease in ccRCC. The frequency of this effect was high in both lung (92-95%) and renal (84%) tumor samples. Moreover, we showed a clear difference (P < 0.05) of the SEMA3B relative mRNA levels in ADC with and without lymph node metastases. We conclude that aberrant expression and methylation of SEMA3B could be suggested as markers of lung and renal cancer progression.

Methylation of CpG-islands in promoter regions as well as interaction of miRNAs with messenger RNAs of target genes are related to multilayer mechanisms regulating gene expression. The goal of this study was to assess a possibility for miRNA gene methylation to influence indirectly activation of their target genes in lung tumors. By using a unified collection of samples of non-small cell lung cancer, it was demonstrated that elevated levels of mRNA for RHOA and NKIRAS1 genes were significantly (Spearman rank correlation, P < 10(-11)) associated both with loss of methylation in their CpG-islands and methylation in a number of miRNA genes, which, according to the miRWalk database, were predicted to possess regulatory functions. Novel potential regulatory miRNAs for RHOA (miR-9-1/-3, -34b/c, -129-2, -125b-1, -375, -1258) and NKIRAS1 (miR-34b/c, -129-2, -125b-1, -193a, -124a-1/-2/-3, -212, -132) genes in lung cancer were identified.

MicroRNAs play an important role in the regulation of expression of many genes and are involved in carcinogenesis. The regulation of miRNA gene expression can involve the methylation of promoter CpG islands. In this work, the methylation of six miRNA genes (mir-107, mir-125b-1, mir-130b, mir-137, mir-375, and mir-1258) in non-small-cell lung cancer (NSCLC) was studied for the first time by methylation-specific PCR using a representative set of specimens (39 cases). Four new genes (mir-125b-1, mir-137, mir-375, and mir-1258) methylated in primary NSCLC tumors were identified with frequencies of 56, 31, 56, and 36%, respectively. The frequencies of miRNA promoter methylation in DNA of tumors and histologically normal tissues differed significantly (P < or = 0.05 by Fisher's test). In lung tissues of 20 donors without a history of cancer, these genes were only methylated in a few cases. It was also shown that the previously unstudied promoter CpG islands of mir-107 and mir-130b were not methylated in NSCLC. The frequencies of mir-125b-1 and mir-137 methylation were shown for the first time to correlate with NSCLC progression (clinical stage and metastasis).

MicroRNA regulates gene expression, is involved in many cellular processes, and plays an important role in the development of cancer. The regulation of the expression of miRNA genes can be achieved by methylating their CpG islands, which is shown in different types of tumors. The methylation of miRNA genes in clear cell renal cell carcinoma (CCRCC) has mainly been studied for the miR-9 and miR-34 families. The methylation of six miRNA genes (miR-124a-2, -124a-3, -9-1, -9-3, -34b/c, -129-2) was investigated with the use of representative set of CCRCC samples (46 cases). Methylation of three genes miR-124a-2, -124a-3, and -129-2 was studied in kidney tumors for the first time. Methylation analysis was performed using methyl specific PCR. It is shown that the frequency of methylation of six genes (miR-124a-2, -124a-3, -9-1, -9-3, -34b/c and -129-2) was significantly higher in tumor samples than in samples of histologically normal tissue (P < 3 x 10(-5) by Fisher's exact test). These results suggest the properties of tumor suppressors for the six miRNA genes indicated in CCRCC. We also found correlations between the methylation frequency of some miRNA genes and signs of the progression of CCRCC (tumor size, clinical stage, loss of differentiation, and metastasis).

To date, there are more than two thousand human miRNAs, each of them may be involved in the regulation of hundreds of protein coding target genes. Methylation of CpG-islands, in turn, affects miRNAs gene expression. Our aim was to evaluate the role of methylation in the regulation of miRNA gene expression and, consequently, in the regulation of expression of target genes in primary lung tumors. Using a common collection of non-small cell lung cancer samples we performed a comprehensive study, including analysis of the methylation status and expression levels of some miRNA genes and their potential target genes on chromosome 3: RAR-beta2 and NKIRAS1. Increased frequency of methylation in lung tumors compared to histologically normal tissue was revealed for miR-9-1 and miR-34b/c genes with significant statistics (P < or = 0.05 by Fisher exact test) and for miR-9-3 and miR-193a was marginally significant (P < or = 0.1). Significant correlation was revealed between alterations of methylation and expression level of miR-9-1 gene (P = 5 x 10(-12) by Spearman) in the lung tumors, this suggests the role of methylation in the regulation of expression of this miRNA genes. Besides, a statistically significant negative correlation (P = 3 x 10(-12)-5 x 10(-13) by Spearman) was found between alterations of expression levels of miR-9-1 and miR-17and RAR-beta2 target gene and also between expression level alterations of miR-17 and NKIRAS1 that was not previously analyzed. The inverse relationship between expression levels of miRNA genes and their target genes is consistent with the known mechanism of suppression of protein coding genes expression under the action of miRNAs. For the first time significant correlations (P = 3 x 10(-10)-4 x 10(-13) by Spearman) were shown between alterations of methylation status of miRNA genes (miR-9-1, miR-9-3, miR-34b/c, miR-193a) and the expression level of RAR-beta2 target gene and between alterations of methylation status of miR-34b/c, and miR-193a and the expression level of NKIRAS1 target gene in the primary lung tumors, which suggests the possibility of indirect effects of methylation of miRNA genes on expression level of target genes.

This study aimed to clarify genetic and epigenetic alterations that occur during lung carcinogenesis and to design perspective sets of newly identified biomarkers. The original method includes chromosome 3 specific NotI-microarrays containing 180 NotI clones associated with genes for hybridization with 40 paired normal/tumor DNA samples of primary lung tumors: 28 squamous cell carcinomas (SCC) and 12 adenocarcinomas (ADC). The NotI-microarray data were confirmed by qPCR and bisulfite sequencing analyses. Forty-four genes showed methylation and/or deletions in more than 15% of non-small cell lung cancer (NSCLC) samples. In general, SCC samples were more frequently methylated/deleted than ADC. Moreover, the SCC alterations were observed already at stage I of tumor development, whereas in ADC many genes showed tumor progression specific methylation/deletions. Among genes frequently methylated/deleted in NSCLC, only a few were already known tumor suppressor genes: RBSP3 (CTDSPL), VHL and THRB. The RPL32, LOC285205, FGD5 and other genes were previously not shown to be involved in lung carcinogenesis. Ten methylated genes, i.e., IQSEC1, RBSP3, ITGA 9, FOXP1, LRRN1, GNAI2, VHL, FGD5, ALDH1L1 and BCL6 were tested for expression by qPCR and were found downregulated in the majority of cases. Three genes (RBSP3, FBLN2 and ITGA9) demonstrated strong cell growth inhibition activity. A comprehensive statistical analysis suggested the set of 19 gene markers, ANKRD28, BHLHE40, CGGBP1, RBSP3, EPHB1, FGD5, FOXP1, GORASP1/TTC21, IQSEC1, ITGA9, LOC285375, LRRC3B, LRRN1, MITF, NKIRAS1/RPL15, TRH, UBE2E2, VHL, WNT7A, to allow early detection, tumor progression, metastases and to discriminate between SCC and ADC with sensitivity and specificity of 80-100%.

Chromosome 3 specific NotI microarrays containing 180 NotI linking clones associated with 188 genes were hybridized to NotI representation probes prepared using matched tumor/normal samples from major epithelial cancers: breast (47 pairs), lung (40 pairs) cervical (43 pairs), kidney (34 pairs of clear cell renal cell carcinoma), colon (24 pairs), ovarian (25 pairs) and prostate (18 pairs). In all tested primary tumors (compared to normal controls) methylation and/or deletions was found. For the first time we showed that the gene LRRC3B was frequently methylated and/or deleted in breast carcinoma - 32% of samples, cervical - 35%, lung - 40%, renal - 35%, ovarian - 28%, colon - 33% and prostate cancer - 44%. To check these results bisulfite sequencing using cloned PCR products with representative two breast, one cervical, two renal, two ovarian and two colon cancer samples was performed. In all cases methylation was confirmed. Expression analysis using RT-qPCR showed that LRRC3B is strongly down-regulated at the latest stages of RCC and ovarian cancers. In addition we showed that LRRC3B exhibit strong cell growth inhibiting activity (more than 95%) in colony formation experiments in vitro in KRC/Y renal cell carcinoma line. All these data suggest that LRRC3B gene could be involved in the process of carcinogenesis as a tumor suppressor gene., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Tumor-suppressor activity was established for RASSF1A gene by in vitro and in vivo including studies of knock-out mutated mice cells. Data on methylation of promoter region and expression decrease revealed mainly in cancer cell lines were reported. Here, analysis of RASSF1A mRNA quantity was performed for the first time in primary epithelial malignant tumors of five various locations from 130 patients by semi-quantitative RT-PCR. Representative sets of kidney, lung and breast carcinomas samples were studied. Preliminary data for RASSF1A expression in ovarian and colorectal carcinomas are also reported. Our system studies showed unexpected expression profiles, namely mRNA level increase more frequently (2-7 times) than decrease in renal, breast, ovarian, and colorectal carcinomas. Increasing RASSF1A mRNA level was revealed significantly more frequently in renal cell carcinoma (24/38, 63% vs. 8/38, 21%, P = 0.0004, by Fisher exact test) and ovarian carcinomas (8/13, 62% vs. 2/13, 15%, P = 0.0114). Only in non-small cell lung cancer decreasing and increasing of RASSF1A expression were observed with equal frequency (16/38, 42%). Noteworthy, for early clinical stages prevalence of increasing expression both in squamous cell lung cancer and in adenocarcinoma was revealed, and for advanced clinical stages evident prevalence of decreasing RASSF1A expression was established. Cases with increasing expression both in early and advanced stages of clear cell renal cell carcinoma were in prevalence, in advanced stages it was proved significantly (P = 0.0094). These data suggested that RASSF1A expression alterations were tumor specific. Mentioned above regularity could point onto ambivalent RASSF1A functions in tumors--a tumor-suppressor gene and a proto-oncogene as well.

Background: CHL1 gene (also known as CALL) on 3p26.3 encodes a one-pass trans-membrane cell adhesion molecule (CAM). Previously CAMs of this type, including L1, were shown to be involved in cancer growth and metastasis., Methodology/principal Findings: We used Clontech Cancer Profiling Arrays (19 different types of cancers, 395 samples) to analyze expression of the CHL1 gene. The results were further validated by RT-qPCR for breast, renal and lung cancer. Cancer Profiling Arrays revealed differential expression of the gene: down-regulation/silencing in a majority of primary tumors and up-regulation associated with invasive/metastatic growth. Frequent down-regulation (>40% of cases) was detected in 11 types of cancer (breast, kidney, rectum, colon, thyroid, stomach, skin, small intestine, bladder, vulva and pancreatic cancer) and frequent up-regulation (>40% of cases)--in 5 types (lung, ovary, uterus, liver and trachea) of cancer. Using real-time quantitative PCR (RT-qPCR) we found that CHL1 expression was decreased in 61% of breast, 60% of lung, 87% of clear cell and 89% papillary renal cancer specimens (P<0.03 for all the cases). There was a higher frequency of CHL1 mRNA decrease in lung squamous cell carcinoma compared to adenocarcinoma (81% vs. 38%, P = 0.02) without association with tumor progression., Conclusions/significance: Our results suggested that CHL1 is involved in the development of different human cancers. Initially, during the primary tumor growth CHL1 could act as a putative tumor suppressor and is silenced to facilitate in situ tumor growth for 11 cancer types. We also suggested that re-expression of the gene on the edge of tumor mass might promote local invasive growth and enable further metastatic spread in ovary, colon and breast cancer. Our data also supported the role of CHL1 as a potentially novel specific biomarker in the early pathogenesis of two major histological types of renal cancer.