[A Group of New Hypermethylated Long Non-Coding RNA Genes Associated with the Development and Progression of Breast Cancer].

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.

DNA Methylation of a Group of Long Non-Coding RNA Genes at Different Stages of Ovarian Cancer Dissemination.

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.

Long Non-Coding RNAs and microRNAs Groups in the Regulation of Expression Level of a Number of Tumor-Associated Genes in Ovarian Cancer.

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.

[Predictive significance of genetic analysis of the development of dry eye disease of different origin]. / Prediktivnaya znachimost' geneticheskogo analiza razvitiya sindroma «sukhogo glaza¼ razlichnogo geneza.

One of the etiological causes of dry eye disease (DED) is systemic autoimmune diseases (AID): primary Sjögren's syndrome (PSS), rheumatoid arthritis (RA); their manifestation may begin with ophthalmic symptoms. The relationship of PSS and RA with genetic factors is proven. The contribution of polymorphic markers of the genes THBS1, MUC1, TRIM21, STAT4, PTPN22 in the development of these diseases is established, as well as their connection with the development of DED. A panel of genetic markers for evaluating the risk of developing DED in PSS and RA is developed, and its sensitivity and specificity is determined. PURPOSE: The aim of the study was to determine the prognostic significance of a panel of polymorphic gene markers in the development of dry eye syndrome in patients with primary Sjögren's syndrome and rheumatoid arthritis over a five-year follow-up period. MATERIAL AND METHODS: Patients with a verified diagnosis of PSS and RA without signs of DED were examined (n=35 and n=42, respectively). The control group included 82 volunteers without AID and DED. The observation period was 5 years. Every year all study subjects underwent an ophthalmological clinical and functional examination. RESULTS: Dry eye disease had developed in groups of patients with AID with predisposing genotypes of polymorphic markers of the genes THBS1, MUC1, TRIM21, STAT4, PTPN22. The peak of DED development in these patients was in the third year of the follow-up. As a result of ROC analysis, it was found that the sensitivity and specificity of determining the predisposing genotypes of polymorphic markers of the THBS1, MUC1, TRIM21, STAT4, PTPN22 genes was 68 and 87%, respectively (p<0.0001). CONCLUSION: Genetic research methods are essential for minimally invasive early diagnosis of dry eye disease, and can subsequently become the basis for a personalized approach to its treatment.

Hypermethylation of Long Non-Coding RNA Genes Group in the Breast Cancer Development and Progression.

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.

Synergy between the Levels of Methylation of microRNA Gene Sets in Primary Tumors and Metastases of Ovarian Cancer Patients.

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.

The Role of Long Non-Coding RNA CCAT1 and SNHG14 in Activation of Some Protein-Coding Genes Associated with the Development of Ovarian Cancer.

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.

Changes in the Level of Methylation of a Group of microRNA Genes as a Factor in the Development and Progression of Non-Small Cell Lung Cancer.

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.

Optimized Marker System for Early Diagnosis of Breast Cancer.

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.

Aberrant Methylation of 21 MicroRNA Genes in Breast Cancer: Sets of Genes Associated with Progression and a System of Markers for Predicting Metastasis.

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.

[Association of polymorphic markers rs7947461 of the TRIM21 gene and rs33996649 of the PTPN22 gene with the risk of developing exogenous dry eye syndrome]. / Assotsiatsiya polimorfnykh markerov rs7947461 gena TRIM21 i rs33996649 gena PTPN22 s riskom razvitiya sindroma sukhogo glaza ekzogennoi etiologii.

In this age of technological advancement, an increasing number of people is being exposed to external risk factors of damaging their ocular surface (wearing contact lenses, electromagnetic radiation from computers, mobile devices, etc.). However, the presence of external factors does not lead to a 100% risk of developing the dry eye disease (DED). The trigger mechanism in the development of autoimmune lesions of the ocular environment in some systemic diseases is known to be associated with molecular genetic factors. The search for molecular genetic disorders is based on the analysis of polymorphic markers of a number of genes responsible for the state of the eye surface. PURPOSE: To study the relationship of polymorphic markers rs7947461 of the TRIM21 gene and rs33996649 of the PTPN22 gene with the risk of developing dry eye syndrome of exogenous etiology. MATERIAL AND METHODS: The study included 57 people with exogenous risk factors for DED development. The control group included volunteers without a history of ophthalmic pathologies (n=75). Genotyping was done by real-time polymerase chain reaction followed by melting curve analysis. Statistical processing of data was done using the Statistica 6.1 RUS software for statistical analysis. RESULTS: In the course of the study, 31 patients of the main group were diagnosed with DED and separated into the 1st subgroup; DED diagnosis was not confirmed in 26 patients, who were put into the 2nd subgroup. The 1st subgroup showed a significant increase in the frequency of predisposing genotypes of the TRIM21 and PTPN22 genes. The relative risk of developing DED turned out to be 2.5 and 4.86 times higher, respectively. In the 2nd subgroup, no statistically significant data was found on the presence of predisposing genotypes of polymorphic markers of the TRIM21 and PTPN22 genes (p=0.3). CONCLUSION: The revealed association of polymorphic markers rs7947461 of the TRIM21 gene and rs33996649 of the PTPN22 gene with the risk of developing DED of exogenous etiology puts these loci as possible markers for diagnosing this pathology.

Hypermethylation of Genes in New Long Noncoding RNA in Ovarian Tumors and Metastases: A Dual Effect.

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.

The Role of ESR1 Gene Polymorphic Markers in the Development of Breast Cancer and Resistance to Tamoxifen Therapy.

We studied the effect of functionally significant polymorphic markers of the ESR1 gene on the risk of breast cancer, tamoxifen resistance, and survival of patients with this type of cancer. The study included 239 primary breast cancer patients without distant metastases. The analysis of genotype frequency distribution for the studied ESR1 gene polymorphic markers showed the association of the rs2228480 and rs2234693 markers with tamoxifen resistance in the group of patients with luminal B type breast cancer. An association of these two polymorphic markers with the risk of tumor development was also revealed; for rs2234693 polymorphic marker, a relationship with the survival of patients was also showed.

[Novel miRNAs as Potential Regulators of PD-1/PD-L1 Immune Checkpoint, and Prognostic Value of MIR9-1 and MIR124-2 Methylation in Ovarian Cancer].

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.

[Clinical significance of methylation of a group of miRNA genes in patients with ovarian 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 (rs> 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.

System of Markers Based on the Methylation of a Group of Proapoptotic Genes in Combination with MicroRNA in the Diagnosis of Breast Cancer.

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.

[Unilateral multifocal uveal melanoma (a clinical and genetic study)]. / Monolateral'naia mul'tifokal'naia uveal'naia melanoma (kliniko-geneticheskoe issledovanie).

The study presents clinical and genetic analysis of a case of unilateral multifocal uveal choroidal melanoma in a patient of 67 years. Results of ophthalmoscopy, echography, fluorescent angiography, optical coherence tomography are described. Molecular genetic testing of peripheral blood samples was performed, including detection of the occurrences of CC genotype in C3435T polymorphism of the gene ABCB1/MDR1 associated with unfavorable vital prognosis. Analysis of the genes GNAQ and GNA11 revealed two mutually exclusive mutations in the genes GNAQG183A and GNAQA209C showing genetic heterogeneity of the two tumor lesions. Organ preservation treatment of unilateral multifocal uveal melanoma was proven possible with brachytherapy method. Uveal melanoma with multicentric growth is of interest to ophthalmologists because it requires differential diagnostics from a variety of diseases including metastases in the choroid, such as metastases of uveal melanoma and skin melanoma, as well as other intraocular neoplasms.

[Hypermethylation of miR-107, miR-130b, miR-203a, miR-1258 Genes Associated with Ovarian Cancer Development and Metastasis].

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.

Association between polymorphic markers in candidate genes and the risk of manifestationof endocrine ophthalmopathy in patients with Graves' disease.

AIM: To analyze the association between the polymorphic markers in CTLA4, TNF, IL10 and IL16 genes and the risk of manifestation of endocrine ophthalmopathy (EO) in patients with Graves' disease (GD). MATERIALS AND METHODS: Case-control study included 248 patients with GD. Using polymerase chain reaction we studied the distribution of alleles and genotypes of polymorphic markers such as A60G (rs3087243) in CTLA4 gene, G(-308)A (rs1800629) in TNF gene, G(-1082)A (rs1800896) in IL10 gene, T3249C (rs4778641) in IL16 gene among 141 patients with Graves' disease and EO and 107 patients with GD without EO. RESULTS: The frequencies of A alleles and the AA genotypes were significantly increased and the frequencies of G alleles and the GG genotype polymorphic markers rs3087243 of CTLA4 gene and rs1800896 of IL10 gene, as well as the GG genotype polymorphic marker rs1800629 of TNF gene were reduced in patients with GD and EO. The polymorphism in CTLA4 gene was also associated with the activity and the severity of EO. The comparative analysis of the allele and genotype frequency distribution of polymorphic markers of IL16 gene did not show the significant difference. CONCLUSION: The risk of manifestation and the development of EO in patients with Graves' disease can be caused by not only environmental, but also genetic risk factors.

[Methylation of the genes for the microRNAs miR-129-2 and miR-9-1, changes in their expression, and activation of their potential target genes in clear cell renal cell carcinoma].

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.