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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

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

Background. Renal cell carcinoma (RCC) is asymptomatic up to severe stages and is characterized by a high mortality rate, reaching 90 % with the development of a metastatic process. Objective : to determine the group of microRNA genes, the methylation of which is associated with the progression of the disease, in particular, with metastasis. Materials and methods . Methylation-specific polymerase chain reaction in a representative sample of RCC patients (98 cases) showed an increase in the methylation status of 6 microRNA genes (MIR9-1, MIR9-3, MIR34b/c, MIR130b, MIR1258, MIR107) in tumor DNA samples relative to matched samples of histologically unchanged tissue. Results. For 4 genes (MIR9-1, MIR107, MIR130b, MIR1258), a significant association of methylation with late (III-IV) stages, tumor size, loss of differentiation, and metastasis to lymph nodes or distant organs was shown. These 4 genes were used to compose a potential metastatic prognosis marker system with a clinical sensitivity of 68 % and a specificity of 84 % (area under curve 0.83), which will be applied in the final development of a system for personalized therapy of RCC patients. Conclusion. The association of methylation of the MIR1258 with RCC metastasis has been shown for the first time and is of independent interest as a new promising marker for the prognosis of metastatic relapses.

We studied the association of polymorphic markers of cell cycle control genes (Arg72Pro of the TP53 gene, T(-410)G of the MDM2 gene, and Ser31Arg of the CDKN1A gene) in ovarian cancer and progression-free survival following platinum-based chemotherapy. Tumor tissue samples obtained from 49 patients who had undergone chemotherapy were examined. Patients received standard platinum-based chemotherapy and were observed until disease progression. Polymorphic markers of genes were evaluated by PCR-RFLP and real-time PCR. In patients carrying the G allele of the T(-410)G marker of the MDM2 gene, a decreasing trend was observed in median progression-free survival. An increase in the median progression-free survival was observed in carriers of the Pro allele of the TP53 gene (p=0.045). Furthermore, a stronger association was noted with carriers of the minor Pro/Pro homozygous genotype relative to the Arg/Arg genotype (p=0.007). In the subgroup of patients who underwent optimal or complete cytoreductive surgery, carriage of the minor Arg allele of the Ser31Arg marker (CDN1A gene) was associated with a decrease in the median progression-free survival time (p=0.004).

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.

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

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 (p0.001, 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.

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 (p0.001, 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 (p0.005, 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.

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.

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.

The “gold standard” of diagnosis of colorectal cancer (CRC) is a colonoscopy. Despite the high reliability, this method is not applicable in large-scale population screening or in estimation of the disease dynamics in a particular patient. In this study, we conducted an investigation of aberrant methylation in the APC, RASSF1A and ITGA4 genes. The study included 150 pairs of tumor tissue samples with known mutation status of the RAS family genes and the surrounding histologically unchanged tissue of patients with rectal adenocarcinoma treated at the Republican Clinical Oncology Dispensary of the Ministry of Health of the Republic of Tatarstan. The methylation profiles were studied using MethyLight PCR. The most difference in the methylation between tumor and healthy tissue was observed for the ITGA4 gene (sensitivity 78%, specificity 92.7%). For the APC gene sensitivity was 32%, specificity -93.3%, for the RASSF1 gene sensitivity was 85.3%, specificity - 56.7%. Previous data on the aberrant methylation of the SEPT9 and VIM genes and new data on the APC, RASSF1A and ITGA4 genes were compared with the mutations status in the KRAS and NRAS genes. The DNA of tumor samples was significantly more often methylated in the SEPT9 (P= 0.0018) and ITGA4 (P = 0.0044) genes in the group of patients carrying mutations in the KRAS or NRAS genes, in contrast to the DNA of tumor samples of non-carriers. In statistical analysis of the effectiveness of the diagnostic test system, it was shown that our model, which includes five methylation markers (APC, RASSF1A, ITGA4, SEPT9 and VIM), has the best sensitivity and specificity (82.7% and 97.3%, respectively). The obtained model of the diagnostic test system is proposed to be used for diagnostic problems.

Excessive hormone secretion during hypercorticism and acromegaly results in significant disturbances in bone remodeling, decrease in bone quality, and bone fractures following small traumas. However, the mechanisms of the development of such changes are not clear. In the present study, we examined specimens of bone tissue from patients with endogenous hypercorticism (increased cortisol secretion) and acromegaly (increased growth hormone secretion) obtained during transnasal adenomectomy. Our main purpose was to analyze the expression of genes responsible for osteogenesis in the bone tissue specimens from patients with hypercorticism and acromegaly, targeting an assessment of pathogenetic aspects associated with bone complications. The study included 19 specimens of bone tissue from patients with pituitary tumors (samples with acromegaly, Cushing disease, and inactive pituitary adenomas; the latter served as a control group). We revealed 14 genes (ACP5, ALPL, BGLAP, BMP7, CD40, COL1A1, COL1A2, IGF1, IGFBP2, IL6, LEP, LTA, MMP2, WNT10B) which appeared to be the most important and require further detailed study. The present study confirmed the key role of the Wnt-signaling pathway in the osteogenic process. In addition, we present new data on molecular mechanisms of development of skeletal complications in the case of cortisol and growth hormone oversecretion in humans.

Activating mutations in the EGFR gene influence cell proliferation, angiogenesis, and increases metastatic ability of non-small cell lung cancer (NSCLC) cells; they have a significant impact on the choice of medical therapy of NSCLC. The use of targeted therapy with tyrosine kinase inhibitors requires performance of appropriate genetic tests in NSCLC patients. The aim of this study was to develop a real-time PCRbased diagnostic test-system for rapid and cost-effective analysis of EGFR mutations in paraffin blocks and plasma and to perform comparative estimation of diagnostic characteristics features of real-time wild type blocking PCR and digital PCR. The study included 156 patients with different degrees of lung adenocarcinoma differentiation. A simple and efficient real-time PCR-based method for detection of L858R activating mutation and del19 deletion in the EGFR gene in DNA isolated from paraffin blocks or blood has been developed. The test system for EGFR mutations has been validated using 411 samples of paraffin blocks. The proposed system demonstrated high efficiency for DNA testing from paraffin blocks: a concordance with results of testing by means a Therascreen® EGFR RGQ PCR Kit (Qiagen, Germany) was 100%. Applicability of this test system has been also demonstrated for detection of mutations in plasma.

In the 21st century is an acute problem to find effective and inexpensive methods for the early detection of lung cancer. Patients suspected of having a malignant disease of the lungs, generally undergo clinical studies such as CT scans of the chest and bronchoscopy. The latter is mainly used to confirm the diagnosis. However, even when the signs, symptoms and radiological findings indicate that clinical diagnosis of malignant lung disease is evident, additional invasive procedures for obtaining the biological material suitable for the final confirmation of the presence of malignant cells. Currently, there is a clear understanding of the need to find biomarkers able to detect pre-clinical stage of cancer cells using minimally invasive procedures. In this paper, for the DNA of patients with different types of lung cancer samples, with a view to a better understanding of the causes of lung cancer pathology gene methylation analysis was conducted for SHOX2, TGFBR1 and MIR-375.

The increase in diabetes was noted at the turn of the 21st century. Patients with type 2 diabetes (T2DM) make up the majority of patients. Diabetes is a multifactorial disease. It arises from adverse effects of environmental factors on the body of genetically susceptible peoples. According to modern concepts, T2DM is a polygenic disease. Each of the involved genes contributes to the risk of developing of this disease. In our study, the association between polymorphic genetic markers rs7756992, rs9465871, rs7754840, and rs10946398 in the CDKAL1 gene and rs1111875 in the HHEX/IDE locus and T2DM in the Russian population were studied. Four hundred forty patients with type 2 diabetes and 264 healthy individuals without any signs of the disease were examined. The comparative analysis of distribution of genotypes and allele frequencies points to an association between polymorphic genetic markers rs7756992, rs9465871, and rs10946398 in the CDKAL1 gene and this disease. For the other polymorphic genetic markers (rs7754840 in the CDKAL1 gene and rs1111875 in the HHEX/IDE locus), no statistically significant associations are found. On the basis of these data, we can conclude that the CDKAL1 gene is associated with development of T2DM. For the HHEX/IDE locus, such an association is absent.

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.

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 presence of activating mutations in the EGFR gene influences cell proliferation, angiogenesis, and increases metastatic ability; it has a significant impact on the choice of medical therapy of non-small cell lung cancer (NSCLC). The use of targeted therapy with tyrosine kinase inhibitors requires performance of appropriate genetic tests. The aim of this study was to design a real-time PCR-based diagnostic kit for fast and cheap of EGFR mutations testing in paraffin blocks and plasma, and kit validation using samples from patients with NSCLC, and also comparative estimation of diagnostic features of real-time PCR with wild type blocking and digital PCR for mutation testing in blood plasma. The study included 156 patients with various types of adenocarcinoma differentiation. It was designed a simple and efficient real-time PCR-based method of detecting L858R activating mutation and del19 deletion in the EGFR gene for DNA isolated from paraffin blocks. Kit for EGFR mutations was validated using 411 samples of paraffin blocks. The proposed system showed high efficiency for DNA testing from paraffin blocks: a concordance with results of testing with therascreen® EGFR RGQ PCR Kit ("Qiagen", Germany) was 100%. It has been shown the possibility of using this test system for the detection of mutations in plasma.Mutatsii v gene EGFR vliiaiut na proliferatsiiu kletok, angiogenez i sposobnost' nemelkokletochnogo raka legkikh (NMRL) k metastazirovaniiu, a takzhe na vybor lekarstvennoĭ terapii. Dlia targetnoĭ terapii, provodimoĭ s ispol'zovaniem ingibitorov tirozinkinazy, neobkhodimo predvaritel'noe geneticheskoe testirovanie bol'nykh NMRL. Tsel' dannoĭ raboty sostoiala v razrabotke diagnosticheskoĭ test-sistemy dlia bystrogo i ékonomichnogo analiza mutatsiĭ v gene EGFR na osnove metoda PTsR v real'nom vremeni s ispol'zovaniem parafinovykh blokov i plazmy krovi. Provedena sravnitel'naia otsenka diagnosticheskikh kharakteristik PTsR v real'nom vremeni s blokirovaniem nukleotidnoĭ posledovatel'nosti dikogo tipa i tsifrovoĭ PTsR. V issledovanie vkliucheny156 patsientov s adenokartsinomoĭ legkogo razlichnoĭ stepeni differentsirovki. Razrabotan prostoĭ i proizvoditel'nyĭ metod vyiavleniia s pomoshch'iuPTsR v real'nom vremeni aktiviruiushcheĭ mutatsii L858R i deletsiĭ del19 gena EGFR v DNK, vydelennoĭ iz parafinovykh blokov ili plazmy krovi. Test-sistema validirovana na 411 obraztsakh parafinovykh blokov. Predlozhennaia sistema pokazala vysokuiu éffektivnost' analiza DNK iz parafinovykh blokov: konkordantnost' s rezul'tatami analiza, provedennogo s pomoshch'iu nabora therascreen® EGFR RGQ PCR Kit (“Qiagen”, Germaniia), sostavila 100%. Pokazana vozmozhnost' ispol'zovaniia dannoĭ test-sistemy dlia vyiavleniia mutatsiĭ v plazme krovi.

It is an acute problem for the 21st century to find effective and inexpensive methods for early detection of lung cancer. Patients, suspected of having a malignant disease of lungs, generally undergo clinical studies such as CT scans of the chest and bronchoscopy. The latter is mainly used to confirm the diagnosis. However, even when the signs, symptoms and radiological findings indicate that clinical diagnosis of malignant lung disease is evident, additional invasive procedures for obtaining the biological material suitable for the final confirmation of the presence of malignant cells are required. Currently, there is a clear understanding of the need to find biomarkers able to detect pre-clinical stage of cancer cells using minimally invasive procedures.

The study of hereditary predisposition to multifactorial diseases is essential for diagnosis and selection of the optimal treatment. The study of polymorphisms of candidate genes whose products are involved in the pathogenesis of multifactorial diseases is of great clinical importance. Aim. The aim of this study was to investigate the association of rs2241766 and rs1501299 polymorphisms in the ADIPOQ gene, rs2275737 and rs2275738 polymorphisms in the ADIPOR1 gene and rs11061971 and rs16928751 polymorphisms in the ADIPOR2 gene with the development of type 2 diabetes mellitus (T2DM) in the Russian population. Materials and methods. The study included a group of 500 patients with T2DM diagnosed based on standard diagnostic criteria (T2DM+). The control group (T2DM-) was a random sample of 500 patients with no evidence of the disease and was matched to the T2DM+ group for gender, age and body mass index. The determination of alleles and genotypes was performed using real-time polymerase chain reaction with TaqMan probes. The X 2 test and contingency tables were used to compare the distribution of allele and genotype frequencies. A p-value of Results. Comparative analysis of the distribution of alleles and genotypes indicated an association between T2DM and the disease gene polymorphic marker rs11061971 ADIPOR2 (р = 0.004 for the distribution of alleles, р = 0.011 for the distribution of genotypes). The presence of allele A and genotype AA decreased the risk of development of T2DM (OR = 0.76 and 0.75, respectively), whereas the T allele carriers and TT genotype were associated with an increased risk of developing T2DM (OR = 1.31 and 1.63, respectively). There was no statistically significant association between T2DM and polymorphic markers of ADIPOQ or ADIPOR1 genes. Conclusions. Based on this data, polymorphism of the ADIPOR2 gene in the Russian population is associated with the development of T2DM, but there is no association between T2DM and polymorphism of the ADIPOQ or ADIPOR1 genes.

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

To test FTO, KCNJ11, SLC30A8, and CDKN2B for association with type 2 diabetes mellitus (DM), the allele and genotype frequency distributions were established for several polymorphisms of the genes. A comparison of allele and genotype frequencies between DM patients and healthy subjects implicated CKNJ11, SLC30A8, and CDKN2B, but not FTO, in the disease. The genes that affect insulin production and secretion in pancreatic β cells proved to play a main role in type 2 DM in the Russian population.

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.

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, p0.01, 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 (p0.05) 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.

To study the association with diabetes mellitus type 2 we performed analysis of the distribution of frequencies of alleles and genotypes two polymorphic markers of TCF7L2 gene. The study included groups of T2DM patients and unrelated controls of Russian origin. Analysis of the distribution of frequencies of alleles and genotypes of the polymorphic marker rs7903146 of TCF7L2 gene showed the presence of association with T2DM in Russian population, while for the marker rs12255372 was not found statistically significant associations with type 2 diabetes. We can conclude that in Russian population TCF7L2 gene is associated with development of T2DM.

To study the distribution of alleles and genotypes of polymorphic markers of genes CYP2C9 and VKORC1 of Russian patients who live in Moscow, and in order to assess the influence of genetic factors on warfarin therapy 400 patients have been genotyped. The dosage of warfarin which is required for achievement of INR target values has been different among owners of different geno- types of polymorphic markers of genes CYP2C9 . Meanwhile the highest average dose has been required for genotype *1/*1 and the lowest – for owners of alleles *2 and *3 . For polymorphism G(- 1639)A of the gene VKORC1 the dosage of warfarin which is required for achievement of the INR target values, has been different among owners of different genotypes. The highest average dose has been required for genotype GG, and the lowest – for genotype AA. The results will allow to work out more accurate algorithm of choosing of the initial dose of warfarin depending on the genotypes of polymorphic markers of genes CYP2C9 and VKORC1.

The increase in diabetes was noted at the turn of the 21st century. Patients with type 2 diabetes (T2DM) make up the majority of patients. Diabetes is a multifactorial disease. It arises from adverse effects of environmental factors on the body of genetically susceptible peoples. According to modern concepts, T2DM is a polygenic disease. Each of the involved genes contributes to the risk of developing of this disease. In our study, the association between polymorphic genetic markers rs7756992, rs9465871, rs7754840, and rs10946398 in the CDKAL1 gene and rs1111875 in the HHEX/IDE locus and T2DM in the Russian population were studied. Four hundred forty patients with type 2 diabetes and 264 healthy individuals without any signs of the disease were examined. The comparative analysis of distribution of genotypes and allele frequencies points to an association between polymorphic genetic markers rs7756992, rs9465871, and rs10946398 in the CDKAL1 gene and this disease. For the other polymorphic genetic markers (rs7754840 in the CDKAL1 gene and rs1111875 in the HHEX/IDE locus), no statistically significant associations are found. On the basis of these data, we can conclude that the CDKAL1 gene is associated with development of T2DM. For the HHEX/IDE locus, such an association is absent.

To date, there are more than 2000 known human miRNAs, each of which may be involved in the regulation of hundreds of protein-coding target genes. In turn, the methylation of CpG islands affects the miRNA gene expression. Our aim was to evaluate the role of methylation in the regulation of miRNA gene expression and, consequently, in the regulation of the expression of target genes in primary lung tumors. Using a common collection of samples of non-small-cell lung cancer, we have performed a comprehensive study, including an analysis of the methylation status and level of expression of some miRNA genes and their potential target genes on chromosome 3, i.e., RAR-beta2 and NKIRAS1. The 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 < 0.05 by Fisher’s exact test) and for miR-9-3 and miR-193a was marginally significant (P < 0.1). A significant correlation was revealed between the changes in methylation and level of expression of miR-9-1 gene (P ≈ 5 × 10−12 by Spearman) in lung tumors, which suggests the role of methylation in the regulation of expression of these miRNA genes. Furthermore, a statistically significant negative correlation (P ≈ 3 × 10−12 to 5 × 10−13 by Spearman) was found between changes in the levels of expression of miR-9-1 and miR-17 and RAR-beta2 target genes, as well as between the changes in the level of expression of miR-17 and NKIRAS1 that were not previously analyzed. The inverse relationship between the levels of expression of miRNA genes and their target genes is consistent with the known mechanism of the suppression of the expression of protein-coding genes under the action of miRNA. For the first time, significant correlations (P ≈ 3 × 10−10 to 4 × 10−13 by Spearman) were shown between changes in the methylation status of miRNA genes (miR-9-1, miR-9-3, miR-34b/c, miR-193a) and the level of expression of the RAR-beta2 target gene and changes in the methylation status of miR-34b/c, and miR-193a and the level of expression of the NKIRAS1 target gene in the primary lung tumors, which suggests the possibility of indirect effects of the methylation of miRNA genes on the level of expression of target genes.

Tumor suppressor activity of RASSF1A in vitro and in vivo was established, in particular, in studies of knockout mice cells. Data on methylation of the promoter region and a lower expression of RASSF1A were mostly obtained with cancer cell lines. Here, the RASSF1A mRNA was quantified the first time in primary epithelial malignant tumors of five various locations from 130 patients by semi-quantitative RT-PCR. Representative samples of kidney, lung, and breast carcinomas were examined. Preliminary data were obtained for RASSF1A expression in ovarian and colorectal carcinomas. System studies showed unexpected expression profiles, namely, the mRNA level increased (two- to sevenfold) more frequently than decreased in renal, breast, ovarian, and colorectal carcinomas. A higher RASSF1A mRNA level was significantly more frequent in renal cell carcinomas (24/38, 63% vs 8/38, 21%, P = 0.0004 by Fisher’s exact test) and ovarian carcinomas (8/13, 62% vs 2/13, 15%, P = 0.0114). Equal frequencies of lower and higher RASSF1A expression levels were only observed in non-small cell lung cancer (16/38, 42%). Noteworthy, an increase in expression was more common at early clinical stages of squamous cell lung cancer and adenocarcinoma, while a decrease in RASSF1A expression was more frequent at advanced clinical stages. In clear cell renal cell carcinoma, an increase in RASSF1A expression occurred more often at both early and advanced stages and was significant at advanced stages (P = 0.0094). The findings suggested tumor specificity for changes in RASSF1A expression. The observed regularities may also indicate that RASSF1A has dual functions in tumors, acting as a tumor suppressor and as a protooncogene.

BACKGROUND: Rare variants of the WFS1 gene encoding wolframin cause Wolfram syndrome, a monogenic disease associated with diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. In contrast, common variants of WFS1 showed association with type 2 diabetes (T2D) in numerous Caucasian populations. AIM: In this study, we tested whether the markers rs752854, rs10010131, and rs734312, located in the WFS1 gene, are related to the development of T2D in a Russian population. METHODS: The polymorphic markers were genotyped in Russian diabetic (n = 1,112) and non-diabetic (n = 1,097) patients using a Taqman allele discrimination assay. The correlation between the carriage of disease-associated WFS1 variants and the patients' clinical and metabolic characteristics was studied using ANOVA and ANCOVA. Adjustment for confounding variables such as gender, age, body mass index, obesity, HbA1c, and hypertension was made. RESULTS: Haplotype GAG, consisting of the minor alleles of rs752854, rs10010131, and rs734312, respectively, showed association with decreased risk of T2D (OR = 0.44, 95% CI = 0.32-0.61, p = 4.3 x 10-7). Compared to other WFS1 variants, non-diabetic individuals homozygous for GAG/CAG had significantly increased fasting insulin (padjusted = 0.047) and homeostasis model assessment of β-cell function (HOMA-β) index (padjusted = 0.006). Diabetic patients homozygous for GAG/GAG showed significantly elevated levels of 2-h insulin (padjusted = 0.029) and HOMA-β = 0.011. CONCLUSIONS: Disease-associated variants of WFS1 contribute to the pathogenesis of T2D through impaired insulin response to glucose stimulation and altered β-cell function.

Earlier, methylation of a CpG island in the SEMA3B gene (3p21.31) was observed in cell lines of small-cell and non-small-cell lung carcinoma. According to NCBI (Build 36), that island belonged to intron 1 of the gene. Our study concerns the methylation of two CpG islands, promoter and intronic, in the SEMA3B gene in patients with clear cell renal cell carcinoma (RCC). Methylation-specific PCR and bisulfite sequencing revealed a high frequency of methylation in the promoter CpG island (34/61, 56%) and somewhat lower, in the intronic (17/48, 35%). A significant inverse correlation was found between the SEMA3B mRNA level and methylation of the promoter CpG island in RCC (P < 0.05 according to Fisher’s exact test). The intronic island showed no such correlation. Thus, we suggest that the methylation of the promoter CpG island contributes to the inactivation of the SEMA3B suppressor gene in RCC tissue.

Tumor-specific expression downregulation may be indicative of a gene’s involvement in tumor suppression. For instance, SEMA3B mRNA levels are decreased in many cell lines of small-cell and non-small cell lung cancer, and SEMA3B was shown to suppress the growth of the NSCLC cell line NCI-H1299 and tumor formation in immunodeficient mice. In this work, SEMA3B expression levels were determined in epithelial tumors of different localizations. In cell lines of renal, breast, and ovarian cancer, SEMA3B mRNA levels were frequently (4/11, 36%) decreased as much as 10–250-fold according to semiquantitative RT-PCR assay. SEMA3B expression levels were also determined in primary tumor extracts of kidney, lung, breast, ovarian, and colorectal cancer. In clear cell renal cell carcinoma, SEMA3B expression was decreased 5–1000-fold in 25 of 51 extracts (49%) compared to 5/51 (10%) extracts with increased mRNA levels; the result was highly significant: P < 0.0001 by Fisher’s exact test. SEMA3B was frequently downregulated in ovarian (5/16, 31% vs. 2/16, 12%) and colorectal cancer (6/11, 54% vs. 2/11, 18%). These results suggest that SEMA3B is involved in the suppression of kidney, ovarian, and colon tumor growth.

The short arm of chromosome 3 (3p) contains several critical regions that have increased frequencies of allelic deletions and harbor a set of tumor suppressor genes. In particular, the range of functions performed by RASSF1A (LUCA region, 3p21.31) includes those potentially associated with carcinogenesis. Among 3p genes, RASSF1A has the highest methylation frequency in epithelial tumors of various locations. For the first time, two different methods (methylation-specific PCR and methylation-sensitive restriction analysis) independently showed that the methylation level of the CpG island in the RASSF1A promoter region significantly correlated with grade and clinical stage of clear cell renal cell carcinoma (RCC). An analysis of 23 3p polymorphic markers in a representative set of 80 RCC cases characterized clinically and histologically revealed that RCC progression significantly correlated with the frequency of allelic imbalances in some critical regions of 3p (LUCA and AP20), but not in 3p as a whole. These data suggest that RCC progression is associated with the methylation of the RASSF1A promoter and, possibly, with structural and functional alterations in other 3p genes. In addition, significant correlation between RASSF1A methylation and allelic losses at the nearby polymorphic marker locus suggests the “two hit” model for the inactivation of this tumor suppressor gene in RCC.

The protein encoded by RAR-β2 (retinoic acid receptor) gene is a member of the superfamily, of nuclear receptors of retinoids which are involved in regulation of cell differentiation and proliferation. The level of RAR-β2 mRNA is downregulated in a number of cell lines derived from human epithelial tumors. Inactivation of the RAR-β2 gene is associated with methylation of its promoter region, which is observed in various carcinomas at a frequency of 30–70%. In renal and ovarian tumors, methylation at this region is poorly studied, the data being contradictory. We report a high methylation frequency in the gene promoter region in RCC (59%, 36/61) and a somewhat lower frequency in EOC (30%, 15/50). Methylation frequency in BC (46%, 26/56) is consistent with the published data. Significant correlation of methylation frequency in promoter region of RAR-β2 gene with RCC progression (P ≤ 0.005 by Fisher’s exact test) was established.

Allelic imbalances (AI) of polymorphic markers at the short arm of chromosome 3 (3p) were mapped using DNA samples of renal cell carcinoma (RCC, 80 cases), breast carcinoma (BC, 95 cases), and epithelial ovarian cancer (EOC, 50 cases) at the same dense panel of markers (up to 24 loci). Six regions with the increased AI frequency (versus the average values determined for all the analyzed 3p markers) at RCC, BC or EOC were found in 3p chromosome. Four 3p regions presumably contain tumor-suppressor genes (TSG) involved in the epithelial tumors of various types. Region between D3S2409 and D3S3667 markers in the 3p21.31 region was identified in this study for the first time. The AI peak in D3S2409-D3S3667 region was statistically significant (P < 0.001, according to Fisher) when representative sample set of 95 BC patients was analyzed. The data on increased frequency of polymorphic marker allele amplification suggest that the D3S2409-D3S3667 region contains both putative TSG and protooncogenes.

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.

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.

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).

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.