[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.

[The hyper-methylated genes microRNA as potential markers of clear-cell carcinoma of kidney].

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 profile of group of miRNA genes in clear cell renal cell carcinoma; involvement in cancer progression].

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).

[Methylation of some miRNA genes is involved in the regulation of their target genes RAR-beta2 and NKIRAS1 expression in lung cancer].

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.