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.
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 analyzed with using a representative sample (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 was changed from 37% to 65% in tumor samples and significantly higher in tumor samples than in samples of histologically normal tissue (
P
≤ 3 × 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).
Association of
TP53
Arg72Pro and
MDM2
T309G polymorphic markers with the risk of non-small-cell lung cancer was studied in a Russian population of the Moscow region. The minor Pro/Pro genotype of ...Arg72Pro and the TG genotype of T309G were associated with non-small-cell lung cancer (OR = 5.46,
p
= 8 × 10
−6
and OR = 7.38,
p
= 0.0001, respectively). Both Pro/Pro and TG genotypes were also strongly associated with lung adenocarcinoma (OR = 8.71,
p
= 3 × 10
−6
and OR = 8.13,
p
= 0.003, respectively) and squamous-cell lung carcinoma (OR = 4.2,
p
= 0.001 and OR = 7.02,
p
= 0.002, respectively). Finally, combined susceptible genotypes of
TP53
and
MDM2
polymorphisms Arg72Pro and T309G were reliably associated with non-small-cell lung cancer and both its subtypes (OR = 7.9,
p
= 0.01; OR = 9.12,
p
= 0.02; OR = 7.31,
p
= 0.03, respectively).
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.
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 (
,
,
,
,
,
,
,
,
,
,
,
, and
) were hypermethylated already at the early stages of OvCa, while hypermethylation of
,
,
, and
was pronounced in metastatic tumors, and
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
and
genes, which are EMT drivers. We also identified three miRNA genes (
,
, and
) 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
and
were related to the greatest relative risk of death.
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.
•Hypermethylation of MIR-125b-1, -127, -132, -193a, and -34b was revealed in BC•Hypomethylation of MIR-191 was shown in primary breast tumors•MIR-124-1, 125b-1, 127, 132, 193a, 34b methylation correlated with down-regulation•Levels of miR-127-5p, -124-3p, -375 and DAPK1, BCL2, RASSF1(A) correlated negatively•Hypermethylation of MIR-127 and -125b-1 was associated with breast cancer progression
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.
•Eight novel hypermethylated miRNA genes were revealed in epithelial ovarian cancer.•Hypomethylation of MIR-191 was shown in primary epithelial ovarian tumors.•Hypermethylation correlated with downregulation for MIR-125B-1/-129-2/-132/-137/-193A.•Hypermethylation of 10 miRNA genes was associated with EOC metastasis.•Anti-metastatic properties were observed for non-suppressor miR-203 and miR-375.
The diagnostic significance of molecular markers was assessed for the most common somatic aberrations at the
K-ras, TP53, CDKN2A
, and
MADH4
loci, as well as less common mutations of
BRCA1, BRCA2
, ...and
CHEK2
, arising in preinvasive stages of sporadic adenocarcinoma of the pancreas. The study was performed on paired primary pancreatic adenocarcinoma and normal pancreatic tissue specimens obtained from 37 Russian patients. Surgical adenocarcinoma specimens were subjected to manual microdissection. Mutations of
K-ras
codon 12 were found in 24 tumor specimens (0.65), but not in normal pancreatic tissue specimens. Mutations of
BRCA1
(185delAG, 300T > G, 4153delA, 4158A > G, 5382insC),
BRCA2
(695insT, 6174delT), and
CHEK2
(1100delC) were not found. The informativeness of allelic losses did not differ significantly among the three tumor suppressor loci and was 60% for
TP53
(
GDB186817
) and
CDKN2A
(
D9S974
+
D9S162
) and 65.7% for
MADH4
(
D18S363
+
D18S474
) (
t
= 0.48). The
CDKN2A
locus had the highest LOH frequency of 0.95. For
TP53
and
MADH4
the LOH frequency was 0.62 and 0.70, respectively. In 80% of adenocarcinomas, at least one locus was characterized with LOH. The overall informativeness of the combined data on
K-ras
mutations and loss of heterozygosity at 9p, 17p, and 18q was 85.7%. Only 9% of the tumors were characterized with microsatellite instability.
Somatic mutations in the
KRAS
gene are important markers of some types of tumors, for example, pancreatic cancer, and may be useful in early diagnostics. A biochip has been developed which allows ...deter-mining most frequent mutations in 12, 13, and 61 codons of the
KRAS
gene. To increase the sensitivity of the method and to enable the analysis of minor fractions of tumor cells in clinical samples, the method of blocking wild type sequence PCR amplification by LNA-oligonucleotides has been used. The product of LNA-clamp PCR was further hybridized with oligonucleotide probes, immobilized on the biochip. The biochip was tested with 42 clinical DNA samples from patients with pancreatic cancer, mostly duct adenocarcinomas. As reference methods, RFLP analysis and sequencing were used. The developed approach allows detecting somatic mutations in the
KRAS
gene if the portion of tumor cells with mutation is at least 1% of the whole cell population.
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.
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