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.
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.
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.
—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 methylation 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.
Ovarian cancer (OC) develops asymptomatically and escapes diagnosis until advanced stages, the feature contributing to a higher mortality rate. New prospects of OC diagnosis and treatment have been ...opened in studies of the gene regulation mechanisms that involve long noncoding RNAs (lncRNAs) and identification of the lncRNA genes that are inhibited via methylation of the promoter region. A set of 122 samples of primary OC tumors was examined by methylation specific real-time PCR to assess the methylation level of the lncRNA genes
PLUT
,
SNHG1
,
SNHG6
,
SNHG12,
and
TINCR
. A significant increase in their methylation levels was observed in OC (
p
< 0.001 by the nonparametric Mann–Whitney test). The methylation levels of
SNHG6
,
SNHG12
, and
TINCR
were found to correlate significantly (
p
< 0.05) with the stage of the tumor process, the histological grade, and metastasis. Downregulation of
SNHG6
,
SNHG12
, and
TINCR
was detected by real-time RT–qPCR, and a significant correlation between methylation and expression was demonstrated for
SNHG6
and
TINCR
(
r
s
≤ –0.5,
p
< 0.001). The respective lncRNA genes were assumed to provide potential epigenetic markers of OC.
We identified a group of miRNA genes whose methylation is associated with ovarian cancer metastasis. Based on these data, new markers and the systems of markers predicting tumor dissemination were ...selected. Using methylation-specific PCR and a representative set of 54 ovarian cancer samples, we identified 10 microRNA genes (
MIR-124a-2
,
MIR-127
,
MIR-125b-1
,
MIR-129-2
,
MIR-137
,
MIR-193a
,
MIR-203a
,
MIR-34b/c
,
MIR-130b
, and
MIR-1258
) whose methylation is associated with tumor metastasis. The greatest association was established for 4 genes:
MIR-137
,
MIR-193a
,
MIR-34b/c
, and
MIR-130b
(
p
<0.01). ROC analysis revealed 3 most optimal marker systems including 4-5 miRNA genes and characterized by high sensitivity (82-94%) and specificity (76-86%) at AUC=0.89-0.92. Methylation of any three genes from these systems is sufficient to predict metastasis with the specified accuracy. Detection of the group of hypermethylated miRNA genes with predictive value for ovarian cancer metastasis is of great importance for personalized treatment of the patients.
MicroRNA and methylation are important epigenetic mechanisms in the pathogenesis of cancer. The role of a group of microRNA hypermethylated genes in the pathogenesis of ovarian cancer was studied and ...their diagnostic and prognostic potential was evaluated. Studies on a representative sample of 54 ovarian cancer specimens with the use of methyl-specific PCR resulted in detection of five microRNA genes (
MIR-9-1
,
MIR-9-3
,
MIR-107
,
MIR-1258
, and
MIR-130b
) methylated in the majority of tumor specimens in comparison with paired specimens of histologically intact tissue (37-57%
vs.
4-9%,
p
<0.01). Methylation of three genes (
MIR-9-1
,
MIR-9-3
, and
MIR-130b
) was significantly (
p
≤0.05) associated with the parameters of ovarian cancer progress (clinical stage, differentiation degree, tumor size, and presence of metastases). These findings attest to oncosuppressive role of the studied microRNA genes (
MIR-9-1
,
MIR-9-3
,
MIR-107
,
MIR-1258
, and
MIR-130b
) in the pathogenesis and progress of ovarian cancer and indicated their prognostic potential.
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.
Changes in the levels of expression of proapoptotic genes
APAF1
and
DAPK1
and antiapoptotic gene
BCL2
were studied by real time PCR in specimens of tumors and histologically intact tissue from 28 ...patients with breast cancer. The expression of
APAF1
and
DAPK1
was below the normal in the majority of tumor samples (
p
<0.05), while the level of
BCL2
mRNA more often surpassed the normal (
p
<0.1). Study of the same sample of specimens by methylspecific PCR showed predominance of
APAF1
and
DAPK1
hypermethylation (
p
<0.05 and
p
<0.1, respectively) and more frequent hypomethylation of
BCL2
. A significant correlation between changes in the levels of expression and methylation (
r
=0.40-0.49;
p
<0.05) was detected for all three genes (
APAF1
,
DAPK1
, and
BCL2
). The results suggest that methylation play an important role in the regulation of these apoptosis system genes in breast cancer
