Epigenetic analysis in placentas from sickle cell disease patients reveals a hypermethylation profile.

Pregnancy in Sickle Cell Disease (SCD) women is associated to increased risk of clinical and obstetrical complications. Placentas from SCD pregnancies can present increased abnormal findings, which may lead to placental insufficiency, favoring adverse perinatal outcome. These placental abnormalities are well known and reported, however little is known about the molecular mechanisms, such as epigenetics. Thus, our aim was to evaluate the DNA methylation profile in placentas from women with SCD (HbSS and HbSC genotypes), compared to uncomplicated controls (HbAA). We included in this study 11 pregnant women with HbSS, 11 with HbSC and 21 with HbAA genotypes. Illumina Methylation EPIC BeadChip was used to assess the whole placental DNA methylation. Pyrosequencing was used for array data validation and qRT-PCR was applied for gene expression analysis. Our results showed high frequency of hypermethylated CpGs sites in HbSS and HbSC groups with 73.5% and 76.2% respectively, when compared with the control group. Differentially methylated regions (DMRs) also showed an increased hypermethylation status for the HbSS (89%) and HbSC (86%) groups, when compared with the control group methylation data. DMRs were selected for methylation validation (4 DMRs-HbSS and 3 DMRs the HbSC groups) and after analyses three were validated in the HbSS group, and none in the HbSC group. The gene expression analysis showed differential expression for the PTGFR (-2.97-fold) and GPR56 (3.0-fold) genes in the HbSS group, and for the SPOCK1 (-2.40-fold) and ADCY4 (1.80-fold) genes in the HbSC group. Taken together, these data strongly suggest that SCD (HbSS and HbSC genotypes) can alter placental DNA methylation and lead to gene expression changes. These changes possibly contribute to abnormal placental development and could impact in the clinical course, especially for the fetus, possibly leading to increased risk of abortion, fetal growth restriction (FGR), stillbirth, small for gestational age newborns and prematurity.

DNA Methylation Markers from Negative Surgical Margins Can Predict Recurrence of Oral Squamous Cell Carcinoma.

The identification of molecular markers in negative surgical margins of oral squamous cell carcinoma (OSCC) might help in identifying residual molecular aberrations, and potentially improve the prediction of prognosis. We performed an Infinium MethylationEPIC BeadChip array on 32 negative surgical margins stratified based on the status of tumor recurrence in order to identify recurrence-specific aberrant DNA methylation (DNAme) markers. We identified 2512 recurrence-associated Differentially Methylated Positions (DMPs) and 392 Differentially Methylated Regions (DMRs) which were enriched in cell signaling and cancer-related pathways. A set of 14-CpG markers was able to discriminate recurrent and non-recurrent cases with high specificity and sensitivity rates (AUC 0.98, p = 3 × 10-6; CI: 0.95-1). A risk score based on the 14-CpG marker panel was applied, with cases classified within higher risk scores exhibiting poorer survival. The results were replicated using tumor-adjacent normal HNSCC samples from The Cancer Genome Atlas (TCGA). We identified residual DNAme aberrations in the negative surgical margins of OSCC patients, which could be informative for patient management by improving therapeutic intervention. This study proposes a novel DNAme-based 14-CpG marker panel as a promising predictor for tumor recurrence, which might contribute to improved decision-making for the personalized treatment of OSCC cases.

Association of DNA sequence-independent genetic regulatory mechanisms with apical periodontitis: A scoping review.

OBJECTIVE: Different studies in the last decade have proposed that gene expression alterations that are independent of the DNA sequence may also play an important role in periapical disease. The present study aimed to assess the available evidence supporting a relationship between these alterations and apical periodontitis through a scoping review. DESIGN: Specific strategies were developed for different databases (MEDLINE via PubMed, Cochrane Library, Scopus, Web of Science, and Virtual Health Library) and a search performed by March 1st, 2019. The evidence sources were selected according to the eligibility criteria and underwent a critical appraisal of methodological quality. RESULTS: The initial search retrieved 212 references, with eight eligible articles after the removal of replicates and application of exclusion criteria. Five studies identified altered DNA methylation on inflammatory response genes (FOXP3, CXCL3, FADD, MMP2, MMP9, IFNG, IL4, IL12) on AP patients. Three others identified the alterations on the expression of several microRNAs (miR-29b, 106b, 125b, 143, 146a, 155, 198) during AP. No evidence was identified regarding mechanisms of histone methylation, or of epigenetic heritability or stability. CONCLUSIONS: There is available evidence for the involvement of different genetic regulatory mechanisms independent of changes in DNA sequence in the development or severity of apical periodontitis. However, due to methodological limitations, further research must be performed before novel therapies and diagnostic tools for AP may arise from these data.

Esophageal squamous cell carcinoma transcriptome reveals the effect of FOXM1 on patient outcome through novel PIK3R3 mediated activation of PI3K signaling pathway.

Esophageal squamous cell carcinoma (ESCC) presents poor prognosis, and patients diagnosed with this tumor currently lack target treatments. Therefore, in order to identify potential targets for ESCC treatment, we carried out a transcriptome analysis with ESCC and paired nonmalignant surrounding mucosa samples, followed by a master regulator analysis, and further explored the role of the identified central regulatory genes through in vivo and in vitro assays. Among the transcription factors deregulated/enriched in ESCC, we focused on FOXM1 because of its involvement in the regulation of critical biological processes. A new transcriptome analysis performed with ESCC cell lineage TE-1 showed that the modulation of FOXM1 expression resulted in PIK3R3 expression changes, whereas chromatin immunoprecipitation assay revealed that FOXM1 was capable of binding onto PIK3R3 promoter, thus demonstrating that PIK3R3 is a new FOXM1 target. Furthermore, FOXM1 overexpression resulted in the activation of PIK3/AKT signaling pathway through PIK3R3-mediated AKT phosphorylation. Finally, the analysis of the clinic-pathological data of ESCC patients revealed that overexpression of both FOXM1 and PIK3R3 was associated with poor prognosis, but only the latter was an independent prognosis factor for ESCC patients. In conclusion, our results show that FOXM1 seems to play a central role in ESCC carcinogenesis by upregulating many oncogenes found overexpressed in this tumor. Furthermore, PIK3R3 is a novel FOXM1 target that triggers the activation of the PI3K/AKT pathway in ESCC cells.

Intrinsic LINE-1 Hypomethylation and Decreased Brca1 Expression are Associated with DNA Repair Delay in Irradiated Thyroid Cells.

Exposure to ionizing radiation greatly increases the risk of developing papillary thyroid carcinoma (PTC), especially during childhood, mainly due to gradual inactivation of DNA repair genes and DNA damages. Recent molecular characterization of PTC revealed DNA methylation deregulation of several promoters of DNA repair genes. Thus, epigenetic silencing might be a plausible mechanism for the activity loss of tumor suppressor genes in radiation-induced thyroid tumors. Herein, we investigated the impact of ionizing radiation on global methylation and CpG islands within promoter regions of homologous recombination (HR) and non-homologous end joining (NHEJ) genes, as well as its effects on gene expression, using two well-established normal differentiated thyroid cell lines (FRTL5 and PCCL3). Our data reveal that X-ray exposure promoted G2/M arrest in normal thyroid cell lines. The FRTL5 cells displayed a slower kinetics of double-strand breaks (DSB) repair and a lower long interspersed nuclear element-1 (LINE-1) methylation than the PCCL3 cells. Nevertheless, acute X-ray exposure does not alter the expression of genes involved in HR and NHEJ pathways, apart from the downregulation of Brca1 in thyroid cells. On the other hand, HR and NHEJ gene expressions were upregulated in radiation-induced senescent thyroid cells. Taken together, these data suggest that FRTL5 cells intrinsically have less efficient DNA DSB repair machinery than PCCL3 cells, as well as genomic instability, which could predispose the FRTL5 cells to unrepaired DSB lesions and, therefore, gene mutations.

Alterações epigenéticas e suas consequências funcionais em carcinoma epidermóide de esôfago

O carcinoma epidermóide de esôfago (CEE) é um dos dez tipos de tumores mais frequentes no mundo e apresenta baixa sobrevida, consequência principalmente de um diagnóstico tardio e da falta de um tratamento eficaz. Atualmente, um grande enfoque tem sido dado às alterações epigenéticas como potenciais biomarcadores em câncer. Entretanto, para CEE, muito pouco é conhecido em termos de tais alterações Desta forma, nosso estudo procurou estabelecer uma assinatura de metilação do DNA para CEE e investigar as consequências funcionais de tais alterações. Para isso, foram incluídos no estudo 10 pacientes com CEE (tumor e tecido normal adjacente) e 4 indivíduos saudáveis que doaram uma biópsia de tecido esofágico. Tais amostras foram submetidas à análise pela plataforma GoldenGate(Illumina) que investiga o perfil de metilação do promotor de cerca de 800 genes relacionados a câncer. A comparação de classes pareada identificou 37 sítios CpG diferencialmente metilados, correspondentes a 34 genes, entre tumor e tecido normal adjacente. Com base nestes genes, o componente inflamatório e a adesão celular foram apontados como as principais vias celulares alteradas. A análise dos tumores e tecido normal adjacente em comparação ao tecido esofágico de indivíduos saudáveis nos mostrou ainda que a hipermetilação de TFF1 e a hipometilação de BCL3 já estavam presentes na mucosa adjacente, o que pode representar alterações precoces. Para validar os resultados obtidos com a plataforma GoldenGate, avaliamos por pirosequenciamento o perfil de metilação de três genes encontrados hipermetilados nos tumores, dois genes hipometilados, dois genes para os quais não foram encontradas alterações e do gene TFF1 em uma nova série de amostras. Nesta mesma série, avaliamos ainda o perfil de metilação dos elementos repetitivos LINE1 por pirosequenciamento, o que nos mostrou que os tumores apresentam uma hipometilação global em comparação ao tecido não tumoral...

Esophageal cancer (EC) is one of the ten most incidenttumors worldwide. The mortality rates for this type of tumor are very similar to the incidence rates, which is a direct consequence of a late diagnosis and a poor treatment. Esophageal squamous cell carcinoma (ESCC) is believed to arise from esophageal mucosa through accumulation of both genetic and epigenetic changes. Alterations on DNA methylation have been pointed as early changes during tumor development. The aim of this study was to examine the global deregulation of methylation states in ESCC and identify DNA methylation signatures associated with different stages of the disease. We performed a bead array analysis of more than 800 cancer-related genes in a series of 10 ESCC samples, 10 matched surrounding tissues, and 4 healthy esophageal mucosa. The comparison between tumors and matched surrounding tissue revealed 37 differentially methylated CpG sites, corresponding to 34 genes, and these CpG sites were significantly enriched in genes related to several pathways including the cell communication pathway and the inflammatory component. In addition, by comparing the samples from ESCC patients with healthy esophageal mucosa, we identified TFF1 hypermethylation and BCL3hypomethylation as potential early markers of ESCC. Pyrosequencing was used for validation of DNA methylation changes as well as for the assessment of global methylation by LINE1 assay. This analysis revealed a global hypomethylation in esophageal tumors, what is accordance with previous reports for other cancer types. We also investigated the functional consequences of BCL3 hypomethylation, a gene supposed to be involved in cell proliferation and apoptosis control. Very interestingly, this gene possesses three CpG islands and it is induced by IL6 (a gene found hypomethylated in our tumor samples)...

Alterações epigenéticas e suas consequências funcionais em carcinoma epidermóide de esôfago / [Epigenetic changes and their functional consequences in esophageal squamous cell carcinoma]

O carcinoma epidermóide de esôfago (CEE) é um dos dez tipos de tumoresmais frequentes no mundo e apresenta baixa sobrevida, consequênciaprincipalmente de um diagnóstico tardio e da falta de um tratamento eficaz.Atualmente, um grande enfoque tem sido dado às alterações epigenéticas comopotenciais biomarcadores em câncer. Entretanto, para CEE, muito pouco éconhecido em termos de tais alterações Desta forma, nosso estudo procurouestabelecer uma assinatura de metilação do DNA para CEE e investigar asconsequências funcionais de tais alterações. Para isso, foram incluídos no estudo 10 pacientes com CEE (tumor e tecido normal adjacente) e 4 indivíduos saudáveis que doaram uma biópsia de tecido esofágico. Tais amostras foram submetidas à análise pela plataforma GoldenGate (Illumina) que investiga o perfil de metilação do promotor de cerca de 800 genes relacionados a câncer. A comparação de classes pareada identificou 37 sítios CpG diferencialmente metilados, correspondentes a 34 genes, entre tumor e tecido normal adjacente. Com base nestes genes, o componente inflamatório e a adesão celular foram apontados como as principais vias celulares alteradas. A análise dos tumores e tecido normal adjacente em comparação ao tecido esofágico de indivíduos saudáveis nos mostrou ainda que a hipermetilação de TFF1 e a hipometilação de BCL3 já estavam presentes na mucosa adjacente, o que pode representar alterações precoces. Para validar os resultados obtidos com a plataforma GoldenGate, avaliamos por pirosequenciamento o perfil de metilação de três genes encontrados hipermetilados nos tumores, dois genes hipometilados, dois genes para os quais não foram encontradas alterações e do gene TFF1 em uma nova série de amostras...

Esophageal cancer (EC) is one of the ten most incident tumors worldwide. Themortality rates for this type of tumor are very similar to the incidence rates, which is a direct consequence of a late diagnosis and a poor treatment. Esophageal squamous cell carcinoma (ESCC) is believed to arise from esophageal mucosa throughaccumulation of both genetic and epigenetic changes. Alterations on DNAmethylation have been pointed as early changes during tumor development. The aimof this study was to examine the global deregulation of methylation states in ESCC and identify DNA methylation signatures associated with different stages of the disease. We performed a bead array analysis of more than 800 cancer-related genes in a series of 10 ESCC samples, 10 matched surrounding tissues, and 4 healthy esophageal mucosa. The comparison between tumors and matched surrounding tissue revealed 37 differentially methylated CpG sites, corresponding to 34 genes,and these CpG sites were significantly enriched in genes related to several pathways including the cell communication pathway and the inflammatory component. Inaddition, by comparing the samples from ESCC patients with healthy esophagealmucosa, we identified TFF1 hypermethylation and BCL3 hypomethylation as potential early markers of ESCC. Pyrosequencing was used for validation of DNAmethylation changes as well as for the assessment of global methylation by LINE1 assay. This analysis revealed a global hypomethylation in esophageal tumors, what is accordance with previous reports for other cancer types...