RESUMO
Hepatocellular carcinoma (HCC) development is associated with altered modifications in DNA methylation, changing transcriptional regulation. Emerging evidence indicates that DNA methyltransferase 1 (DNMT1) plays a key role in the carcinogenesis process. This study aimed to investigate how pirfenidone (PFD) modifies this pathway and the effect generated by the association between c-Myc expression and DNMT1 activation. Rats F344 were used for HCC development using 50 mg/kg of diethylnitrosamine (DEN) and 25 mg/kg of 2-Acetylaminofluorene (2-AAF). The HCC/PFD group received simultaneous doses of 300 mg/kg of PFD. All treatments lasted 12 weeks. On the other hand, HepG2 cells were used to evaluate the effects of PFD in restoring DNA methylation in the presence of the inhibitor 5-Aza. Histopathological, biochemical, immunohistochemical, and western blot analysis were carried out and our findings showed that PFD treatment reduced the amount and size of tumors along with decreased Glipican-3, ß-catenin, and c-Myc expression in nuclear fractions. Also, this treatment improved lipid metabolism by modulating PPARγ and SREBP1 signaling. Interestingly, PFD augmented DNMT1 and DNMT3a protein expression, which restores global methylation, both in our in vivo and in vitro models. In conclusion, our results suggest that PFD could slow down HCC development by controlling DNA methylation.
Assuntos
Carcinoma Hepatocelular , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , Antígeno Nuclear de Célula em Proliferação , Piridonas , Animais , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Piridonas/farmacologia , Ratos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Humanos , Células Hep G2 , Antígeno Nuclear de Célula em Proliferação/metabolismo , Masculino , Ratos Endogâmicos F344 , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Dietilnitrosamina , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/genéticaRESUMO
BACKGROUND: Acute leukemia involving lymphocytic and myeloid cells is cancer with a high mortality rate. Swift and timely diagnosis might be a potential approach to improving patient prognosis and survival. The microRNA (miRNA) signatures are emerging nowadays for their promising diagnostic potential. MiRNA levels from bone marrow can be used as prognostic biomarkers. METHODS: The current study was designed to evaluate if the microRNAs and tumor suppressor genes (TSGs) profiling of hematopoietic bone marrow could help in acute leukemia early detection. Also, we assessed the DNA methyltransferase 3A (DNMT3A) expression and its possible epigenetic effects on miRNAs plus TSGs expression levels. The expression levels of ten miRNAs and four TSGs involved in acute lymphocytic leukemia (ALL) as well as acute myeloid leukemia (AML) were quantified in 43 and 40 bone marrow samples of ALL and AML patients in comparison with cancer-free subjects via real-time quantitative PCR (RT-qPCR). The receiver-operating-characteristic (ROC) analysis of miRNAs was performed in the study groups. Further, the correlation between the DNMT3A and TSGs was calculated. RESULTS: Significant differences were detected in the bone marrow expression of miRNAs and TSGs (P < 0.05) between acute leukemia patients and healthy group. ROC analysis confirmed the ability of miR-30a, miR-101, miR-132, miR-129, miR-124, and miR-143 to discriminate both ALL and AML patients with an area under the ROC curve of ≥ 0.80 (P < 0.001) and high accuracy. The correlation between DNMT3A and P15/P16 TSGs revealed that DNMT3A plays a vital role in epigenetic control of TSGs expression. Our findings indicated that the downregulation of bone marrow miRNAs and TSGs was accompanied by acute leukemia development. CONCLUSIONS: The authors conclude that this study could contribute to introducing useful biomarkers for acute leukemia diagnosis.
Assuntos
Leucemia Mieloide Aguda , MicroRNAs , Leucemia-Linfoma Linfoblástico de Células Precursoras , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Medula Óssea/patologia , Detecção Precoce de Câncer , Genes Supressores de Tumor , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , MicroRNAs/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , PrognósticoRESUMO
RATIONALE: Cannabis sativa is the most widely used drug by adolescents globally. The recreational use of synthetic cannabinoids by teenagers has also grown in recent years. Despite the wrong perception that exposure to these drugs does not cause harm, repeated exposure to cannabinoids at early stages of life compromises important maturation processes and brain development. Chronic early cannabinoid use has been related to a higher risk of psychiatric outcomes, including cocaine addiction. Evidence suggests that exposure to natural and synthetic cannabinoids during adolescence modifies molecular and behavioral effects of cocaine in adulthood. Responses to cocaine are regulated by epigenetic mechanisms, such as DNA methylation, in the brain's reward regions. However, the involvement of these processes in modulation of the vulnerability to the effects of cocaine induced by prior exposure to cannabinoids remains poorly understood. OBJECTIVES: Investigate whether exposure to the synthetic cannabinoid WIN55,212-2 during adolescence modulates anxiety- and depression-like behavior, memory, and cocaine reward in adult mice. We also evaluated whether exposure to cannabinoids during adolescence modulates the expression of enzymes that are involved in DNA methylation. RESULTS: Exposure to WIN55,212-2 during adolescence did not alter anxiety- or depressive-like behavior. However, prior exposure to cannabinoids inhibited cocaine-induced conditioned place preference without modulating cocaine-induced hyperlocomotion, accompanied by an increase in expression of the enzyme DNA methyltransferase 3a (DNMT3a) in the prefrontal cortex. CONCLUSIONS: Our findings suggest that exposure to WIN55,212-2 during adolescence leads to changes in DNMT3a expression, and this pathway appears to be relevant to modulating the rewarding effects of cocaine.
Assuntos
Canabinoides , Cocaína , Animais , Canabinoides/farmacologia , Cocaína/farmacologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Camundongos , Córtex Pré-Frontal/metabolismo , RecompensaRESUMO
PURPOSE: Altered miRNAs play a crucial role in the emergence of the breast cancer stem cell (BCSC) phenotype. The interplay between miRNAs and methylation enzymes has been documented. One of the most aggressive breast cancer cell lines, MDA-MB-231, has expressed much more DNMT3B than DNMT3A. This study aims to evaluate the ability of miR-203 restoration and miR-150 inhibition to regulate DNMT3B and DNMT3A to modify the methylation level of BCSC-associated genes. METHODS: MDA-MB-231 cells were transfected with miR-203 mimic or miR-150 inhibitor or DNMT3B siRNA, and downstream analysis was performed by flow cytometry, real-time PCR and Western blotting. RESULTS: DNMT3A and DNMT3B are regulated both by miR-203a-3p and miR-150-5p. Transfection with miR-203 mimic and miR-150 inhibitor significantly reduced the CD44+CD24- subpopulation and down-regulated the expression of CD44 mRNA by increasing promoter methylation levels. SiRNA knockdown of DNMT3B increased the CD44+CD24- subpopulation and the expression of CD44 and ALDH1A3 by decreasing methylation density. The inhibition of miR-150 down-regulated OCT3/4 and SOX2 expression without affecting methylation levels, while miR-203 restoration and miR-150 inhibition down-regulated NANOG expression by elevating the methylation level. A positive-feedback loop was found between miR-203 and its target DNMT3B, as restoring miR-203 suppressed DNMT3B, while knocking down DNMT3B up-regulated miR-203. The restoration of miR-203 and knockdown of DNMT3B decreased methylation levels and increased the expression of miR-141 and miR-200c. CONCLUSIONS: The study concluded that miR-203 and miR-150 play a role in the regulation of genes involved in BCSC methylation, including other miRNAs, by targeting DNMT3B and DNMT3A.
Assuntos
Neoplasias da Mama/genética , DNA (Citosina-5-)-Metiltransferases/fisiologia , DNA Metiltransferase 3A/fisiologia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Feminino , Humanos , Metilação , Células-Tronco Neoplásicas , Células Tumorais Cultivadas , DNA Metiltransferase 3BRESUMO
OBJECTIVE: To present a clinical case of a patient with Tatton-Brown-Rahman syndrome, to provide evidence of the importance of supplying patients with appropriate dental care, emphasizing in behavioral management. STUDY DESIGN: Clinical case report. RESULTS: This 7-year-old child, had a history of persistent ductus arteriousus, autism spectrum behavior, language disorders, dyslalias, hearing disorder, hypotonic musculature, and joint hyperlaxity. The main facial and oral diagnoses were dolichocephaly, convex profile, atypical swallowing, mixed breathing, class II malocclusion, mandibular retrognathism, maxillary prognathism, caries lesions, and biofilm associated gingivitis. A comprehensive treatment was carried out from the stage of adaptation to dental care, control of dental biofilm, motivation, and teaching of oral hygiene with appropriate strategies to the child's age and cognitive abilities. Also, resin restorations, habits management and malocclusion with the use of the modified upper and lower Sanders orthopedic device. The child began with a definitely negative behavior at dental appointments, and evolved to negative on Frankl's Behavior Rating Scale. CONCLUSION: Dentists must manage behavior management protocols, in order to avoid situations of rejection of treatment in patients with TBRS, and thus avoid sedation or general anesthesia. Prevention is the priority for these patients supported by recreational-educational strategies.
Assuntos
Anormalidades Múltiplas , Cárie Dentária , Deficiência Intelectual , Criança , DNA (Citosina-5-)-Metiltransferases , Assistência Odontológica , Face , HumanosAssuntos
DNA (Citosina-5-)-Metiltransferases , Decitabina/farmacologia , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Proteínas de Neoplasias , Animais , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Transgênicos , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/enzimologia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMO
We aimed to investigate the effects of aging and different exercise modalities on aversive memory and epigenetic landscapes at brain-derived neurotrophic factor, cFos, and DNA methyltransferase 3 alpha (Bdnf, cFos, and Dnmt3a, respectively) gene promoters in hippocampus of rats. Specifically, active epigenetic histone markers (H3K9ac, H3K4me3, and H4K8ac) and a repressive mark (H3K9me2) were evaluated. Adult and aged male Wistar rats (2 and 22 months old) were subjected to aerobic, acrobatic, resistance, or combined exercise modalities for 20 min, 3 times a week, during 12 weeks. Aging per se altered histone modifications at the promoters of Bdnf, cFos, and Dnmt3a. All exercise modalities improved both survival rate and aversive memory performance in aged animals (n = 7-10). Exercise altered hippocampal epigenetic marks in an age- and modality-dependent manner (n = 4-5). Aerobic and resistance modalities attenuated age-induced effects on hippocampal Bdnf promoter H3K4me3. Besides, exercise modalities which improved memory performance in aged rats were able to modify H3K9ac or H3K4me3 at the cFos promoter, which could increase gene transcription. Our results highlight biological mechanisms which support the efficacy of all tested exercise modalities attenuating memory deficits induced by aging.
Assuntos
Envelhecimento/fisiologia , Aprendizagem da Esquiva , Epigênese Genética , Hipocampo/metabolismo , Memória , Condicionamento Físico Animal , Acetilação , Animais , Cromatina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Masculino , Metilação , Regiões Promotoras Genéticas/genética , Ratos Wistar , Taxa de SobrevidaRESUMO
BACKGROUND: Several genetic and epigenetic alterations are related to the development and progression of Gastric Cancer (GC), one of those being the deregulated microRNA (miRNA) expression profile. miRNAs are small noncoding RNAs that negatively regulate the expression of thousands of genes, including oncogenes and tumor suppressor genes. Our group identified, in previous studies, some miRNAs that are differentially expressed in GC when compared to the gastric mucosa without cancer, including hsa-miR-29c and hsa-miR-135b. The aim of the study was to modulate the expression of the miRNAs hsa-miR-29c-5p and hsa-miR-135b-5p and evaluate the expression of their target genes in 2D and 3D cell cultures. METHODS: hsa-miR-29c-5p and hsa-miR-135b-5p expression profiles were modulated by transfecting mimics and antimiRs, respectively, in 2D and 3D cell cultures. The expression of the proteins coded by the genes CDC42, DNMT3A (target genes of hsa-miR-29c-5p) and APC (target gene of hsa-miR-135b-5p) were measured by Western Blot. RESULTS: Results showed that mimics and antimiRs transfection significantly altered the expression of both miRNAs, increasing the expression of hsa-miR-29c-5p and reducing the expression of hsa-miR-135b-5p, especially in the 3D culture of the cell lines. When analyzing the proteins expression, we observed that AGP01 and AGP03 cell lines transfected with mimics had a reduction in the levels of CDC42 and DNMT3A and all three cell lines transfected with antimiRs had an increase in the expression of the protein APC. CONCLUSION: We concluded that three-dimensional culture can be a more representative in vitro model that resembles better the in vivo reality. Our results also showed that hsa-miR-29c-5p is an important regulator of CDC42 and DNMT3A genes in the intestinal subtype gastric cancer and hsa-miR-135b-5p regulates the APC gene in both intestinal and diffuse subtypes of GC. Dysregulation in their expression, and consequently in their respectively signaling pathways, shows how these miRNAs can influence the carcinogenesis of different histological subtypes of gastric cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , Genes APC , MicroRNAs/genética , Interferência de RNA , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Linhagem Celular Tumoral , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Humanos , Modelos Biológicos , Gradação de Tumores , Estadiamento de Neoplasias , TranscriptomaRESUMO
Acute myeloid leukemia is characterized by its high biological and clinical heterogeneity, which represents an important barrier for a precise disease classification and accurate therapy. While epigenetic aberrations play a pivotal role in acute myeloid leukemia pathophysiology, molecular signatures such as change in the DNA methylation patterns and genetic mutations in enzymes needed to the methylation process can also be helpful for classifying acute myeloid leukemia. Our study aims to unveil the relevance of DNMT3A and TET2 genes in global and specific methylation patterns in acute myeloid leukemia. Peripheral blood samples from 110 untreated patients with acute myeloid leukemia and 15 healthy control individuals were collected. Global 5-methylcytosine and 5-hydroxymethylcytosine in genomic DNA from peripheral blood leukocytes were measured by using the MethylFlashTM Quantification kits. DNMT3A and TET2 expression levels were evaluated by real-time quantitative polymerase chain reaction. The R882A hotspot of DNMT3A and exons 6-10 of TET2 were amplified by polymerase chain reaction and sequenced using the Sanger method. Methylation patterns of 16 gene promoters were evaluated by pyrosequencing after treating DNA with sodium bisulfite, and their transcriptional products were measured by real-time quantitative polymerase chain reaction.Here, we demonstrate altered levels of 5-methylcytosine and 5-hydroxymethylcytosine and highly variable transcript levels of DNMT3A and TET2 in peripheral blood leukocytes from acute myeloid leukemia patients. We found a mutation prevalence of 2.7% for DNMT3A and 11.8% for TET2 in the Mexican population with this disease. The average overall survival of acute myeloid leukemia patients with DNMT3A mutations was only 4 months. In addition, we showed that mutations in DNMT3A and TET2 may cause irregular DNA methylation patterns and transcriptional expression levels in 16 genes known to be involved in acute myeloid leukemia pathogenesis. Our findings suggest that alterations in DNMT3A and TET2 may be associated with acute myeloid leukemia prognosis. Furthermore, alterations in these enzymes affect normal methylation patterns in acute myeloid leukemia- specific genes, which in turn, may influence patient survival.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogênicas/genética , DNA Metiltransferase 3A , Análise Mutacional de DNA , Dioxigenases , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The use of light emitting diodes (LED) as a therapeutic resource for wound healing has increased over the last years; however, little is still known about the molecular pathways associated to LED exposure. In the present study, we verified the effects of LED therapy on DNA methylation and expression of the DNA methyltransferase (Dnmt) genes, Dnmt1 and Dnmt3a, in an in vivo model of epithelial wound healing. Male Wistar rats were submitted to epithelial excision in the dorsal region and subsequently distributed within the experimental groups: group 1, animals that received irradiation of 0.8 J/cm(2) of LED (604 nm); group 2, animals that received 1.6 J/cm(2) of LED (604 nm); control (CTL), animals not submitted to therapeutic intervention. LED applications were performed during 7 days, and tissues from the periphery of the wound area were obtained for molecular analysis. The Image-J software was used for analysis of the wound area. DNA methylation was evaluated by ELISA-based method and gene expressions were quantified by real-time PCR. Decrease on global DNA methylation profile was observed in all experimental groups (CTL, 1, and 2) revealing the participation of DNA methylation in the healing process. Significant decrease in the wound area accompanied by increase in the Dnmt3a expression was associated to group 2. Based on our findings, we propose that DNA methylation is an important molecular mechanism associated to wound healing and that irradiation with 1.6 J/cm(2) of LED evokes an increase in the expression of the Dnmt3a that might associates to the efficiency of the epithelial wound healing.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos da radiação , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Terapia a Laser , Pele/patologia , Cicatrização/genética , Cicatrização/efeitos da radiação , Animais , DNA (Citosina-5-)-Metiltransferase 1 , DNA Metiltransferase 3A , Modelos Animais de Doenças , Masculino , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Pele/efeitos da radiaçãoRESUMO
Methylation of CpG islands in promoter gene regions is frequently observed in lymphomas. DNA methylation is established by DNA methyltransferases (DNMTs). DNMT1 maintains methylation patterns, while DNMT3A and DNMT3B are critical for de novo DNA methylation. Little is known about the expression of DNMTs in lymphomas. DNMT3A and 3B genes can be regulated post-transcriptionally by miR-29 family. Here, we demonstrated for the first time the overexpression of DNMT1 and DNMT3B in Burkitt lymphoma (BL) tumor samples (69% and 86%, respectively). Specifically, the treatment of two BL cell lines with the DNMT inhibitor 5-aza-dC decreased DNMT1 and DNMT3B protein levels and inhibited cell growth. Additionally, miR-29a, miR-29b and miR-29c levels were significantly decreased in the BL tumor samples. Besides, the ectopic expression of miR-29a, miR-29b and miR-29c reduced the DNMT3B expression and miR-29a and miR-29b lead to increase of p16(INK4a) mRNA expression. Altogether, our data suggest that deregulation of DNMT1, DNMT3B and miR29 may be involved in BL pathogenesis.