RESUMO
Prostate cancer (PCa) seriously jeopardizes men's health worldwide. Dihydroartemisinin, which is an effective antimalarial agent, has shown potential anticancer effects in various human cancer cell lines, including PCa cells. However, the mechanisms underlying the anticancer activity of dihydroartemisinin are not fully understood. Ubiquitin-like with plant homeodomain and ring finger domain 1 (UHRF1) is highly expressed in a variety of tumors and is negatively correlated with the prognosis of various tumors. We reported previously that UHRF1 is downregulated during apoptosis induced by dihydroartemisinin in PC-3 PCa cells. In this study, we transfected PC-3 cells with lentiviruses containing UHRF1 or shRNA-UHRF1. Then, the cells were treated with dihydroartemisinin at different concentrations. Our data showed that overexpression of UHRF1 promoted cell proliferation and migration in PC-3 cells, inhibited cell apoptosis, increased cell proportion in G2 phase, increased DNA methyltransferase 1 and decreased p16INK4A expression at mRNA and protein levels. Downregulation of UHRF1 produces the opposite results. Moreover, the phenomena caused by overexpression of UHRF1 were inhibited after dihydroartemisinin treatment. Compared with control cells, cells overexpressing UHRF1 can resist the proapoptotic and antiproliferative effects of dihydroartemisinin to a certain extent. The effects of UHRF1 knockdown were further aggravated by dihydroartemisinin treatment, but no statistically significant effect was observed with increasing drug concentration. Our results suggested that dihydroartemisinin decreases proliferation and migration but enhances apoptosis of PCa cells, likely by downregulating UHRF1 and upregulating p16INK4A.
Assuntos
Artemisininas/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Neoplasias da Próstata/patologia , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1/efeitos dos fármacos , Relação Dose-Resposta a Droga , Técnicas de Silenciamento de Genes , Humanos , MasculinoRESUMO
Aurora-A has attracted a great deal of interest as a potential therapeutic target for patients with CRC. However, the outcomes of inhibitors targeting Aurora-A are not as favorable as expected, and the basis behind the ineffectiveness remains unknown. Here, we found that signal transducer and activator of transcription 1 (STAT1) was highly expressed in colorectal cancer (CRC) xenograft mouse models that were resistant to alisertib, an Aurora-A inhibitor. Unexpectedly, we found that alisertib disrupted Aurora-A binding with ubiquitin-like with plant homeodomain and ring finger domain 1 (UHRF1), leading to UHRF1 mediated ubiquitination and degradation of DNA methyltransferase 1 (DNMT1), which in turn resulted in demethylation of CpG islands of STAT1 promoter and STAT1 overexpression. Simultaneous silencing Aurora-A and UHRF1 prevented STAT1 overexpression and effectively inhibited CRC growth. Hence, concomitant targeting Aurora-A and UHRF1 can be a promising therapeutic strategy for CRC.
Assuntos
Antineoplásicos/farmacologia , Aurora Quinase A/antagonistas & inibidores , Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Inativação Gênica/efeitos dos fármacos , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Animais , Azepinas/farmacologia , Neoplasias Colorretais/genética , Ilhas de CpG/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Modelos Animais de Doenças , Camundongos , Regiões Promotoras Genéticas , Pirimidinas/farmacologia , Fator de Transcrição STAT1/metabolismoRESUMO
Chemical investigation of the South-Pacific marine sponge Suberea clavata led to the isolation of eight new bromotyrosine metabolites named subereins 1-8 (2-9) along with twelve known co-isolated congeners. The detailed configuration determination of the first representative major compound of this family 11-epi-fistularin-3 (11R,17S) (1) is described. Their chemical characterization was achieved by HRMS and integrated 1D and 2D NMR (nuclear magnetic resonance) spectroscopic studies and extensive comparison with literature data. For the first time, a complete assignment of the absolute configurations for stereogenic centers C-11/17 of the known members (11R,17S) 11-epi-fistularin-3 (1) and 17-deoxyfistularin-3 (10) was determined by a combination of chemical modifications, Mosher's technology, and ECD spectroscopy. Consequently, the absolute configurations of all our new isolated compounds 2-9 were determined by the combination of NMR, Mosher's method, ECD comparison, and chemical modifications. Interestingly, compounds 2-7 were obtained by chemical transformation of the major compound 11-epi-fistularin-3 (1). Evaluation for acetylcholinesterase inhibition (AChE), DNA methyltransferase 1 (DNMT1) modulating activity and antifouling activities using marine bacterial strains are also presented.
Assuntos
Poríferos/metabolismo , Tirosina/análogos & derivados , Animais , Incrustação Biológica/prevenção & controle , Inibidores da Colinesterase/química , Inibidores da Colinesterase/isolamento & purificação , Inibidores da Colinesterase/farmacologia , DNA (Citosina-5-)-Metiltransferase 1/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Espectroscopia de Ressonância Magnética , Oceano Pacífico , Tirosina/química , Tirosina/isolamento & purificação , Tirosina/farmacologiaRESUMO
Casticin (CAS) is a polymethyl flavonoid from Fructus viticis and has multiple pharmacological activities, including anticancer. However, whether the molecular mechanism underlying CAS represses stemness characteristics in hepatocellular carcinoma (HCC) cells involves intervention in the reciprocal negative regulation between DNA methyltransferase 1 (DNMT1) and miR-148a-3p has not yet been reported. In this study, the effect of CAS on stemness characteristics of HCC cells and its mechanism were investigated. Results showed that CAS selectively reduced the viabilities of HCC cells but not L02 cells, as determined by CCK-8 assay. Importantly, the sub-cytotoxic concentrations of CAS could inhibit the stemness characteristics in HCC cells, as demonstrated by the expression of stemness biomarkers (CD44, EpCAM, Bmi1, Nanog, and Oct4), sphere forming assay, RT-qPCR, and Western blotting. In addition, CAS repressed DNMT1 activity and expression and increased miR-148a-3p. The effect of CAS on stemness characteristics was abolished by stable DNMT1 overexpression. MiR-148a-3p overexpression enhanced the reduction of CAS on stemness characteristics. DNMT1 overexpression promoted miR-148a-3p promoter hypermethylation as detected by methylation-specific PCR (MSP), which repressed its expression. Conversely, miR-148a-3p repressed DNMT1 expression by specific site binding to 3'-UTR of DNMT1 mRNA, as determined by luciferase assay. Moreover, the combination of CAS and agomir-148a-3p had robust effects on tumor suppression as compared to the sole activity of either molecule in nude mouse xenograft experiments in vivo. The findings suggested that CAS could inhibit stemness characteristics in HCC cells by interruption of the reciprocal negative regulation between DNMT1 and miR-148a-3p.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Flavonoides/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , MicroRNAs/metabolismo , Antineoplásicos/farmacologia , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1/efeitos dos fármacos , Flavonoides/farmacologia , Citometria de Fluxo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , MicroRNAs/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIM: Early life stress can induce epigenetic changes through genetic and environmental interactions and is a risk factor for depression. Brain-derived neurotrophic factor (BDNF) has been implicated in the pathophysiology of depression and antidepressant drug action. We investigated epigenetic changes at the BDNF exon I promoter in the hippocampus of adult rats subjected to maternal separation (MS) during early life and treated with an antidepressant drug as adults. METHODS: Rat pups were subjected to MS from postnatal day 1 to 21 and received chronic escitalopram (ESC) as adults. We assessed the effects of MS and ESC on BDNF exon I and DNA methyltransferases (DNMT) mRNA levels (quantitative reverse-transcription polymerase chain reaction), acetylated histone H3, and MeCP2 binding to the BDNF promoter I (chromatin immunoprecipitation followed by real-time polymerase chain reaction), and BDNF protein levels (enzyme-linked immunosorbent assay). RESULTS: The levels of BDNF protein, exon I mRNA, histone H3 acetylation, and DNMT1 and DNMT3a mRNA were altered in the MS group compared with the control group. Significant decreases were observed in the BDNF protein, exon I mRNA, and histone H3 acetylation levels and there were significant increases in DNMT1 and DNMT3a mRNA levels. The comparison between the MS + ESC and MS groups revealed significant increases in BDNF protein, exon I mRNA, and histone H3 acetylation levels and significant decreases in MeCP2 and DNMT1 and DNMT3a mRNA levels. CONCLUSION: These findings indicate that MS induced epigenetic changes at the BDNF exon I promoter and these changes were prevented by antidepressant drug treatment during adulthood.
Assuntos
Antidepressivos de Segunda Geração/farmacologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Citalopram/farmacologia , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Epigênese Genética/fisiologia , Hipocampo/metabolismo , Histonas/metabolismo , Privação Materna , Proteína 2 de Ligação a Metil-CpG/metabolismo , RNA Mensageiro/metabolismo , Acetilação , Animais , Fator Neurotrófico Derivado do Encéfalo/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferases/efeitos dos fármacos , DNA Metiltransferase 3A , Epigênese Genética/efeitos dos fármacos , Éxons , Feminino , Hipocampo/efeitos dos fármacos , Histonas/efeitos dos fármacos , Masculino , Proteína 2 de Ligação a Metil-CpG/efeitos dos fármacos , Gravidez , Regiões Promotoras Genéticas , RNA Mensageiro/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Alcohol use disorder is the outcome of both genetic and environmental influences and their interaction via epigenetic mechanisms. The neurotransmitter glutamate is an important regulator of reward circuits and implicated in adaptive changes induced by ethanol intake. The present study aimed at investigating corticolimbic and corticostriatal genetic signatures focusing on the glutamatergic phenotype in relation to early-life stress (ELS) and consequent adult ethanol consumption. A rodent maternal separation model was employed to mimic ELS, and a free-choice paradigm was used to assess ethanol intake in adulthood. Gene expression levels of the Vesicular Glutamate Transporters (Vglut) 1, 2 and 3, as well as two key regulators of DNA methylation, DNA (cytosine-5)-methyltransferase 1 (Dnmt1) and methyl-CpG-binding protein 2 (Mecp2), were analyzed. Brain regions of interest were the ventral tegmental area (VTA), nucleus accumbens (Acb), medial prefrontal cortex (mPFC) and dorsal striatum (dStr), all involved in mediating aspects of ethanol reward. Region-specific Vglut, Dnmt1 and Mecp2 expression patterns were observed. ELS was associated with down-regulated expression of Vglut2 in the VTA and mPFC. Rats exposed to ELS were more sensitive to ethanol-induced changes in Vglut expression in the VTA, Acb, and dStr and in Dnmt1 and Mecp2 expression in the striatal regions. These findings suggest long-term glutamatergic and DNA methylation neuroadaptations as a consequence of ELS, and show an association between voluntary drinking in non-preferring, non-dependent, rodents and different Vglut, Dnmt1 and Mecp2 expression depending on early-life history.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Corpo Estriado/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Etanol/farmacologia , Expressão Gênica/efeitos dos fármacos , Sistema Límbico/efeitos dos fármacos , Privação Materna , Terminações Pré-Sinápticas/efeitos dos fármacos , Estresse Psicológico/genética , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Comportamento de Escolha , Corpo Estriado/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1/genética , Feminino , Ácido Glutâmico/metabolismo , Sistema Límbico/metabolismo , Masculino , Proteína 2 de Ligação a Metil-CpG/efeitos dos fármacos , Proteína 2 de Ligação a Metil-CpG/genética , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Terminações Pré-Sinápticas/metabolismo , Ratos , Ratos Wistar , Recompensa , Estresse Psicológico/metabolismo , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/efeitos dos fármacos , Proteína Vesicular 1 de Transporte de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/efeitos dos fármacos , Proteína Vesicular 2 de Transporte de Glutamato/genética , Proteínas Vesiculares de Transporte de Glutamato/efeitos dos fármacos , Proteínas Vesiculares de Transporte de Glutamato/genéticaRESUMO
This work aims to study the function of curculigoside in osteoporosis and explore whether DNMT1 is closely involved in osteoblast activity. After OB-6 osteoblasts were treated with hydrogen peroxide (H2O2), a curculigoside treatment group was set up and a series of biological tests including MTT, flow cytometry, western blotting, ROS fluorescence intensity, mitochondrial membrane potential, and ELISA experiments were performed to verify the effect of curculigoside on the activity of osteoblasts. Then, alkaline phosphatase (ALP) activity, alizarin red staining, PCR, and western blotting assays were performed to detect the effects of curculigoside on osteoblast function. By constructing DNMT1 knockdown and overexpression OB-6 cell lines, the effect of DNMT1 on osteoblast function was verified. In addition, the expression level of Nrf2 in each group was detected to speculate the mechanism of DNMT1 in osteoporosis. The cell activity and level of bcl-2 and SOD were significantly increased; the cell apoptosis, ROS fluorescence intensity, mitochondrial membrane potential, MDA and level of caspase-3, Bax, and CAT was reduced in curculigoside treatment group compared with H2O2-induced OB-6 osteoblasts. Meanwhile, the ALP activity, number and area of bone mineralized nodules, and gene and protein expression of OSX and OPG were significantly elevated in curculigoside group. Moreover, DNMT1 knockdown had a similar promotion effect on osteoblast function as curculigoside, and DNMT1 overexpression could reverse the promotion effect of curculigoside on osteoblast function. Further mechanistic studies speculated that DNMT1 might play a role in osteoporosis by affecting Nrf2 methylation. Curculigoside enhances osteoblast activity through DNMT1 controls of Nrf2 methylation.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1 , Osteoporose , Animais , Diferenciação Celular , Peróxido de Hidrogênio/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Osteoblastos/metabolismo , Osteoporose/tratamento farmacológico , Osteoporose/genética , Espécies Reativas de Oxigênio/metabolismo , Camundongos , DNA (Citosina-5-)-Metiltransferase 1/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1/metabolismoRESUMO
Promoter methylation represents one of the major epigenetic mechanisms responsible for the regulation of gene expression. Hypomethylating drugs are currently approved for the treatment of myelodysplastic syndromes and acute myeloid leukemia, and some studies have recently been carried out on diffuse large B cell lymphoma (DLBCL). DLBCL is a type of NonHodgkin lymphoma. The aim of the present study was to assess the role of DNA methyltransferase (DNMT)1 in mediating the epigenetic regulation of some key targets previously emerged as hypermethylated in NonHodgkin lymphoma. Reverse transcriptionquantitative PCR, genomewide arrays and methylationspecific PCR were used to determine the level of methylation of specific targets. Gene silencing, gene expression and immunoblotting were used to investigate the role of DNMT1 and DNMT3a in lymphoma cells. The present study showed that lymphoma cell lines displayed a completely different methylation profile on selected targets compared with primary B lymphocytes and peripheral blood mononuclear cells. 5'azacytidine (5AZA) and 5'aza2deoxycitidine (decitabine) exerted their activity through, at least in part, mechanisms independent of DNMT1 downregulation. Despite a global hypomethylating effect of 5AZA and decitabine, DNMT1 was not found to be necessary to maintain the hypermethylation of Krüppellike factor 4 (KLF4), death associated protein 1 (DAPK1) and spastic paraplegia 20 (SPG20). SPG20 was found to be a completely methylated target in all the tested cell lines, but not in peripheral blood mononuclear cells, suggesting its association with malignancy. The highest methylation was clustered upstream of the transcription starting site in a panel of 28 DLBCL cell lines and the results were unaffected by the silencing of DNMT1 expression. These data demonstrated the epigenetic regulation of SPG20 in lymphoid cells and identified a number of novel markers associated with lymphomas that deserve further investigation.
Assuntos
Proteínas de Ciclo Celular/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Proteínas Quinases Associadas com Morte Celular/genética , Decitabina/farmacologia , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/genética , Antimetabólitos Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Proteínas Quinases Associadas com Morte Celular/efeitos dos fármacos , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Fator 4 Semelhante a Kruppel/efeitos dos fármacos , Fator 4 Semelhante a Kruppel/genéticaRESUMO
The pathological characteristics of Parkinson's disease (PD) include dopaminergic neuron damage, specifically disorders caused by dopamine synthesis, in vivo. Plastrum testudinis extract (PTE) and its bioactive ingredient ethyl stearate (PubChem CID: 8122) were reported to be correlated with tyrosine hydroxylase (TH), which is a biomarker of dopaminergic neurons. This suggests that PTE and its small-molecule active ingredient ethyl stearate have potential for development as a therapeutic drug for PD. In this study, we treated 6-hydroxydopamine (6-OHDA)-induced model rats and PC12 cells with PTE. The mechanism of action of PTE and ethyl stearate was investigated by western blotting, bisulfite sequencing PCR (BSP), real-time PCR, immunofluorescence and siRNA transfection. PTE effectively upregulated the TH expression and downregulated the alpha-synuclein expression in both the substantia nigra and the striatum of the midbrain in a PD model rat. The PC12 cell model showed that both PTE and its active monomer ethyl stearate significantly promoted TH expression and blocked alpha-synuclein, agreeing with the in vivo results. BSP showed that PTE and ethyl stearate increased the methylation level of the Snca intron 1 region. These findings suggest that some of the protective effects of PTE on dopaminergic neurons are mediated by ethyl stearate. The mechanism of ethyl stearate may involve disrupting the abnormal aggregation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) with alpha-synuclein by releasing DNMT1, upregulating Snca intron 1 CpG island methylation, and ultimately, reducing the expression of alpha-synuclein.
Assuntos
Antiparkinsonianos/farmacologia , Antiparkinsonianos/uso terapêutico , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Doença de Parkinson Secundária/tratamento farmacológico , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Extratos de Tecidos/química , alfa-Sinucleína/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferase 1/efeitos dos fármacos , Hidroxidopaminas , Masculino , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/metabolismo , Células PC12 , Doença de Parkinson Secundária/induzido quimicamente , Ratos , Ratos Sprague-Dawley , Estearatos/farmacologia , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , alfa-Sinucleína/efeitos dos fármacosRESUMO
Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine, ClF), a second-generation 2'-deoxyadenosine analog, possesses manifold anti-cancer activities. Our previous reports and some of others demonstrate the potential capacity of ClF to regulate the epigenetic machinery. The study presented here is the first to investigate the influence of ClF on modulators of the DNA methylation machinery, including DNMT1 and CDKN1A, in acute lymphoblastic leukemia (ALL) cells. ClF effects on promoter methylation and transcriptional activity of hypermethylated and silenced tumor suppressor genes (TSGs), including APC, CDKN2A, PTEN, and RARB, have been tested as well. Methylation level of the proximal promoter region of APC, CDKN2A, PTEN, and RARB, as well as expression of those TSGs, DNMT1 and CDKN1A, were estimated by using a methylation-sensitive restriction analysis and qPCR, respectively. The Nalm-6 cell line was used as an experimental in vitro model of ALL cells. We observed ClF-mediated inhibition of cellular viability and apoptosis induction of Nalm-6 cells with an increased percentage of cells positive for active Caspase-3. Interestingly, exposure of Nalm-6 cells to CIF at 20 nM concentration for three days has led to a significant DNMT1 downregulation, accompanied by robust CDKN1A upregulation. ClF caused hypomethylation of APC, CDKN2A, and PTEN, with a concomitant increase in their transcript levels. Taken together, our results demonstrate the ability of ClF to reactivate DNA methylation-silenced TSGs in ALL cells. This may implicate translational significance of our findings and support ClF application as a new epigenetic modulator in the anti-leukemia therapy.
Assuntos
Clofarabina/farmacologia , Metilação de DNA/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Clofarabina/uso terapêutico , Inibidor de Quinase Dependente de Ciclina p21/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Regiões Promotoras GenéticasRESUMO
BACKGROUND: miR-342-3p, localized to 14q32, is a tumor suppressor miRNA implicated in carcinogenesis. Given the presence of a promotor-associated CpG island for its host gene, EVL, we hypothesized that intronic miR-342-3p is a tumor suppressor co-regulated with host gene by promoter DNA methylation in B cell lymphoma. RESULTS: By bisulfite pyrosequencing-verified methylation-specific PCR (MSP), EVL/MIR342 methylation was detected in five (50%) lymphoma cell lines but not normal peripheral blood and tonsils. EVL/MIR342 methylation correlated with repression of both miR-342-3p and EVL in cell lines. In completely methylated SU-DHL-16 cells, 5-AzadC treatment resulted in promoter demethylation and re-expression of miR-342-3p and EVL. In 132 primary lymphoma samples, EVL/MIR342 was preferentially methylated in B cell lymphomas (N = 68; 68.7%) than T cell lymphoma (N = 8; 24.2%) by MSP (P < 0.0001). Moreover, EVL/MIR342 methylation was associated with lower miR-342-3p expression in 79 primary NHL (P = 0.0443). In SU-DHL-16 cells, the tumor suppressor function of miR-342-3p was demonstrated by the inhibition of cellular proliferation and increase of cell death upon over-expression of miR-342-3p. Mechanistically, overexpression of miR-342-3p resulted in a decrease of LC3-II, a biomarker of autophagy, which was pro-survival for SU-DHL-16. Pre-treatment with 3-methyladenine, an autophagy inhibitor, abrogated tumor suppression associated with miR-342-3p overexpression. By luciferase assay, MAP1LC3B, a precursor of LC3-II, was confirmed as a direct target of miR-342-3p. Finally, in SU-DHL-16 cells, overexpression of miR-342-3p downregulated the known target DNMT1, with promoter demethylation and re-expression of tumor suppressor E-cadherin. CONCLUSIONS: Intronic miR-342-3p is co-regulated with its host gene EVL by tumor-specific promoter DNA methylation in B cell lymphoma. The tumor suppressor function of miR-342-3p was mediated via inhibition of pro-survival autophagy by targeting MAP1LC3B and downregulation of DNMT1 with demethylation and re-expression of tumor suppressor genes.
Assuntos
Metilação de DNA/genética , Epigênese Genética/genética , Linfoma de Células B/genética , MicroRNAs/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferase 1/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1/genética , Decitabina/farmacologia , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Feminino , Inativação Gênica , Genes Supressores de Tumor/efeitos dos fármacos , Humanos , Linfoma de Células B/tratamento farmacológico , Masculino , Proteínas Associadas aos Microtúbulos , Regiões Promotoras Genéticas/efeitos dos fármacosRESUMO
Histone modifications, specifically in the lysine residues of histone H3, have been implicated in lifespan regulation in several model organisms. Our previous studies showed that growth hormone (GH) treatment during early life can dramatically influence lifespan in long-lived Ames dwarf mice. However, the effects of this hormonal intervention on epigenetic modifications have never been examined. In this study, we sought to compare tissue-specific histone H3 lysine methylation and acetylation markers in Ames dwarf and wild type (WT) mice and to determine how these markers are affected by early-life GH intervention. Ames dwarf mice exhibited suppressed H3K4me in both hepatic and brain tissues, while showing elevated H3K27me in the brain. Early-life GH intervention significantly altered the histone H3 markers in those tissues. Furthermore, early GH intervention increased expression of histone H3 acetylation at multiple lysine residues in a tissue-specific manner. This included changes in H3K14ac and H3K18ac in the liver and brain, H3K18ac in visceral adipose tissue and H3K9ac, H3K14ac and H3K27ac in subcutaneous adipose tissue. This study serves as an initial, but important step in elucidating the epigenetic mechanisms by which hormonal signals during early life can influence aging and longevity in mammals.