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Esophageal squamous cell carcinoma (ESCC) is associated with the accumulation of genetic and epigenetic changes in the background mucosa. Dysregulated DNA methylation is known to lead to the inactivation of tumor suppressor genes and the activation of oncogenes. To identify the genes whose expression is perturbed by abnormal DNA methylation in ESCC, integrative transcriptomics by serial analysis of gene expression (SAGE) and methylome sequencing by methyl-DNA immunoprecipitation (MeDIP) analysis were performed. We found 159 genes with significantly decreased expression in ESCC compared to that in noncancerous esophageal mucosa. MeDIP-seq analysis identified hypermethylation in the promoter region of 56 of these genes. Using surgically resected tissues of 40 cases, we confirmed that the paired-like homeodomain 1 (PITX1) gene was hypermethylated in ESCC compared to that in normal tissues (P < 0.0001) by pyrosequencing. PITX1 overexpression in ESCC cell lines inhibited cell growth and colony formation, whereas PITX1 knockdown accelerated cell growth. A PITX1-transfected ESCC cell line, KYSE30, formed smaller tumors in nude mice than in mock-transfected cells. Hypermethylation of PITX1 was associated with tumor depth (P = 0.0011) and advanced tumor stage (P = 0.0052) and predicted poor survival in ESCC (hazard ratio, 0.1538; 95% confidence interval, 0.03159-0.7488; P = 0.0169). In this study, we found a novel tumor suppressor gene of ESCC, PITX1, which is silenced by DNA hypermethylation. Downregulation of PITX1 contributes to the growth and progression of ESCC. Hypermethylation of the PITX1 in ESCC correlated with tumor progression and advanced stage cancer, and may predict a poor prognosis.
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Fasting-refeeding in mice induces transient hyperproliferation of colonic epithelial cells, which is dependent on the lactate produced as a metabolite of commensal bacteria. We attempted to manipulate colonic epithelial cell turnover with intermittent fasting to prompt recovery from acute colitis. Acute colitis was induced in C57BL/6 mice by administration of dextran sulfate sodium in the drinking water for 5 days. From day 6, mice were fasted for 36 h and refed normal bait, glucose powder, or lactylated high-amylose starch. On day 9, colon tissues were subjected to analysis of histology and cytokine expression. The effect of lactate on the proliferation of colonocytes was assessed by enema in vivo and primary culture in vitro. Intermittent fasting resulted in restored colonic crypts and less expression of interleukin-1ß and interleukin-17 in the colon than in mice fed ad libitum. Administration of lactate in the colon at refeeding time by enema or by feeding lactylated high-amylose starch increased the number of regenerating crypts. Addition of lactate but not butyrate or acetate supported colony formation of colonocytes in vitro. In conclusion, intermittent fasting in the resolution phase of acute colitis resulted in better recovery of epithelial cells and reduced inflammation.
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ESET/SETDB1, one of the major histone methyltransferases, catalyzes histone 3 lysine 9 (H3K9) trimethylation. ESET is critical for suppressing expression of retroviral elements in embryonic stem cells; however, its role in the immune system is not known. We found that thymocyte-specific deletion of ESET caused impaired T cell development, with CD8 lineage cells being most severely affected. Increased apoptosis of CD8 single-positive cells was observed, and TCR-induced ERK activation was severely inhibited in ESET(-/-) thymocytes. Genome-wide comprehensive analysis of mRNA expression and H3K9 trimethylation revealed that ESET regulates expression of numerous genes in thymocytes. Among them, FcγRIIB, whose signaling can inhibit ERK activation, was strongly and ectopically expressed in ESET(-/-) thymocytes. Indeed, genetic depletion of FcγRIIB in ESET(-/-) thymocytes rescued impaired ERK activation and partially restored defective positive selection in ESET(-/-) mice. Therefore, impaired T cell development in ESET(-/-) mice is partly due to the aberrant expression of FcγRIIB. Collectively, to our knowledge, we identify ESET as the first trimethylated H3K9 histone methyltransferase playing a crucial role in T cell development.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Apoptose , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genoma , Histona-Lisina N-Metiltransferase/deficiência , Histonas/metabolismo , Lisina/metabolismo , Metilação , Camundongos , Regiões Promotoras Genéticas , Receptores de IgG/genética , Receptores de IgG/metabolismo , Timócitos/imunologia , Timócitos/fisiologiaRESUMO
Group 2 innate lymphoid cells (ILC2s) produce a significant amount of interleukin-5 (IL-5), which supports eosinophil responses in various tissues; they also produce IL-13, which induces mucus production and contributes to tissue repair or fibrosis. The ILC2s are activated by alarmins, such as IL-33 released from epithelia, macrophages and natural killer T (NKT) cells in response to infection and allergen exposure, leading to epithelial injury. We examined gene expression in lung ILC2s and found that ILC2s expressed Ifngr1, the receptor for interferon-γ (IFN-γ). Interferon-γ severely inhibited IL-5 and IL-13 production by lung and kidney ILC2s. To evaluate the effects in vivo, we used α-galactosylceramide (α-GalCer) to induce NKT cells to produce IL-33 and IFN-γ. Intraperitoneal injection of α-GalCer in mice induced NKT cell activation resulting in IL-5 and IL-13 production by ILC2s. Administration of anti-IFN-γ together with α-GalCer significantly enhanced the production of IL-5 and IL-13 by ILC2s in lung and kidney. Conversely, cytokine production from ILC2s was markedly suppressed after injection of exogenous IL-33 in Il33(-/-) mice pre-treated with α-GalCer. Hence, IFN-γ induced or already present in tissues can impact downstream pleiotropic functions mediated by ILC2s, such as inflammation and tissue repair.
Assuntos
Imunidade Inata/efeitos dos fármacos , Interferon gama/metabolismo , Rim/metabolismo , Pulmão/metabolismo , Linfócitos/metabolismo , Animais , Células Cultivadas , Galactosilceramidas/farmacologia , Interferon gama/imunologia , Interferon gama/farmacologia , Interleucina-13/imunologia , Interleucina-13/metabolismo , Interleucina-33/deficiência , Interleucina-33/genética , Interleucina-5/genética , Interleucina-5/imunologia , Interleucina-5/metabolismo , Rim/citologia , Rim/efeitos dos fármacos , Rim/imunologia , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Ativação Linfocitária , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Fenótipo , Receptores de Interferon/agonistas , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Receptor de Interferon gamaRESUMO
BACKGROUND AND AIM: Nonalcoholic fatty liver disease (NAFLD) affects 15-40% of the general population; 10-20% of those patients have a more severe form of the disease known as nonalcoholic steatohepatitis (NASH). Cytokeratin-18 (CK18), released during apoptosis and one of the most studied biomarkers in NASH, can be measured by a number of commercially available kits. We compared serum measurements of the CK18 M30 from two different kits using the same cohort to evaluate the reliability between two test kits. METHODS: We measured serum levels of CK18 M30 from 185 patients with biopsy-proven NAFLD from a single center from 2009 to 2015, using two different ELISA kits, Test 1 (T1) and Test 2 (T2). Advanced fibrosis was defined as fibrosis stages 3-4 and NASH defined by NAS score ≥ 5. RESULTS: Mean age was 50.2 years (SD 12.6), 61.1% male and 87% White; 49.6% had NASH and 32.2% advanced fibrosis. There was no significant correlation between measurements from the two kits (p = 0.86, r = 0.01). While T2 predicted NASH and advanced fibrosis, T1 did not. The area under ROC curve for the prediction of NASH was 0.631 for T2 versus 0.500 for T1. CONCLUSIONS: Measurements from two different CK18 M30 test kits did not correlate with each other. One kit showed statistically significantly higher levels of CK18 M30 in patients with advanced fibrosis and NASH, while the other kit did not. With the increasing use of CK18 as a biomarker in NASH, it is important to standardize the different kits as it could greatly bias the results.
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Queratina-18/sangue , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/sangue , Adulto , Área Sob a Curva , Biomarcadores/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Biópsia Guiada por Imagem , Fígado/diagnóstico por imagem , Cirrose Hepática/sangue , Cirrose Hepática/diagnóstico , Cirrose Hepática/patologia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Hepatopatia Gordurosa não Alcoólica/patologia , Modelos de Riscos Proporcionais , Curva ROC , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , UltrassonografiaRESUMO
Long interspersed element-1 (L1) is a transposable element that can move within the genome, potentially leading to genome diversity and modified gene function. Although L1 activity in somatic cells is normally suppressed through DNA methylation, some L1s are activated in tumors including colorectal carcinoma. However, how L1-retrotransposition (L1-RTP) is involved in gastrointestinal disorders remains to be elucidated. We hypothesized that L1-RTP in somatic cells might contribute to colitis-associated cancer (CAC). To address this, we employed an experimental model of CAC using transgenic L1-reporter mice carrying a human L1-EGFP reporter gene. Mice were subjected to repeated cycles of colitis induced by administration of dextran sodium sulfate (DSS) in drinking water with injection of carcinogen azoxymethane (AOM). L1-RTP levels were measured by a quantitative polymerase chain reaction targeting the newly inserted reporter EGFP in various tissues and cell types, including samples obtained by laser microdissection and cell sorting with flow cytometry. DNA methylation levels of the human L1 promoter were analyzed by bisulfite pyrosequencing. AOM+DSS-treated mice exhibited significantly higher levels of L1-RTP in whole colon tissue during the acute phase of colitis when compared with control naïve mice. L1-RTP levels in whole colon tissue were positively correlated with the histological severity of colitis and degree of neutrophil infiltration into the lamina propria (LP), but not with tumor development in the colon. L1-RTP was enriched in LP mesenchymal cells rather than epithelial cells (ECs), myeloid, or lymphoid cells. DNA methylation levels of the human L1 promoter region showed a negative correlation with L1-RTP levels. L1-RTP was absent from most tumors found in 22-week-old mice. In conclusion, we demonstrated that L1-RTP was induced in the mouse CAC mucosa in accordance with the acute inflammatory response; however, retrotransposition appears not to have direct relevance to colitis-induced cancer initiation.
Assuntos
Colite/genética , Neoplasias Intestinais/genética , Elementos Nucleotídeos Longos e Dispersos , Animais , Metilação de DNA , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , CamundongosRESUMO
Periplakin (PPL), a member of the plakin family of proteins that localizes to desmosomes and intermediate filaments, is downregulated in human esophageal squamous cell carcinoma (ESCC). Little is known, however, about the molecular mechanism underlying the regulation of PPL expression and the contribution of PPL loss to the malignant property of the cancer is unclear. We demonstrated that PPL mRNA expression was significantly reduced in ESCC tissues compared with that in normal tissues. Therefore, we hypothesized that CpG hypermethylation is the cause of the downregulation of PPL. Bisulfite-pyrosequencing of 17 cases demonstrated that the frequency of PPL methylation was higher in ESCC tissues than in normal tissues. When human ESCC cell lines were treated with 5-aza-2'-deoxycytidine (5-aza-dC), a DNA-methyltransferase inhibitor, PPL transcription was induced. Human KYSE270 ESCC cells do not stratify under ordinary culture conditions and rarely produce desmosomes; however, the forced expression of PPL promoted cell stratification. PPL induction also promoted adhesion to extracellular matrix but delayed cell migration. The abundance of desmosome-like structures was greatly increased in PPL transfectant as determined by transmission electron microscopy. Very low expression of another desmosome protein EVPL in ESCC, even in PPL transfectant, also supported the significant role of PPL in desmosome formation and cell stratification. Our results first indicate that the downregulation of PPL mediated by DNA hypermethylation, which may play an important role in the loss of ESCC stratification and likely in metastatic phenotype.
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Carcinoma de Células Escamosas/genética , Metilação de DNA , Neoplasias Esofágicas/genética , Plaquinas/genética , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/ultraestrutura , Linhagem Celular Tumoral , Ilhas de CpG , Desmossomos , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/ultraestrutura , Carcinoma de Células Escamosas do Esôfago , Esôfago/metabolismo , Esôfago/ultraestrutura , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa/metabolismo , Mucosa/ultraestrutura , Plaquinas/metabolismo , Regiões Promotoras GenéticasRESUMO
Tumor necrosis factor (TNF)-α is a major effector in various inflammatory conditions. TNF-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily that promotes inflammatory tissue damage through its receptor, FGF-inducible molecule 14 (Fn14). Since both TWEAK and TNF-α have been shown to mediate pathological responses through inter-dependent or independent pathways by in vitro, the potential interplay of these pathways was investigated in a mouse colitis model. Acute colitis was induced by rectal injection of trinitrobenzene sulfonic acid (TNBS), with administration of control IgG, TNF receptor (TNFR)-Ig chimeric protein, anti-TWEAK monoclonal antibody, or the combination of TNFR-Ig and anti-TWEAK antibody. On day 4, disease severity was evaluated and gene expression profiling was analyzed using whole colon tissue. NF-κB activation was investigated with Western blot. Levels of transcript of TWEAK, Fn14 and NF-κB-related molecules were measured in purified colon epithelial cells (ECs). As a result, activation of the canonical (p50/RelA), but not noncanonical (p100/RelB)-mediated pathway was the hallmark of inflammatory responses in this model. Inflammation induced upregulation of Fn14 only in ECs but not in other cell types. Combination treatment of TNFR-Ig and anti-TWEAK antibody synergistically reduced disease severity in comparison with the control antibody or single agent treatment. Gene expression profile of the colon indicated downregulation of canonical NF-κB pathway with combination treatment. In conclusion, synergistic activation of canonical NF-κB by TWEAK and TNF-α is critical for the induction of inflammatory tissue damage in acute inflammation.
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Colite/patologia , NF-kappa B/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fatores de Necrose Tumoral/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Colite/induzido quimicamente , Colo/citologia , Colo/patologia , Citocina TWEAK , Perfilação da Expressão Gênica , Inflamação/imunologia , Inflamação/patologia , Mucosa Intestinal/citologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/biossíntese , Subunidade p50 de NF-kappa B/biossíntese , Receptores do Fator de Necrose Tumoral/biossíntese , Receptor de TWEAK , Fator de Transcrição RelA/biossíntese , Ácido TrinitrobenzenossulfônicoRESUMO
Intestinal regulatory T cells (Treg cells) are necessary for the suppression of excessive immune responses to commensal bacteria. However, the molecular machinery that controls the homeostasis of intestinal Treg cells has remained largely unknown. Here we report that colonization of germ-free mice with gut microbiota upregulated expression of the DNA-methylation adaptor Uhrf1 in Treg cells. Mice with T cell-specific deficiency in Uhrf1 (Uhrf1(fl/fl)Cd4-Cre mice) showed defective proliferation and functional maturation of colonic Treg cells. Uhrf1 deficiency resulted in derepression of the gene (Cdkn1a) that encodes the cyclin-dependent kinase inhibitor p21 due to hypomethylation of its promoter region, which resulted in cell-cycle arrest of Treg cells. As a consequence, Uhrf1(fl/fl)Cd4-Cre mice spontaneously developed severe colitis. Thus, Uhrf1-dependent epigenetic silencing of Cdkn1a was required for the maintenance of gut immunological homeostasis. This mechanism enforces symbiotic host-microbe interactions without an inflammatory response.
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Colite/imunologia , Colo/imunologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Epigênese Genética , Proteínas Nucleares/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Células Cultivadas , Clostridium/imunologia , Colite/genética , Colo/microbiologia , Metilação de DNA , Perfilação da Expressão Gênica , Interleucina-2 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microbiota/imunologia , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno , Simbiose/imunologia , Ubiquitina-Proteína Ligases , Regulação para CimaRESUMO
Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8-/-) and wild-type (WT) mice. We found that CCR8-/- PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca2+ flux and CCL1-driven PMφ aggregation. Similar to CCR8-/- PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. CCR8-/- PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8-/- mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of CCR8-/- mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.
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Citocinas/biossíntese , Macrófagos Peritoneais/metabolismo , Receptores CCR8/metabolismo , Animais , Quimiocina CCL1/antagonistas & inibidores , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Colite/genética , Colite/imunologia , Colite/metabolismo , Modelos Animais de Doenças , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Espaço Intracelular/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Camundongos Knockout , Transporte Proteico , Receptores CCR8/antagonistas & inibidores , Receptores CCR8/deficiência , Receptores CCR8/genética , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/metabolismoRESUMO
BACKGROUND: Viral protein R (Vpr), a protein of human immunodeficiency virus type-1 (HIV-1) with various biological functions, was shown to be present in the blood of HIV-1-positive patients. However, it remained unclear whether circulating Vpr in patients' blood is biologically active. Here, we examined the activity of blood Vpr using an assay system by which retrotransposition of long interspersed element-1 (L1-RTP) was detected. We also investigated the in vivo effects of recombinant Vpr (rVpr) by administrating it to transgenic mice harboring human L1 as a transgene (hL1-Tg mice). Based on our data, we discuss the involvement of blood Vpr in the clinical symptoms of acquired immunodeficiency syndrome (AIDS). RESULTS: We first discovered that rVpr was active in induction of L1-RTP. Biochemical analyses revealed that rVpr-induced L1-RTP depended on the aryl hydrocarbon receptor, mitogen-activated protein kinases, and CCAAT/enhancer-binding protein ß. By using a sensitive L1-RTP assay system, we showed that 6 of the 15 blood samples from HIV-1 patients examined were positive for induction of L1-RTP. Of note, the L1-RTP-inducing activity was blocked by a monoclonal antibody specific for Vpr. Moreover, L1-RTP was reproducibly induced in various organs, including the kidney, when rVpr was administered to hL1-Tg mice. CONCLUSIONS: Blood Vpr is biologically active, suggesting that its monitoring is worthwhile for clarification of the roles of Vpr in the pathogenesis of AIDS. This is the first report to demonstrate a soluble factor in patients' blood active for L1-RTP activity, and implies the involvement of L1-RTP in the development of human diseases.
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Produtos do Gene vpr/sangue , Produtos do Gene vpr/metabolismo , HIV-1/enzimologia , Elementos Nucleotídeos Longos e Dispersos , Recombinação Genética , Adulto , Animais , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Adulto JovemRESUMO
Oral food intake influences the morphology and function of intestinal epithelial cells and maintains gastrointestinal cell turnover. However, how exactly these processes are regulated, particularly in the large intestine, remains unclear. Here we identify microbiota-derived lactate as a major factor inducing enterocyte hyperproliferation in starvation-refed mice. Using bromodeoxyuridine staining, we show that colonic epithelial cell turnover arrests during a 12- to 36-h period of starvation and increases 12-24 h after refeeding. Enhanced epithelial cell proliferation depends on the increase in live Lactobacillus murinus, lactate production and dietary fibre content. In the model of colon tumorigenesis, mice exposed to a carcinogen during refeeding develop more aberrant crypt foci than mice fed ad libitum. Furthermore, starvation after carcinogen exposure greatly reduced the incidence of aberrant crypt foci. Our results indicate that the content of food used for refeeding as well as the timing of carcinogen exposure influence the incidence of colon tumorigenesis in mice.
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Alimentos , Mucosa Intestinal/citologia , Lactatos/metabolismo , Lactobacillus/metabolismo , Inanição , Animais , Proliferação de Células , Mucosa Intestinal/microbiologia , CamundongosRESUMO
BACKGROUND: SRY (sex-determining region Y)-box 2 (SOX2) is a crucial transcription factor for the maintenance of embryonic stem cell pluripotency and the determination of cell fate. Previously, we demonstrated that SOX2 plays important roles in growth inhibition through cell cycle arrest and apoptosis, and that SOX2 expression is frequently down-regulated in gastric cancers. However, the mechanisms underlying loss of SOX2 expression and its target genes involved in gastric carcinogenesis remain largely unknown. Here, we assessed whether microRNAs (miRNAs) regulate SOX2 expression in gastric cancers. Furthermore, we attempted to find downstream target genes of SOX2 contributing to gastric carcinogenesis. METHODOLOGY/PRINCIPAL FINDINGS: We performed in silico analysis and focused on miRNA-126 (miR-126) as a potential SOX2 regulator. Gain- and loss-of function experiments and luciferase assays revealed that miR-126 inhibited SOX2 expression by targeting two binding sites in the 3'-untranslated region (3'-UTR) of SOX2 mRNA in multiple cell lines. In addition, miR-126 was highly expressed in some cultured and primary gastric cancer cells with low SOX2 protein levels. Furthermore, exogenous miR-126 over-expression as well as siRNA-mediated knockdown of SOX2 significantly enhanced the anchorage-dependent and -independent growth of gastric cancer cell lines. We next performed microarray analysis after SOX2 over-expression in a gastric cancer cell line, and found that expression of the placenta-specific 1 (PLAC1) gene was significantly down-regulated by SOX2 over-expression. siRNA- and miR-126-mediated SOX2 knockdown experiments revealed that miR-126 positively regulated PLAC1 expression through suppression of SOX2 expression in gastric cancer cells. CONCLUSIONS: Taken together, our results indicate that miR-126 is a novel miRNA that targets SOX2, and PLAC1 may be a novel downstream target gene of SOX2 in gastric cancer cells. These findings suggest that aberrant over-expression of miR-126 and consequent SOX2 down-regulation may contribute to gastric carcinogenesis.
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Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/fisiologia , Fatores de Transcrição SOXB1/antagonistas & inibidores , Neoplasias Gástricas/etiologia , Regiões 3' não Traduzidas , Sítios de Ligação , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas da Gravidez/genética , Fatores de Transcrição SOXB1/genética , Neoplasias Gástricas/genéticaRESUMO
Ectopic expression of CDX2, a caudal-related homeobox protein, is known to be associated with the development of intestinal metaplasia in the stomach and gastric carcinogenesis. Previously, we reported that DNA methylation was partly responsible for CDX2 silencing in gastric cancer (GC). However, the mechanism underlying the aberrant expression of CDX2 during malignant transformation remained unclear. MicroRNAs (miRNAs) are small non-coding RNAs that function as post-transcriptional regulators. To elucidate the role of miRNAs in CDX2 downregulation in GC cells, putative miRNAs, such as miR-9, were computationally predicted. After exogenous pre-miR-9 precursor transfection, the luciferase activity of a reporter vector containing a part of the 3'-UTR of CDX2 was downregulated in HEK-293T cells. The inverse correlation between the miR-9 and CDX2 protein levels was demonstrated in GC cell lines. By means of miR-9 overexpression and knockdown techniques, the expression levels of the CDX2 protein and downstream target genes (p21, MUC2 and TFF3) were responsively altered in MKN45 and NUGC-3 cells. Transfection of an anti-miR-9 molecule significantly inhibited cell growth by promoting G(1) cell cycle arrest in MKN45 cells similarly to the effect of CDX2 overexpression. Moreover, examination of the miR-9 levels in primary GC tissues revealed that the amounts of miR-9 in the CDX2-negative group were significantly higher than those in the CDX2-positive group (p = 0.004). Therefore, miR-9 might repress CDX2 expression via the binding site in the 3'-UTR, resulting in the promotion of cell proliferation in GCs.
Assuntos
Regiões 3' não Traduzidas/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , MicroRNAs/genética , Neoplasias Gástricas/genética , Western Blotting , Fator de Transcrição CDX2 , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA , Regulação para Baixo , Citometria de Fluxo , Proteínas de Homeodomínio/metabolismo , Humanos , Luciferases/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
Aberrant expression of microRNA (miRNA) has been reported in various cancers. To clarify the role of miRNA in gastric carcinogenesis, we performed miRNA microarray analysis and investigated expression changes of miRNAs in a 5-aza-2'-deoxycytidine (5-aza-CdR)-treated gastric cancer cell line, KATO-III. On microarray analysis, five miRNAs were found to be upregulated (>3-fold) after 5-aza-CdR treatment compared with untreated cells. Among them, miR-181c and miR-432AS exhibited CpG islands in their upstream sequences on computational analysis, and their upregulation was verified by reverse transcription-polymerase chain reaction analyses. In particular, miR-181c upregulation was found not only in KATO-III but also in two other gastric and one colorectal cancer cell line with 5-aza-CdR treatment. Decreased expression of miR-181c was observed in 9 of 16 primary gastric carcinoma (GC) cases compared with the corresponding non-cancerous stomach tissues. Hypermethylation signals in the upstream region of miR-181c were observed in some cultured and primary GC cells with negative or low miR-181c expression. Transfection of the precursor miR-181c molecule induced decreased growth of two gastric cancer cell lines, KATO-III and MKN45. As for targets of miR-181c, oncogenic NOTCH4 and KRAS were identified by complementary DNA microarray analysis after precursor miR-181c molecule transfection, computational searches of miRNA target databases and reporter assaying using the 3'-untranslated regions of the two genes. These results indicate that miR-181c may be silenced through methylation and play important roles in gastric carcinogenesis through its target genes, such as NOTCH4 and KRAS.
Assuntos
Inativação Gênica , MicroRNAs/genética , Neoplasias Gástricas/etiologia , Regiões 3' não Traduzidas , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Ilhas de CpG , Metilação de DNA , Decitabina , Humanos , MicroRNAs/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Receptor Notch4 , Receptores Notch/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Proteínas ras/genéticaRESUMO
BACKGROUND & AIMS: The calcium channel voltage-dependent alpha2delta subunit consists of 4 genes, CACNA2D1 to CACNA2D4, of which CACNA2D2 and CACNA2D3 are located on 3p21.3 and 3p21.1, respectively. Here, we examined the relation between alpha2delta subunit gene alterations and gastric carcinogenesis. METHODS: The expression and methylation status of the alpha2delta subunit genes were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and methylation-specific PCR in gastric cancers (GCs). The effects of CACNA2D3 expression were examined by cell proliferation and adhesion assays, and they predicted target gene alterations. RESULTS: Aberrant methylation of CACNA2D1 and CACNA2D3 mostly corresponded to their expression status in GC cell lines. CACNA2D1/3 methylation was detected in 10 (12.5%) and 24 (30%) of the 80 GC cases, respectively, but no CACNA2D2 methylation was seen in 32 cases. CACNA2D3 methylation was more frequently found in diffuse type than in intestinal type (16/38 [42.1%] vs 8/42 [19.0%]; P = .025) GCs. Among the 53 patients with advanced GCs, patients with cancers showing CACNA2D3 methylation had a significantly shorter survival time than patients without this methylation (P = .003). Exogenous CACNA2D3 expression strongly inhibited cell growth and adhesion and up-regulated p21 and p27 expression in HEK-293T and NUGC4 cells. Inverse effects were seen by CACNA2D3 small interfering RNA treatment in the CACNA2D3-positive cell lines, indicating that CACNA2D3 may have tumor suppressive functions. CONCLUSIONS: Loss of CACNA2D3 expression through aberrant promoter hypermethylation may contribute to gastric carcinogenesis, and CACNA2D3 methylation is a useful prognostic marker for patients with advanced GC.