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Methylation, the most common chemical modification of cellular components such as DNA, RNA, and proteins, impacts biological processes including transcription, RNA processing, and protein dynamics. Although abnormal expression of methyltransferase can lead to various diseases including cancers, little is known about the relationship between methyltransferase and cancers. Here we aimed to understand the role of methyltransferase in cancer metastasis. We found that elevated methyltransferase-like 9 (METTL9) is closely associated with the acquisition of metastatic activity in human scirrhous gastric cancers. The stable knockdown of METTL9 via an shRNA vector technique in our original metastatic cells from scirrhous gastric cancer patients significantly inhibited migration and invasion. In metastatic cells, METTL9 protein is predominantly localized in mitochondria, and the METTL9 knockdown significantly reduced mitochondrial Complex I activity. METTL9 can be a candidate of molecular targets to inhibit peritoneal dissemination of scirrhous gastric cancers. This report is the first to describe the relationship between METTL9 and cancer metastasis.
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Constitutive expression of human telomerase reverse transcriptase (hTERT) with DNA methylation of its promoter is a common phenomenon in tumor cells. We recently found that the transcriptional factor Krüppel-like factor 2 (KLF2) binds to the CpG sequences in the hTERT promoter and inhibits hTERT gene expression in normal resting T-cells. The human T-cell line Kit 225 in the resting phase induced by the deprivation of interleukin (IL)-2 showed no decrease in the expression of hTERT, despite the high expression of KLF2. To elucidate the mechanisms of deregulation of hTERT expression in T-cells, we examined the relationship between DNA methylation and KLF2 binding to the hTERT promoter. The hTERT promoter was methylated in Kit 225 cells, resulting in the inhibition of the binding of KLF2 to the promoter. DNA demethylation by the reagent Zebularine recovered KLF2 binding to the hTERT promoter, followed by the downregulation of its gene expression. These findings indicate that the repressive effect of KLF2 on hTERT gene expression is abolished by DNA methylation in T-cell lines.
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OBJECTIVES: Scirrhous gastric cancer, which accounts for approximately 10% of all gastric cancers, often disseminates to the peritoneum, leading to intractable cases with poor prognosis. There is an urgent need for new treatment approaches for this difficult cancer. METHODS: We previously established an original cell line, HSC-60, from a scirrhous gastric cancer patient and isolated a peritoneal-metastatic cell line, 60As6, in nude mice following orthotopic inoculations. In the present study, we focused on the expression of long noncoding ribonucleic acid (RNA) (lncRNA) in the cell lines and investigated the mechanism on peritoneal dissemination. RESULTS: We demonstrated that an lncRNA, HOX transcript antisense RNA (HOTAIR), is expressed significantly more highly in 60As6 than HSC-60 cells. Then, using both HOTAIR knockdown and overexpression experiments, we showed that high-level expression of HOTAIR promotes epithelial-mesenchymal transition (EMT) in 60As6 cells. By luciferase assay, we found that HOTAIR directly targets and binds to miR-217, and that miR-217 directly binds to Zinc finger E-box-binding homeobox 1 (ZEB1). The knockdown of HOTAIR in 60As6 cells significantly reduced the invasion activity and peritoneal dissemination - and significantly prolonged the survival - in the orthotopic tumor mouse model. CONCLUSION: An EMT-associated pathway (the HOTAIR-miR-217-ZEB1 axis) appears to inhibit peritoneal dissemination and could lead to a novel therapeutic strategy against scirrhous gastric cancer in humans.
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Adenocarcinoma Esquirroso/genética , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , Peritônio/patologia , RNA Longo não Codificante/genética , Neoplasias Gástricas/genética , Adenocarcinoma Esquirroso/secundário , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Gástricas/secundárioRESUMO
Background and Aims: The Lewis Score (LS) and Capsule Endoscopy Crohn's Disease Activity Index (CECDAI) are the two currently used small bowel capsule endoscopy (SBCE) scoring systems for Crohn's disease (CD). The present study describes a new scoring system for evaluation of small bowel CD, especially mucosal inflammation. Methods: In this cross-sectional study, 108 CD patients underwent 196 SBCEs. The small bowel lesions were scored using our new Crohn's Disease Activity in Capsule Endoscopy (CDACE). CDACE is the sum of scores for location of inflammation, range of inflammation, and stenosis, with a value ranging from 0 to 1643. We analyzed the relation between CDACE and LS, CECDAI, CDAI, and CRP values and evaluated the inter-rater reliability of CDACE using the intraclass correlation coefficient (ICC) (2.1). Results: The mean (±SD) values of LS, CECDAI, and CDACE were 501 ± 1177, 5.8 ± 5.4 and 431 ± 356, respectively. CDACE correlated significantly with LS and CECDAI (ρ = 0.737, P < 0.0001 for LS and ρ = 0.915, P < 0.0001 for CECDAI). CDACE also correlated significantly with CDAI (ρ = 0.36) and CRP (ρ = 0.23). The ICC (2.1) was 0.829, indicating strong agreement among readers. Conclusions: CDACE is a potentially useful SBCE scoring system for small bowel CD, as it represents the extent and spread of small bowel mucosal inflammation and stenosis.
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Tau aggregates in neurons of brain lesions is a hallmark pathology of tauopathies, including Alzheimer's disease (AD). Recent studies suggest that the RNA-binding protein TIA1 initiates Tau aggregation by inducing the formation of stress granules (SGs) containing Tau. SGs are stress-inducible cytoplasmic protein aggregates containing many RNA-binding proteins that has been implicated as an initial site of multiple pathogenic protein aggregates in several neurodegenerative diseases. In this study, we found that ubiquitin-specific protease 10 (USP10) is a critical factor for the formation of Tau/TIA1/USP10-positive SGs. Proteasome inhibition or TIA1-overexpression in HT22 neuronal cells induced the formation of TIA1/Tau-positive SGs, and the formations were severely attenuated by depletion of USP10. In addition, the overexpression of USP10 without stress stimuli in HT22 cells induced TIA1/Tau/USP10-positive SGs in a deubiquitinase-independent manner. In AD brain lesions, USP10 was colocalized with Tau aggregates in the cell body of neurons. The present findings suggest that USP10 plays a key role in the initiation of pathogenic Tau aggregation in AD through SG formation.
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Grânulos Citoplasmáticos/metabolismo , Neurônios/metabolismo , Ubiquitina Tiolesterase/metabolismo , Proteínas tau/metabolismo , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND AND STUDY AIMS: The aim of this study was tp compare the diagnostic efficiency of the PillCam SB3 capsule endoscopy (CE) system with the older system, PillCam SB2, taking into consideration the experience of the image reader. PATIENTS AND METHODS: Small intestinal CE was conducted on 64 patients around May 2014 when the SB3 was introduced in our hospital. Data obtained from 20 patients (SB2: 10 and SB3: 10) based on transit time were assessed by junior (experience: 20 images), intermediate (>â50), and expert readers (>â600). RESULTS: Reading time with the CE down to the end of the small intestine was shorter in the SB3 group for each reader (SB2 vs. SB3: junior, 40.2â±â10.1 vs. 23.7â±â6.7 [ P â=â0.0009]; intermediate, 21.4â±â4.9 vs. 10.3â±â2.9 [ P â=â0.0003]; expert, 23.2â±â5.6 vs. 11.1â±â2.9âmin [ P â=â0.0002]). Interpretation agreement rates between the findings by junior and intermediate readers and those by the expert reader were 84.6 % and 92.3â%, respectively. For the junior reader, rates of agreement using the SB2 and SB3 systems with those by the expert reader were 85.7â% and 83.3â%, respectively; no significant difference was noted between the two systems. Similarly, for the intermediate reader, the respective agreement rates using the SB2 and SB3 systems were 85.7â% and 100â%, respectively. CONCLUSIONS: The PillCam SB3 reduces the time burden on readers irrespective of their experience.
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OBJECTIVES: Scirrhous gastric cancers grow rapidly, and frequently invade the peritoneum. Such peritoneal dissemination properties markedly reduce patient survival. Thus, an effective means for inhibiting peritoneal dissemination is urgently required. METHODS: We previously established a cell line, HSC-58, from a scirrhous gastric cancer patient, and further successfully isolated a metastatic line, 58As9, in nude mice upon orthotopic inoculation. Using the lines, we examined the mechanism underlying peritoneal dissemination from the viewpoint of microRNA (miRNA) expression. RESULTS: miRNA array and qRT-PCR analysis showed that the expressions of epithelial-mesenchymal transition (EMT)-associated miRNAs such as miR-200c and miR-141 were significantly low in 58As9. Using 58As9 with stably overexpressing miR-200c, miR-141, or both, together with a luciferase reporter assay, we found that miR-200c targeted zinc finger E-box-binding homeobox 1 (ZEB1) and miR-141 targeted ZEB2. The overexpressed lines reversed the EMT status from mesenchymal to epithelial in 58As9, and significantly reduced the invasion activity and peritoneal dissemination for a significant prolongation of survival in the orthotopic tumor models in nude mice. CONCLUSIONS: EMT-associated miRNAs such as miR-200c and miR-141 and their target genes ZEB1/ZEB2 have good potential for antiperitoneal dissemination therapy in patients with scirrhous gastric cancers.
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Adenocarcinoma Esquirroso/patologia , Transição Epitelial-Mesenquimal/genética , Invasividade Neoplásica/genética , Neoplasias Peritoneais/secundário , Neoplasias Gástricas/patologia , Adenocarcinoma Esquirroso/genética , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , MicroRNAs/genética , Neoplasias Peritoneais/genética , Neoplasias Gástricas/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/biossíntese , Homeobox 1 de Ligação a E-box em Dedo de Zinco/biossínteseRESUMO
Membrane-associated guanylate kinase with inverted orientation protein 1 (MAGI-1) is a cytoplasmic scaffold protein that interacts with various signaling molecules; it negatively controls the cell growth of various types of cells and positively controls cell-cell interaction. In T cells, MAGI-1 has been shown to inhibit Akt activity through its interaction with PTEN and MEK1. In this study we found that MAGI-1 expression is decreased in multiple (9 out of 15) human T-cell leukemia cell lines, including adult T-cell leukemia (ATL), T-cell acute lymphoblastic leukemia and chronic T-cell lymphocytic leukemia. The overexpression of MAGI-1 protein in a MAGI-1-low ATL cell line reduced cellular growth. While the overexpression of MAGI-1 protein in a MAGI-1-low ATL cell line reduced the Akt and MEK activities, the knockdown of MAGI-1 in a MAGI-1-high ATL cell line augmented the Akt and MEK activities. Collectively, the findings of the present study suggest that the decreased expression of MAGI-1 in human T cells contributes to the development of several types of T-cell leukemia, partly through the stimulation of the Akt and MEK pathways.
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Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Expressão Gênica , Leucemia de Células T/genética , Leucemia de Células T/patologia , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/patologia , Proteínas Adaptadoras de Transdução de Sinal , Moléculas de Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Guanilato Quinases , Humanos , MAP Quinase Quinase 1 , Proteína Oncogênica v-akt , Transdução de SinaisRESUMO
Self-renewal, replication, and differentiation of hematopoietic stem cells (HSCs) are regulated by cytokines produced by niche cells in fetal liver and bone marrow. HSCs must overcome stresses induced by cytokine deprivation during normal development. In this study, we found that ubiquitin-specific peptidase 10 (USP10) is a crucial deubiquitinase for mouse hematopoiesis. All USP10 knockout (KO) mice died within 1 year because of bone marrow failure with pancytopenia. Bone marrow failure in these USP10-KO mice was associated with remarkable reductions of long-term HSCs (LT-HSCs) in bone marrow and fetal liver. Such USP10-KO fetal liver exhibited enhanced apoptosis of hematopoietic stem/progenitor cells (HSPCs) including LT-HSCs but not of lineage-committed progenitor cells. Transplantation of USP10-competent bone marrow cells into USP10-KO mice reconstituted multilineage hematopoiesis. These results suggest that USP10 is an essential deubiquitinase in hematopoiesis and functions by inhibiting apoptosis of HSPCs including LT-HSCs.
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Apoptose , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Ubiquitina Tiolesterase/metabolismo , Anemia/patologia , Animais , Medula Óssea/patologia , Ciclo Celular , Linhagem da Célula , Citocinas/deficiência , Fígado/citologia , Fígado/embriologia , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina Tiolesterase/deficiênciaRESUMO
A 56-year-old man was diagnosed with aplastic anemia and paroxysmal nocturnal hemoglobinuria at 43 years of age and treatment with cyclosporin A was started. Liver cirrhosis, ascites, and thrombus in the hepatic veins were found at 56 years of age and Budd-Chiari syndrome (BCS) was diagnosed according to angiography findings. He was treated with diuretics and paracentesis was performed several times, but with limited efficacy. A Denver® peritoneovenous shunt (PVS) was inserted into the right jugular vein; his ascites and renal function improved immediately and his general condition has remained good for 12 months since starting the above treatment regimen. A PVS is a treatment option for ascites due to BCS.
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Anemia Aplástica/complicações , Ascite/cirurgia , Síndrome de Budd-Chiari/cirurgia , Hemoglobinúria Paroxística/cirurgia , Trombose Venosa/cirurgia , Ascite/complicações , Síndrome de Budd-Chiari/complicações , Hemoglobinúria Paroxística/complicações , Humanos , Cirrose Hepática/complicações , Masculino , Pessoa de Meia-Idade , Derivação Peritoneovenosa , Resultado do Tratamento , Trombose Venosa/complicaçõesRESUMO
Tax1 encoded by the human T-cell leukemia virus type 1 (HTLV-1) has been believed to dysregulate the expression of cellular genes involved in cell survival and mortality, leading to the development of adult T-cell leukemia (ATL). The function of Tax1 in ATL development however is still controversial, primarily because Tax1 induces cell cycle progression and apoptosis. To systemically understand cell growth phase-dependent induction of cell survival or cell death by Tax1, we established a single experimental system using an interleukin 2 (IL-2)-dependent human T-cell line Kit 225 that can be forced into resting phase by IL-2 deprivation. Introduction of Tax1 and HTLV-2 Tax (Tax2B) decreased mitochondrial activity alongside apoptosis in growing cells but not in resting cells. Cell cycle profile analysis indicated that Tax1 and Tax2B were likely to perturb the S phase in growing cells. Studies with Tax1 mutants and siRNA for NF-κB/RelA revealed that Tax1-mediated cell growth inhibition and apoptosis in growing Kit 225 cells depend on RelA. Interestingly, inactivation of the non-canonical NF-κB and p38 MAPK pathways relieved Tax1-mediated apoptosis, suggesting that the Tax1-NF-κB-p38 MAPK axis may be associated with apoptosis in growing cells. Inflammatory mediators such as CCL3 and CCL4, which are involved in oncogene-induced senescence (OIS), were induced by Tax1 and Tax2B in growing cells. In contrast, RelA silencing in resting cells reduced mitochondrial activity, indicating that NF-κB/RelA is also critical for Tax1-mediated cell survival. These findings suggest that Tax1-mediated cell survival and death depend on the cell growth phase. Both effects of Tax1 may be implicated in the long latency of HTLV-1 infection.
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Produtos do Gene tax , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Linfócitos T/citologia , Linfócitos T/virologia , Adulto , Ciclo Celular/genética , Morte Celular , Proliferação de Células , Sobrevivência Celular , Quimiocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas Mutantes/metabolismo , Linfócitos T/enzimologia , Fator de Transcrição RelA/metabolismoRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) is a causative retrovirus of adult T-cell leukemia and HTLV-1-associated myelopathy. Unlike HTLV-1, the same group of retrovirus HTLV-2 has not been found to be associated with these diseases. HTLV-1 and HTLV-2 encode transforming proteins Tax1 and Tax2, and a few distinct activities of Tax1 from those of Tax2 have been proposed to contribute to the HTLV-1-specific pathogenesis of disease. One significant difference of Tax1 from Tax2 is the activation of transcription factor NF-κB2/p100/p52. We found that Tax1 but not Tax2 induces the expression of OX40 ligand (OX40L) in a human T-cell line. To induce the OX40L expression, Tax1 but not Tax2 was observed to interact with NF-κB2/p100/p52 and RelB and the distinct interaction activity was mediated by the Tax1 amino acid region of 225-232. In addition, Tax1 but not Tax2 or Tax1/225-232 interacted with p65, p50, and c-Rel; however, the interactions were much less than those noted with NF-κB2/p100/p52 and RelB. OX40L is a T-cell costimulatory molecule of the tumor necrosis factor family, and its signal plays a critical role in establishing adaptive immunity by inducing the polarized differentiation of T-cells to cells such as T helper type 2 and T follicular helper cells. Therefore, the present findings suggest that Tax1 might alter the immune response to HTLV-1 and/or differentiation of HTLV-1-infected T-cells via OX40L induction, thereby acting as a factor mediating the distinct phenotypes and pathogenesis of HTLV-1 from that of HTLV-2.
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Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Vírus Linfotrópico T Tipo 2 Humano/fisiologia , Subunidade p52 de NF-kappa B/metabolismo , Ligante OX40/biossíntese , Células HEK293 , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Vírus Linfotrópico T Tipo 2 Humano/imunologia , Humanos , Células Jurkat , Linfócitos T/imunologia , Linfócitos T/virologiaRESUMO
The transcription factor caudal-type homeobox 1 (CDX1) is a key regulator of differentiation in the normal colon and in colorectal cancer (CRC). CDX1 activates the expression of enterocyte genes, but it is not clear how the concomitant silencing of stem cell genes is achieved. MicroRNAs (miRNAs) are important mediators of gene repression and have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in CRC, remain poorly understood. Here, we identified microRNA-215 (miR-215) as a direct transcriptional target of CDX1 by using high-throughput small RNA sequencing to profile miRNA expression in two pairs of CRC cell lines: CDX1-low HCT116 and HCT116 with stable CDX1 overexpression, and CDX1-high LS174T and LS174T with stable CDX1 knockdown. Validation of candidate miRNAs identified by RNA-seq in a larger cell-line panel revealed miR-215 to be most significantly correlated with CDX1 expression. Quantitative ChIP-PCR and promoter luciferase assays confirmed that CDX1 directly activates miR-215 transcription. miR-215 expression is depleted in FACS-enriched cancer stem cells compared with unsorted samples. Overexpression of miR-215 in poorly differentiated cell lines causes a decrease in clonogenicity, whereas miR-215 knockdown increases clonogenicity and impairs differentiation in CDX1-high cell lines. We identified the genome-wide targets of miR-215 and found that miR-215 mediates the repression of cell cycle and stemness genes downstream of CDX1. In particular, the miR-215 target gene BMI1 has been shown to promote stemness and self-renewal and to vary inversely with CDX1. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in CRC.
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Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/citologia , Diferenciação Celular , Linhagem Celular Tumoral , Colo/metabolismo , Ilhas de CpG , Perfilação da Expressão Gênica , Células HCT116 , Humanos , Complexo Repressor Polycomb 1/metabolismo , Análise de Sequência de RNA , TransfecçãoRESUMO
In normal human T cells, telomerase activity is strictly regulated. T cells are thought to express telomerase to avoid replicative senescence, unlike most normal somatic cells with definite replicative lifespan. T cells in blood and tissues are usually in a state of quiescence without expression of the limiting catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT). In contrast to activation, repression of hTERT transcription has not been studied well. Our previous studies have found an hTERT promoter element with repressive function. Here we identified KLF2, which represses hTERT transcription by binding to the putative promoter element. KLF2 and hTERT exhibited reciprocal mRNA expression patterns in primary human T cells. In activated T cells, KLF2 binding to the hTERT promoter was eliminated, relieving the repression of hTERT transcription found in resting T cells. Our results suggest that KLF2 is involved in strict repression of hTERT expression through binding to the promoter in primary human T cells.
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Fatores de Transcrição Kruppel-Like/fisiologia , Linfócitos T/enzimologia , Telomerase/metabolismo , Transcrição Gênica/fisiologia , Sequência de Bases , Western Blotting , Domínio Catalítico , Células Cultivadas , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/genética , Telomerase/química , Telomerase/genéticaRESUMO
Human cancers are driven by genetic mutations which cause aberrant activation of pro-growth pathways. Although cancers are uniquely dependent on the pro-growth signaling from oncogenic pathways, efforts to directly target these have been largely unsuccessful. One of the most common and drug resistant oncogenic drivers in colon cancer is the GTPase KRAS. It has been shown that colon cancers with KRAS driver mutations are also 'addicted' to proteins outside of the KRAS pathway due to aberrant re-wiring of cell signaling. A number of genes with a synthetic lethal relationship to mutant KRAS have been previously identified by RNAi screens. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, and their expression is frequently dysregulated in cancers. Recently, we have used an innovative functional miRNA screening approach to identify miRNAs that inhibit the survival of KRAS-mutant cells but not KRAS-wild-type cells. MiR-126 was one of the miRNAs that displayed this selective effect. We found that miR-126 induced synthetic lethality in KRAS-Mutant cells via the down-regulation of the polo-like kinase signaling network and a number of genes specifically necessary for the growth of KRAS-Mutant tumors. This study offers a new way forward for exploiting the regulatory power of miRNAs to specifically target aberrant cell signaling in cancer.
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We have recently developed a biochemical approach to isolate miRNA-bound mRNAs and have used this method to identify the genome-wide mRNAs regulated by the tumor suppressor miRNA miR-34a. This method involves transfection of cells with biotinylated miRNA mimics, streptavidin pulldown, RNA isolation, and qRT-PCR. The protocol in this chapter describes these steps and the issues that should be considered while designing such pulldown experiments.
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MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transfecção/métodos , Genes Supressores de Tumor , Células HeLa , Humanos , MicroRNAs/genética , RNA Mensageiro/genética , Estreptavidina/químicaRESUMO
The RECQ protein family of helicases has critical roles in protecting and stabilizing the genome. Three of the 5 known members of the human RecQ family are genetically linked with cancer susceptibility syndromes, but the association of the most abundant human RecQ homolog, RECQ1, with cellular transformation is yet unclear. RECQ1 is overexpressed in a variety of human cancers, indicating oncogenic functions. Here, we assessed genome-wide changes in gene expression upon knockdown of RECQ1 in HeLa and MDA-MB-231 cells. Pathway analysis suggested that RECQ1 enhances the expression of multiple genes that play key roles in cell migration, invasion, and metastasis, including EZR, ITGA2, ITGA3, ITGB4, SMAD3, and TGFBR2. Consistent with these results, silencing RECQ1 significantly reduced cell migration and invasion. In comparison to genome-wide annotated promoter regions, the promoters of genes downregulated upon RECQ1 silencing were significantly enriched for a potential G4 DNA forming sequence motif. Chromatin immunoprecipitation assays demonstrated binding of RECQ1 to the G4 motifs in the promoters of select genes downregulated upon RECQ1 silencing. In breast cancer patients, the expression of a subset of RECQ1-activated genes positively correlated with RECQ1 expression. Moreover, high RECQ1 expression was associated with poor prognosis in breast cancer. Collectively, our findings identify a novel function of RECQ1 in gene regulation and indicate that RECQ1 contributes to tumor development and progression, in part, by regulating the expression of key genes that promote cancer cell migration, invasion and metastasis.
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Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , RecQ Helicases/genética , Transcriptoma , Linhagem Celular Tumoral , Proliferação de Células/genética , Dano ao DNA/genética , Reparo do DNA/genética , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica/genética , RecQ Helicases/metabolismoRESUMO
MicroRNAs (miRNAs) regulate the expression of hundreds of genes. However, identifying the critical targets within a miRNA-regulated gene network is challenging. One approach is to identify miRNAs that exert a context-dependent effect, followed by expression profiling to determine how specific targets contribute to this selective effect. In this study, we performed miRNA mimic screens in isogenic KRAS-Wild-type (WT) and KRAS-Mutant colorectal cancer (CRC) cell lines to identify miRNAs selectively targeting KRAS-Mutant cells. One of the miRNAs we identified as a selective inhibitor of the survival of multiple KRAS-Mutant CRC lines was miR-126. In KRAS-Mutant cells, miR-126 over-expression increased the G1 compartment, inhibited clonogenicity and tumorigenicity, while exerting no effect on KRAS-WT cells. Unexpectedly, the miR-126-regulated transcriptome of KRAS-WT and KRAS-Mutant cells showed no significant differences. However, by analyzing the overlap between miR-126 targets with the synthetic lethal genes identified by RNAi in KRAS-Mutant cells, we identified and validated a subset of miR-126-regulated genes selectively required for the survival and clonogenicity of KRAS-Mutant cells. Our strategy therefore identified critical target genes within the miR-126-regulated gene network. We propose that the selective effect of miR-126 on KRAS-Mutant cells could be utilized for the development of targeted therapy for KRAS mutant tumors.
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Neoplasias Colorretais/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Immunoblotting , Camundongos , Camundongos Nus , Mutação , Proteínas Proto-Oncogênicas p21(ras) , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The tumor suppressor p21 acts as a cell cycle inhibitor and has also been shown to regulate gene expression by functioning as a transcription corepressor. Here, we identified p21-regulated microRNAs (miRNAs) by sequencing small RNAs from isogenic p21(+/+) and p21(-/-) cells. Three abundant miRNA clusters, miR-200b-200a-429, miR-200c-141, and miR-183-96-182, were downregulated in p21-deficient cells. Consistent with the known function of the miR-200 family and p21 in inhibition of the epithelial-mesenchymal transition (EMT), we observed EMT upon loss of p21 in multiple model systems. To explore a role of the miR-183-96-182 cluster in EMT, we identified its genome-wide targets and found that miR-183 and miR-96 repressed common targets, including SLUG, ZEB1, ITGB1, and KLF4. Reintroduction of miR-200, miR-183, or miR-96 in p21(-/-) cells inhibited EMT, cell migration, and invasion. Conversely, antagonizing miR-200 and miR-183-96-182 cluster miRNAs in p21(+/+) cells increased invasion and elevated the levels of VIM, ZEB1, and SLUG mRNAs. Furthermore, we found that p21 forms a complex with ZEB1 at the miR-183-96-182 cluster promoter to inhibit transcriptional repression of this cluster by ZEB1, suggesting a reciprocal feedback loop.
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Inibidor de Quinase Dependente de Ciclina p21/genética , Transição Epitelial-Mesenquimal , Proteínas de Homeodomínio/genética , MicroRNAs/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular , Movimento Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HEK293 , Proteínas de Homeodomínio/metabolismo , Humanos , Immunoblotting , Integrina beta1/genética , Integrina beta1/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Família Multigênica , Mutação , Oligonucleotídeos Antissenso/genética , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) Tax (Tax1) plays crucial roles in leukemogenesis in part through activation of NF-κB. In this study, we demonstrated that Tax1 activated an NF-κB binding (gpκB) site of the gp34/OX40 ligand gene in a cell type-dependent manner. Our examination showed that the gpκΒ site and authentic NF-κB (IgκB) site were activated by Tax1 in hematopoietic cell lines. Non-hematopoietic cell lines including hepatoma and fibroblast cell lines were not permissive to Tax1-mediated activation of the gpκB site, while the IgκB site was activated in those cells in association with binding of RelB. However RelA binding was not observed in the gpκB and IgκB sites. Our results suggest that HTLV-1 Tax1 fails to activate the canonical pathway of NF-κB in non-hematopoietic cell lines. Cell type-dependent activation of NF-κB by Tax1 could be associated with pathogenesis by HTLV-1 infection.