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Centromeres are the specialized regions of the chromosomes that direct faithful chromosome segregation during cell division. Despite their functional conservation, centromeres display features of rapidly evolving DNA and wide evolutionary diversity in size and organization. Previous work found that the noncanonical B-form DNA structures are abundant in the centromeres of several eukaryotic species with a possible implication for centromere specification. Thus far, systematic studies into the organization and function of non-B-form DNA in plants remain scarce. Here, we applied the oat system to investigate the role of non-B-form DNA in centromeres. We conducted chromatin immunoprecipitation sequencing using an antibody to the centromere-specific histone H3 variant (CENH3); this accurately positioned oat centromeres with different ploidy levels and identified a series of centromere-specific sequences including minisatellites and retrotransposons. To define genetic characteristics of oat centromeres, we surveyed the repeat sequences and found that dyad symmetries were abundant in oat centromeres and were predicted to form non-B-DNA structures in vivo. These structures including bent DNA, slipped DNA, Z-DNA, G-quadruplexes, and R-loops were prone to form within CENH3-binding regions. Dynamic conformational changes of predicted non-B-DNA occurred during the evolution from diploid to tetraploid to hexaploid oat. Furthermore, we applied the single-molecule technique of AFM and DNA:RNA immunoprecipitation with deep sequencing to validate R-loop enrichment in oat centromeres. Centromeric retrotransposons exhibited strong associations with R-loop formation. Taken together, our study elucidates the fundamental character of non-B-form DNA in the oat genome and reveals its potential role in centromeres.
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Avena , Retroelementos , Avena/genética , Avena/metabolismo , Centrômero/genética , Centrômero/metabolismo , Histonas/genética , Histonas/metabolismo , PoliploidiaRESUMO
Genetic association between E3 ubiquitin ligase SMURF2 and colorectal cancer (CRC) has been identified, while the mechanism remains undefined. Tumor-promoting gene YY1 represents a downstream factor of SMURF2. The study was designed to evaluate the effect of SMURF2 on the malignant phenotypes of CRC cells and the underlying mechanism. The expression pattern of SMURF2 and YY1 in CRC clinical tissues and cells was characterized by immunohistochemistry (IHC) and Western blot. Gain- and loss-of-function experiments were conducted to assess the effect of SMURF2 and YY1 on the behaviors of CRC cells. After bioinformatics analysis, the relationship between YY1 and SENP1 as well as between SENP1 and c-myc was determined by luciferase reporter and ChIP assays. Rescue experiments were performed to show their involvement during CRC progression. Finally, in vivo models of tumor growth were established for validation. SMURF2 was lowly expressed and YY1 was highly expressed in CRC tissues and cells. YY1 overexpression resulted in promotion of CRC cell proliferation, migration, and invasion, which could be reversed by SMURF2. Furthermore, SMURF2 could induce ubiquitination-mediated degradation of YY1, which bound to the SENP1 promoter and upregulated SENP1 expression, leading to enhancement of c-myc expression. The in vivo data revealed the suppressive role of SMURF2 gain-of-function in tumor growth through downregulation of YY1, SENP1, or c-myc. Altogether, our data demonstrate the antitumor activity of SMURF2 in CRC and the anti-tumor mechanism associated with degradation of YY1 and downregulation of SENP1/c-myc.
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Neoplasias Colorretais , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proliferação de Células/genética , Regulação para Baixo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismoRESUMO
Colorectal carcinoma (CRC) poses heavy burden to human health and has an increasing incidence. Currently, the existing biomarkers for CRC bring about restrained clinical benefits. GSK3ß is reported to be a novel therapeutic target for this disease but with undefined molecular mechanisms. Thus, we aimed to investigate the regulatory effect of GSK3ß on CRC progression via FTO/MZF1/c-Myc axis. Firstly, the expression patterns of GSK3ß, FTO, MZF1 and c-Myc were determined after sample collection. Lowly expressed GSK3ß but highly expressed FTO, MZF1 and c-Myc were found in CRC. After transfection of different overexpressed and interference plasmids, the underlying mechanisms concerning GSK3ß in CRC cell functions were analysed. Additionally, the effect of GSK3ß on FTO protein stability was assessed followed by detection of MZF1 m6A modification and MZF1-FTO interaction. Mechanistically, GSK3ß mediated ubiquitination of demethylase FTO to reduce FTO expression. Besides, GSK3ß inhibited MZF1 expression by mediating FTO-regulated m6A modification of MZF1 and then decreased the proto-oncogene c-Myc expression, thus hampering CRC cell proliferation. We also carried out in vivo experiment to verify the regulatory effect of GSK3ß on CRC via FTO-mediated MZF1/c-Myc axis. It was found that GSK3ß inhibited CRC growth in vivo which was reversed by overexpressing c-Myc. Taken together, our findings indicate that GSK3ß suppresses the progression of CRC through FTO-regulated MZF1/c-Myc axis, shedding light onto a new possible pathway by which GSK3ß regulates CRC.
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Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Neoplasias Colorretais/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Modelos Biológicos , Proto-Oncogene Mas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Matrix metalloproteinases (MMPs) are evolutionarily conserved extracellular matrix proteinases. Genetic analysis of the Drosophila MMPs, Mmp1 and Mmp2, in vivo reveal that they play vital roles in tissue remodeling. Although the catalytic domain (CD) undertakes most MMP functions, few studies have sought to demonstrate the biochemical properties of the CDs of fly MMPs. Here, we identified the overexpression, purification, and refolding of the CDs of Drosophila Mmp1 and Mmp2 for biochemical studies. Zymography assays and substrate degradation analysis showed that both Mmp1-CD and Mmp2-CD were able to digest casein, gelatin, fibronectin, collagen (types I, IV, and V), while Mmp2-CD showed much higher degradation activity compared with Mmp1-CD. Moreover, human collagen III could be degraded by Mmp1-CD but not Mmp2-CD, and rat collagen I and laminin could be degraded by Mmp2-CD but not Mmp1-CD, suggesting that Drosophila Mmp1 and Mmp2 might have overlapping yet distinct substrate specificity. Using synthetic fluorescent substrates, we further demonstrated that the enzymatic activity of Mmp1-CD and Mmp2-CD could be inhibited by human tissue inhibitors of metalloproteinases (TIMPs). These results reveal the context of the cooperative yet distinct roles of Mmp1 and Mmp2 in tissue remodeling.
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Proteínas de Drosophila , Metaloproteinase 1 da Matriz , Metaloproteinase 2 da Matriz , Animais , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster , Metaloproteinase 1 da Matriz/biossíntese , Metaloproteinase 1 da Matriz/química , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/isolamento & purificação , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Especificidade por SubstratoRESUMO
To improve the neural network detection accuracy of the electric power bushings in infrared images, a modified algorithm based on the You Only Look Once version 2 (YOLOv2) network is proposed to achieve better recognition results. Specifically, YOLOv2 corresponds to a convolutional neural network (CNN), although its rotation invariance is poor, and some bounding boxes (BBs) exhibit certain deviations. To solve this problem, the standard Hough transform and image rotation are utilized to determine the optimal recognition angle for target detection, such that an optimal recognition effect of YOLOv2 on inclined objects (for example, bushing) is achieved. With respect to the problem that the BB is biased, the shape feature of the bushing is extracted by the Gap statistic algorithm, based on K-means clustering; thereafter, the sliding window (SW) is utilized to determine the optimal recognition area. Experimental verification indicates that the proposed rotating image method can improve the recognition effect, and the SW can further modify the BB. The accuracy of target detection increases to 97.33%, and the recall increases to 95%.
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High-sensitive metasurface-based sensors are essential for effective substance detection and insightful bio-interaction studies, which compress light in subwavelength volumes to enhance light-matter interactions. However, current methods to improve sensing performance always focus on optimizing near-field response of individual meta-atom, and fingerprint recognition for bio-substances necessitates several pixelated metasurfaces to establish a quasi-continuous spectrum. Here, a novel sensing strategy is proposed to achieve Terahertz (THz) refractive sensing, and fingerprint recognition based on surface waves (SWs). Leveraging the long-range transmission, strong confinement, and interface sensitivity of SWs, a metasurface-supporting SWs excitation and propagation is experimentally verified to achieve sensing integrations. Through wide-band information collection of SWs, the proposed sensor not only facilitates refractive sensing up to 215.5°/RIU, but also enables the simultaneous resolution of multiple fingerprint information within a continuous spectrum. By covering 5 µm thickness of polyimide, quartz and silicon nitride layers, the maximum phase change of 91.1°, 101.8°, and 126.4° is experimentally obtained within THz band, respectively. Thus, this strategy broadens the research scope of metasurface-excited SWs and introduces a novel paradigm for ultrasensitive sensing functions.
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Continual high expression of cysteine proteases calpain I and II have been implicated in tumorigenicity; conversely, N-acetyl-leu-leunorleucinal (ALLN), which inhibits calpain I and II, should also influence tumor growth and carcinogenesis. To explore the role of ALLN against colon cancer and in promoting apoptosis, we used colon cancer HCT116 cell lines, p53 or Bax-deficient HCT116 cell lines. Cell viability and tumor growth decreased in a concentration-dependent manner when treated with 0-26µM ALLN. Treatment with ALLN induced apoptosis in HCT116 cell; however, flow cytometry showed that apoptosis significantly decreased in Bax-deficient HCT116 cell lines, but not in p53-deficient HCT116 cell lines. In addition, the ALLN-induced apoptosis response was through Bax translocation from cytosol to mitochondria. In this study we showed intraperitoneally injected ALLN to inhibit colon tumor formation in nude mice, and found ALLN to inhibit tumor growth in colon cancer cells, mainly through apoptosis that depends on translocation of Bax to a mitochondrial endogenous pathway; this implies a molecular mechanism for ALLN against human colon cancer. These results suggest that ALLN could become a novel agent for prevention of colon cancer.
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Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Leupeptinas/farmacologia , Proteína X Associada a bcl-2/fisiologia , Linhagem Celular Tumoral , HumanosRESUMO
Pea (Pisum sativum L.) is an important legume crop for both food and feed. Bruchids (Callosobruchus spp.) are destructive insect pests of pea in the field and during storage. In this study, we identified a major quantitative trait locus (QTL) controlling seed resistance to C. chinensis (L.) and C. maculatus (Fab.) in field pea using F2 populations derived from a cross between PWY19 (resistant) and PHM22 (susceptible). QTL analysis in the two F2 populations grown in different environments consistently identified a single major QTL, qPsBr2.1, controlling the resistance to both bruchid species. qPsBr2.1 was mapped onto linkage group 2 between DNA markers 18339 and PSSR202109 and explained 50.91% to 70.94% of the variation in resistance, depending on the environment and bruchid species. Fine mapping narrowed down qPsBr2.1 to a genomic region of 1.07 Mb on chromosome 2 (chr2LG1). Seven annotated genes were found in this region, including Psat2g026280 (designated as PsXI), which encodes a xylanase inhibitor and was considered as a candidate gene for bruchid resistance. PCR amplification and sequence analysis of PsXI suggested the presence of an insertion of unknown length in an intron of PWY19, which causes variation in the open reading frame (ORF) of PsXI. Moreover, the subcellular localization of PsXI differed between PWY19 and PHM22. These results together suggested that PsXI encoding xylanase inhibitor is responsible for the bruchid resistance of the field pea PWY19.
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Serum response factor (SRF) regulates differentiation and proliferation by binding to RhoA-actin-activated MKL or Ras-MAPK-activated ELK transcriptional coactivators, but the molecular mechanisms responsible for SRF regulation remain unclear. Here, we show that Nemo-like kinase (NLK) is required for the promotion of SRF/ELK signaling in human and mouse cells. NLK was found to interact with and phosphorylate SRF at serine residues 101/103, which in turn enhanced the association between SRF and ELK. The enhanced affinity of SRF/ELK antagonized the SRF/MKL pathway and inhibited mouse myoblast differentiation in vitro. In a skeletal muscle-specific Nlk conditional knockout mouse model, forming muscle myofibers underwent hypertrophic growth, resulting in an increased muscle and body mass phenotype. We propose that both phosphorylation of SRF by NLK and phosphorylation of ELKs by MAPK are required for RAS/ELK signaling, confirming the importance of this ancient pathway and identifying an important role for NLK in modulating muscle development in vivo.
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Targeted cancer therapies have revolutionized treatment but their efficacies are limited by the development of resistance driven by clonal evolution within tumors. We developed "CAPTURE", a single-cell barcoding approach to comprehensively trace clonal dynamics and capture live lineage-coupled resistant cells for in-depth multi-omics analysis and functional exploration. We demonstrate that heterogeneous clones, either preexisting or emerging from drug-tolerant persister cells, dominated resistance to vemurafenib in BRAFV600E melanoma. Further integrative studies uncovered diverse resistance mechanisms. This includes a previously unrecognized and clinically relevant mechanism, chromosome 18q21 gain, which leads to vulnerability of the cells to BCL2 inhibitor. We also identified targetable common dependencies of captured resistant clones, such as oxidative phosphorylation and E2F pathways. Our study provides new therapeutic insights into overcoming therapy resistance in BRAFV600E melanoma and presents a platform for exploring clonal evolution dynamics and vulnerabilities that can be applied to study treatment resistance in other cancers.
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Molecular classification has improved diagnosis and treatment for patients with malignant gliomas. However, classification has relied on individual assays that are both costly and slow, leading to frequent delays in treatment. Here, we propose the use of DNA methylation, as an emerging clinical diagnostic platform, to classify gliomas based on major genomic alterations and provide insight into subtype characteristics. We show that using machine learning models, DNA methylation signatures can accurately predict somatic alterations and show improvement over existing classifiers. The established Unified Diagnostic Pipeline (UniD) we develop is rapid and cost-effective for genomic alterations and gene expression subtypes diagnostic at early clinical phase and improves over individual assays currently in clinical use. The significant relationship between genetic alteration and epigenetic signature indicates broad applicability of our approach to other malignancies.
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Metilação de DNA , Glioma , Metilação de DNA/genética , Epigênese Genética , Epigenômica , Glioma/genética , HumanosRESUMO
Treatment-resistant glioma stem cells are thought to propagate and drive growth of malignant gliomas, but their markers and our ability to target them specifically are not well understood. We demonstrate that podoplanin (PDPN) expression is an independent prognostic marker in gliomas across multiple independent patient cohorts comprising both high- and low-grade gliomas. Knockdown of PDPN radiosensitized glioma cell lines and glioma-stem-like cells (GSCs). Clonogenic assays and xenograft experiments revealed that PDPN expression was associated with radiotherapy resistance and tumor aggressiveness. We further demonstrate that knockdown of PDPN in GSCs in vivo is sufficient to improve overall survival in an intracranial xenograft mouse model. PDPN therefore identifies a subset of aggressive, treatment-resistant glioma cells responsible for radiation resistance and may serve as a novel therapeutic target.
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OBJECTIVE: To develop a targeting protein for Xenopus kinesin-like protein 2 (TPX2) C' terminal SBP-3 x Flag-tagged HCT 116 cell model. METHODS: Homologous arms were amplified by polymerase chain reaction (PCR), and then the adeno-associated virus (AAV) -targeting vector of TPX2 was constructed. HCT 116 cells were targeted after the viruses were packaged. Positive cell clones with neomycin resistance gene were obtained by G418 and PCR screening. Finally, the neomycin gene cassette was excised after the targeted clones were infected with adenovirus expressing Cre-recombinase, and the TPX2 C' terminal SBP and 3 x Flag endogenous double-tagged HCT 116 cells were obtained by PCR screening. RESULTS: Two positive cell clones with neomycin resistance gene were obtained by PCR screening. The positive clones with neomycin resistance gene excised were obtained by Cre adenovirus infection, and the knock-in of SBP-3 x Flag gene was verified by Western blot analysis. CONCLUSION: The TPX2 C' terminal SBP-3 x Flag tagged HCT 116 cell model was successfully established.
Assuntos
Proteínas de Ciclo Celular/genética , Neoplasias Colorretais/patologia , Células HCT116 , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Neoplasias Colorretais/genética , Dependovirus/genética , Marcação de Genes , Vetores Genéticos , HumanosRESUMO
Cancer cell-derived extracellular vesicles (EVs) have been reported to promote the progression of colorectal cancer (CRC), although the regulatory mechanism remains uncharacterized. In this study, we investigated the role of microRNA-25 (miR-25)/sirtuin 6 (SIRT6) in the contribution of EVs derived from CRC cells to progression of CRC. In a co-culture system with EVs from HCT116 and NCM460 cells, the viability, migratory, and invasive properties of SW480 and SW620 cells were evaluated by cell counting kit-8 (CCK-8) and Transwell assays. Luciferase, chromatin immunoprecipitation (ChIP), and RNA immunoprecipitation (RIP) assays were conducted to verify the interaction among miR-25, SIRT6, lin-28 homologB (Lin28b), and neuropilin-1 (NRP-1). It was established that HCT116 cell-derived EVs promoted the malignant properties of SW480 cells and SW620 cells by delivering miR-25. SIRT6 was targeted by miR-25, whereas SIRT6 inhibited NRP-1 through downregulation of Lin28b. The tumor-bearing nude mouse experiments substantiated that HCT116 cell-derived EVs transferred miR-25 to facilitate tumor formation and metastasis by inhibiting SIRT6. In summary, our study clarifies the involvement of miR-25-targeted SIRT6 inhibition and SIRT6-mediated inhibition of the Lin28b/NRP-1 axis in CRC cell-derived EVs to CRC progression and metastasis.
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General plasmonic systems to realize plasmonically induced transparency (PIT) effect only exist one single PIT mainly because they only allow one single coupling pathway. In this study, we propose a distinct graphene resonator-based system, which is composed of graphene nanoribbons (GNRs) coupled with dielectric grating-loaded graphene layer resonators, to achieve two switchable PIT effects. By designing crossed directions of the resonators, the proposed system exists two different PIT effects characterized by different resonant positions and linewidths. These two PIT effects result from two separate and polarization-selective coupling pathways, allowing us to switch the PIT from one to the other by simply changing the polarization direction. Parametric studies are carried to demonstrate the coupling effects whereas the two-particle model is applied to explain the physical mechanism, finding excellent agreements between the numerical and theoretical results. Our proposal can be used to design switchable PIT-based plasmonic devices, such as tunable dual-band sensors and perfect absorbers.
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An amendment to this paper has been published and can be accessed via the original article.
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The transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2) is one of the master regulators that control hundreds of genes containing antioxidant response elements (AREs). The NRF2-ARE pathway plays a complex role in colorectal cancer (CRC). NRF2 activity is known to be regulated by KEAP1-CUL3 E3 ligase-mediated ubiquitination, indicating the importance of deubiquitination regulation. However, the deubiquitinase (DUB) of NRF2 remains unknown. Here, by screening a DUB library, we identified DUB3 as a DUB that remarkably stabilized NRF2. Further experiments demonstrated that DUB3 promoted NRF2 stability and transcriptional activity by decreasing the K48-linked ubiquitination of NRF2. Coimmunoprecipitation studies revealed interactions between NRF2 and DUB3, as well as between KEAP1 and DUB3, indicating that NRF2, DUB3, and KEAP1 formed a large functional complex. Importantly, ectopic expression of DUB3 caused NRF2-dependent chemotherapy resistance in colon cancer cell lines. Thus, to the best of our knowledge, our findings are the first to identify DUB3 as a NRF2 DUB and may provide a new strategy against chemotherapy resistance in CRC and other NRF2-related diseases.
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Neoplasias Colorretais/patologia , Enzimas Desubiquitinantes/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Endopeptidases/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/fisiologia , Sistemas CRISPR-Cas/genética , Proliferação de Células/fisiologia , Neoplasias Colorretais/tratamento farmacológico , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/genética , Paclitaxel/uso terapêutico , Ativação Transcricional/genética , Ubiquitinação/fisiologiaRESUMO
A series of novel water-soluble 4-quinolone-3-carboxamides was prepared and evaluated as antiproliferative agents. Preliminary results indicated that most compounds tested in this study showed potent antiproliferative potencies against human tumor cell lines, and compound 8k was found to be the most potent antiproliferative agents with IC50 value of lower than 10 µM against nine human tumor cell lines. These results suggested that (1) the alkylamino side chain substituent was the advisable pharmacophoric group for the enhanced antiproliferative activities; (2) the length of the alkylamino side chain moiety also affected their antiproliferative potencies, and three methylene units were more favorable; (3) introducing arylated alkyl substituent into N1-position of quinolone facilitated antiproliferative activities of this class of compounds. Further investigations on mechanism of action of this class of compound demonstrated that the representative compound 8k could trigger p53/Bax-independent colorectal cancer cell apoptosis via inducing ROS accumulation.
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Antineoplásicos/farmacologia , Quinolonas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Quinolonas/síntese química , Quinolonas/química , Solubilidade , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Água/químicaRESUMO
Interleukin-1ß (IL-1ß) is a key proinflammatory cytokine that initiates several signaling cascades, including those involving CCAAT/enhancer binding proteins (C/EBPs). The mechanism by which IL-1ß propagates a signal that activates C/EBP has remained elusive. Nemo-like kinase (NLK) is a mitogen-activated protein kinase (MAPK)-like kinase associated with many pathways and phenotypes that are not yet well understood. Using a luciferase reporter screen, we found that IL-1ß-induced C/EBP activation was positively regulated by NLK. Overexpression of NLK activated C/EBP and potentiated IL-1ß-triggered C/EBP activation, whereas knockdown or knockout of NLK had the opposite effect. NLK interacted with activating transcription factor 5 (ATF5) and inhibited the proteasome-dependent degradation of ATF5 in a kinase-independent manner. Consistently, NLK deficiency resulted in decreased levels of ATF5. NLK cooperated with ATF5 to activate C/EBP, whereas NLK could not activate C/EBP upon knockdown of ATF5. Moreover, TAK1, a downstream effector of IL-1ß that acts upstream of NLK, mimicked the ability of NLK to stabilize ATF5 and activate C/EBP. Thus, our findings reveal the TAK1-NLK pathway as a novel regulator of basal or IL-1ß-triggered C/EBP activation though stabilization of ATF5.
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Fatores Ativadores da Transcrição/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Regulação Enzimológica da Expressão Gênica , Interleucina-1beta/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Animais , Sequência de Bases , Linhagem Celular Tumoral , Células HEK293 , Humanos , Luciferases/metabolismo , Camundongos , Camundongos Knockout , Microscopia Confocal , Dados de Sequência Molecular , Fosforilação , Plasmídeos/metabolismo , Interferência de RNA , TransfecçãoRESUMO
We collected paired samples of tumor and adjacent normal colorectal tissues from 22 patients with colorectal carcinoma to compare the differences in the expression of lysine specific demethylase 1 (LSD1) in these two tissues. The results showed that in 19 paired samples (86.4%), LSD1 is more highly expressed in tumor tissue than in normal tissue. To explore the role of LSD1 in colorectal tumorigenesis, we used somatic cell gene targeting to generate an LSD1 knockout (KO) HCT 116 human colorectal cancer cell line as a research model. The analysis of phenotypic changes showed that LSD1 KO colorectal cancer cells are less tumorigenic, both in vivo and in vitro. The differential expression analysis of the cells by mRNA sequencing (RNA-Seq) yielded 2,663 differentially expressed genes, and 28 of these genes had highly significant differences (Q <0.01). We then selected the 4 colorectal cancer-related genes ADM, DKK1, HAS3 and SMURF2 for quantitative real-time PCR verification. The results showed that the differences in the expression of ADM, DKK1 and HAS3 were consistent with those measured using the RNA-Seq data. As DKK1 was the gene with the most significant differential expression, we analyzed the key proteins of the DKK1-related Wnt/ß-catenin signaling pathway and found that, after knocking out LSD1, the amount of free ß-catenin translocated to the nucleus was significantly reduced and that the transcription of the signaling pathway target gene c-Myc was down-regulated. Our studies show that LSD1 activates the Wnt/ß-catenin signaling pathway by down-regulating the pathway antagonist DKK1, which may be one of the mechanisms leading to colorectal tumorigenesis.