RESUMO
Lysine-specific histone demethylase 1 (LSD1), which demethylates mono- or di- methylated histone H3 on lysine 4 (H3K4me1/2), is essential for early embryogenesis and development. Here we show that LSD1 is dispensable for mouse embryonic stem cell (ESC) self-renewal but is required for mouse ESC growth and differentiation. Reintroduction of a catalytically-impaired LSD1 (LSD1MUT) recovers the proliferation capability of mouse ESCs, yet the enzymatic activity of LSD1 is essential to ensure proper differentiation. Indeed, increased H3K4me1 in Lsd1 knockout (KO) mouse ESCs does not lead to major changes in global gene expression programs related to stemness. However, ablation of LSD1 but not LSD1MUT results in decreased DNMT1 and UHRF1 proteins coupled to global hypomethylation. We show that both LSD1 and LSD1MUT control protein stability of UHRF1 and DNMT1 through interaction with HDAC1 and the ubiquitin-specific peptidase 7 (USP7), consequently, facilitating the deacetylation and deubiquitination of DNMT1 and UHRF1. Our studies elucidate a mechanism by which LSD1 controls DNA methylation in mouse ESCs, independently of its lysine demethylase activity.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Diferenciação Celular , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , Histona Desmetilases , Camundongos Knockout , Células-Tronco Embrionárias Murinas , Ubiquitina-Proteína Ligases , Animais , Histona Desmetilases/metabolismo , Histona Desmetilases/genética , Camundongos , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Células-Tronco Embrionárias Murinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 1/genética , Histonas/metabolismo , Proliferação de Células , UbiquitinaçãoRESUMO
BACKGROUND: Cholangiocarcinoma (CCA) is a highly malignant tumor characterized by a lack of effective targeted therapeutic strategies. The protein UHRF1 plays a pivotal role in the preservation of DNA methylation and works synergistically with DNMT1. Posttranscriptional modifications (PTMs), such as ubiquitination, play indispensable roles in facilitating this process. Nevertheless, the specific PTMs that regulate UHRF1 in CCA remain unidentified. METHODS: We confirmed the interaction between STUB1 and UHRF1 through mass spectrometry analysis. Furthermore, we investigated the underlying mechanisms of the STUB1-UHRF1/DNMT1 axis via co-IP experiments, denaturing IP ubiquitination experiments, nuclearâcytoplasmic separation and immunofluorescence experiments. The downstream PLA2G2A gene, regulated by the STUB1-UHRF1/DNMT1 axis, was identified via RNA-seq. The negative regulatory mechanism of PLA2G2A was explored via bisulfite sequencing PCR (BSP) experiments to assess changes in promoter methylation. The roles of PLA2G2A and STUB1 in the proliferation, invasion, and migration of CCA cells were assessed using the CCK-8 assay, colony formation assay, Transwell assay, wound healing assay and xenograft mouse model. We evaluated the effects of STUB1/UHRF1 on cholangiocarcinoma by utilizing a primary CCA mouse model. RESULTS: This study revealed that STUB1 interacts with UHRF1, resulting in an increase in the K63-linked ubiquitination of UHRF1. Consequently, this facilitates the nuclear translocation of UHRF1 and enhances its binding affinity with DNMT1. The STUB1-UHRF1/DNMT1 axis led to increased DNA methylation of the PLA2G2A promoter, subsequently repressing its expression. Increased STUB1 expression in CCA was inversely correlated with tumor progression and overall survival. Conversely, PLA2G2A functions as a tumor suppressor in CCA by inhibiting cell proliferation, invasion and migration. CONCLUSIONS: These findings suggest that the STUB1-mediated ubiquitination of UHRF1 plays a pivotal role in tumor progression by epigenetically silencing PLA2G2A, underscoring the potential of STUB1 as both a prognostic biomarker and therapeutic target for CCA.
Assuntos
Neoplasias dos Ductos Biliares , Proteínas Estimuladoras de Ligação a CCAAT , Colangiocarcinoma , Metilação de DNA , Progressão da Doença , Ubiquitina-Proteína Ligases , Ubiquitinação , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Camundongos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Animais , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/metabolismo , Masculino , Proliferação de Células , Feminino , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genéticaRESUMO
Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) still faces great challenges due to uncontrollable inflammation disorders, complicated causes of occurrence, and high mortality. Small-activating RNA (saRNA) has emerged as a novel and powerful gene-activating tool that may be useful in the treatment of ALI/ARDS. However, effective saRNA therapy is still challenged by the lack of effective and safe gene delivery vehicles. In this study, we develop a type of artificial neutrophil that is used to deliver saRNAs for ALI/ARDS treatment. The saRNA targeting CCAAT-enhancer binding protein α (CEBPA-saRNA) is complexed with H1 histone and further camouflaged with neutrophil membranes (NHR). Interestingly, we are the first to find that the H1 histone possesses the most effective binding capability to saRNA, compared to other subtypes. The prepared NHR shows excellent physicochemical properties, effective cellular uptake by the inflammatory M1 macrophages, and efficient activation of CEBPA, leading to significant M2 polarization. NHR shows an extended circulation lifetime and high-level accumulation in the inflamed lungs. The in vivo experiments indicate that NHR ameliorates ALI in a mouse model. This type of artificial neutrophil shows powerful inflammatory inhibition both in vitro and in vivo, which opens a new avenue for the treatment of ALI/ARDS.
Assuntos
Lesão Pulmonar Aguda , Neutrófilos , Síndrome do Desconforto Respiratório , Animais , Neutrófilos/metabolismo , Camundongos , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/patologia , Humanos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Camundongos Endogâmicos C57BL , Histonas/metabolismo , Histonas/químicaRESUMO
Embryonic stem cells are crucial for studying developmental biology due to their self-renewal and pluripotency capabilities. This research investigates the differentiation of mouse ESCs into adipocytes, offering insights into obesity and metabolic disorders. Using a monolayer differentiation approach over 30 days, lipid accumulation and adipogenic markers, such as Cebpb, Pparg, and Fabp4, confirmed successful differentiation. RNA sequencing revealed extensive transcriptional changes, with over 15,000 differentially expressed genes linked to transcription regulation, cell cycle, and DNA repair. This study utilized Robust Rank Aggregation to identify critical regulatory genes like PPARG, CEBPA, and EP300. Network analysis further highlighted Atf5, Ccnd1, and Nr4a1 as potential key players in adipogenesis and its mature state, validated through RT-PCR. While key adipogenic factors showed plateaued expression levels, suggesting early differentiation events, this study underscores the value of ESCs in modeling adipogenesis. These findings contribute to our understanding of adipocyte differentiation and have significant implications for therapeutic strategies targeting metabolic diseases.
Assuntos
Adipócitos , Adipogenia , Diferenciação Celular , Células-Tronco Embrionárias Murinas , Animais , Adipogenia/genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Diferenciação Celular/genética , Adipócitos/metabolismo , Adipócitos/citologia , PPAR gama/metabolismo , PPAR gama/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Transcrição Gênica , Regulação da Expressão GênicaRESUMO
Recently, we have demonstrated that mice, cultured embryos in α-minimum essential medium (αMEM) and subsequent fed a high-fat, high-sugar diet, developed steatohepatitis. In this study, we investigated using these samples whether the expression of lipid droplet formation genes in the liver is higher in MEM mice, whether these expressions are regulated by histone acetylation, writers/readers of histone acetylation, and the transcriptional factors of endoplasmic reticulum stress. Mice were produced by two-cell embryos in αMEM or standard potassium simplex-optimized medium (control) in vitro for 48 h, and implanted into an oviduct for spontaneous delivery. MEM and control-mice were fed a high-fat, high-sugar diet for 18 wk, and then liver samples were collected and analyzed by histology, qRT-PCR, and chromatin immunoprecipitation assay. Gene expression of Cidea, Cidec, and Plin4 were higher in MEM mice and histone H3K9 acetylation, BRD4, and CBP were higher in MEM mice than in control mice around those genes. However, the binding of endoplasmic reticulum stress-related transcription factors (ATF4, CHOP and C/EBPα) around those genes in the liver, was not clearly differed between MEM mice and control mice. The increased expression of Cidea, Cidec and Plin4 in the liver, accompanied by the development of steatohepatitis in mice induced is positively associated with increased histone H3K9 acetylation and CBP and BRD4 binding around these genes.
Assuntos
Estresse do Retículo Endoplasmático , Fígado Gorduroso , Histonas , Gotículas Lipídicas , Fígado , Animais , Histonas/metabolismo , Acetilação , Gotículas Lipídicas/metabolismo , Camundongos , Feminino , Fígado/metabolismo , Fígado Gorduroso/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/etiologia , Fator 4 Ativador da Transcrição/metabolismo , Fator 4 Ativador da Transcrição/genética , Dieta Hiperlipídica/efeitos adversos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fator de Transcrição CHOP/metabolismo , Fator de Transcrição CHOP/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genéticaRESUMO
Transcription factors (TFs) have an important role in the regulation of the gene expression network. The role of TFs in the immune response of freshwater crayfish is poorly understood, but leveraging the regulatory mechanisms of immune response could augment the resistance against the invasive oomycete pathogen, Aphanomyces astaci. Previous studies indicated that the TFs CCAAT/enhancer-binding protein (C/EBP) and putative Krüppel homolog-1 protein (Kr-h1) might play a role in immune and stress response of the noble crayfish (Astacus astacus). Here, we aimed to further characterise these two gene products to gain a better understanding of their evolutionary origin, domain organisation and expression patterns across different crayfish tissues. Furthermore, we conducted an immune stimulation experiment to observe the potential changes in the gene expression of C/EBP and Kr-h1 under immune challenge in different crayfish tissues. Our results showed that both C/EBP and Kr-h1 are closely related to other C/EBPs and Kr-h1s in Malacostraca. Gene expression analysis revealed that both TFs are present in all analysed tissues, with higher expression of C/EBP in the gills and Kr-h1 in the abdominal muscle. Immune stimulation with laminarin (mimicking ß-1-3-glucan in the oomycete cell wall) showed an activation of the crayfish immune system, with an overall increase in the total haemocyte count (THC) compared to untreated control and crayfish buffered saline (CBS) treatment. On the gene expression level, an up-regulation of the C/EBP gene was detected in the laminarin treated group in hepatopancreas and heart, while no changes were observed for the Kr-h1 gene. Our results indicate an early change in C/EBP expression in multiple tissues during immune stimulation and suggest its involvement in the immune response of the noble crayfish.
Assuntos
Astacoidea , Animais , Astacoidea/imunologia , Astacoidea/genética , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Regulação da Expressão Gênica , FilogeniaRESUMO
Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency, centromeric instability, and facial anomalies syndrome, characterized by DNA hypomethylation at heterochromatin. It remains unclear why CDCA7-HELLS is the sole nucleosome remodeling complex whose deficiency abrogates the maintenance of DNA methylation. We here identify the unique zinc-finger domain of CDCA7 as an evolutionarily conserved hemimethylation-sensing zinc finger (HMZF) domain. Cryo-electron microscopy structural analysis of the CDCA7-nucleosome complex reveals that the HMZF domain can recognize hemimethylated CpG in the outward-facing DNA major groove within the nucleosome core particle, whereas UHRF1, the critical activator of the maintenance methyltransferase DNMT1, cannot. CDCA7 recruits HELLS to hemimethylated chromatin and facilitates UHRF1-mediated H3 ubiquitylation associated with replication-uncoupled maintenance DNA methylation. We propose that the CDCA7-HELLS nucleosome remodeling complex assists the maintenance of DNA methylation on chromatin by sensing hemimethylated CpG that is otherwise inaccessible to UHRF1 and DNMT1.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Metilação de DNA , Nucleossomos , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Nucleossomos/metabolismo , Nucleossomos/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Microscopia Crioeletrônica , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Ilhas de CpG , Ubiquitinação , Evolução Molecular , DNA/metabolismo , DNA/química , DNA/genética , Dedos de Zinco , Cromatina/metabolismo , Cromatina/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA Helicases/metabolismo , DNA Helicases/genética , DNA Helicases/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/química , Eucariotos/genética , Eucariotos/metabolismo , Ligação Proteica , Histonas/metabolismo , Histonas/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/químicaRESUMO
Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an epigenetic regulator that plays critical roles in tumours. However, the DNA methylation alteration patterns driven by UHRF1 and the related differentially expressed tumour-related genes remain unclear. In this study, a UHRF1-shRNA MCF-7 cell line was constructed, and whole-genome bisulfite sequencing and RNA sequencing were performed. The DNA methylation alteration landscape was elucidated, and DNA methylation-altered regions (DMRs) were found to be distributed in both gene bodies and adjacent regions. The DMRs were annotated and categorized into 488 hypermethylated/1696 hypomethylated promoters and 1149 hypermethylated/5501 hypomethylated gene bodies. Through an integrated analysis with the RNA sequencing data, 217 methylation-regulated upregulated genes and 288 downregulated genes were identified, and these genes were primarily enriched in nervous system development and cancer signalling pathways. Further analysis revealed 21 downregulated oncogenes and 15 upregulated TSGs. We also showed that UHRF1 silencing inhibited cell proliferation and migration and suppressed tumour growth in vivo. Our study suggested that UHRF1 and the oncogenes or TSGs it regulates might serve as biomarkers and targets for breast cancer treatment.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células MCF-7 , Feminino , Proliferação de Células/genética , Animais , Regiões Promotoras Genéticas , Camundongos , Epigênese Genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Movimento Celular/genéticaRESUMO
Objective: To demonstrate the type of CEBPA gene mutations among patients with acute myeloid leukemia (AML), clinical characteristics, and prognostic effect on patient outcomes. Methods: Demographic data, clinical features, laboratory characteristics, and data about treatment and follow-up of 57 patients with CEBPA mutated AML diagnosed at Peking Union Medical College Hospital between April 2016 and November 2022 were collected and analyzed. Results: In total, 57 patients with CEBPA mutation accounted for 16.1% of all the 353 patients with AML, among which 28 patients had CEBPA-bZIPinf and 29 had CEBPA-other. Compared with the CEBPA-other group, the CEBPA-bZIPinf group was younger (54 vs 64 years, P=0.010), de novo AML was more common (P=0.001), and the level of bone marrow blast was higher (68.0% vs 36.3%, P=0.001). Moreover, 24 patients from the CEBPA-bZIPinf group and 19 from the CEBPA-other group received chemotherapy. The one-course complete remission (CR) rate of the CEBPA-bZIPinf group was significantly higher than that of the CEBPA-other (87.5% vs 47.4%, P=0.010) and CEBPA-wt (87.5% vs 50.3%, P=0.002) groups. After a median follow-up of 11 months, the median OS of the CEBPA-bZIPinf group was significantly longer than that of the CEBPA-wt group (not reached vs 22.1 months, P=0.012) . Conclusion: CEBPA-bZIPinf mutated AML is a unique clinical entity, with a younger age of diagnosis, better response to chemotherapy, and better prognosis.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Leucemia Mieloide Aguda , Mutação , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/diagnóstico , Proteínas Estimuladoras de Ligação a CCAAT/genética , Prognóstico , Pessoa de Meia-Idade , Masculino , FemininoRESUMO
OBJECTIVE: To investigate the expression and clinical significance of long noncoding RNA(lncRNA) HEIH in patients with acute myeloid leukemia (AML). METHODS: 50 newly diagnosed AML patients (except M3) admitted to the First Affiliated Hospital of Bengbu Medical College from January 2019 to December 2020 were included in the study, with 30 patients with non-hematological malignancies as controls. The relative expression level of lncRNA HEIH in all patients were detected, the correlation of clinical characteristics, gene mutations, FAB classification, efficacy, prognosis and overall survival (OS) of AML patients with the expression level of lncRNA HEIH were analyzed. RESULTS: The expression level of lncRNA HEIH in AML patients was significantly higher than that in patients with non-hematological malignancies (P <0.01). Moreover, AML patients with high white blood cell count (WBC), CEBPA and FLT3 mutations, poor efficacy, and poor prognosis often showed higher expression of lncRNA HEIH, and patients with high lncRNA HEIH expression showed a shorter overall survival (OS). CONCLUSION: lncRNA HEIH shows an unique molecular biological significance in AML patients, which may provide a new approach for diagnosis, monitoring and targeted therapy of AML.
Assuntos
Leucemia Mieloide Aguda , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , Leucemia Mieloide Aguda/genética , Prognóstico , Mutação , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética , Masculino , Relevância ClínicaRESUMO
Adipogenesis involves intricate molecular mechanisms regulated by various transcription factors and signaling pathways. In this study, we aimed to identify factors specifically induced during adipogenesis in the human preadipocyte cell line, SGBS, but not in the mouse preadipocyte cell line, 3T3-L1. Microarray analysis revealed distinct gene expression profiles, with 1460 genes induced in SGBS cells and 1297 genes induced in 3T3-L1 cells during adipogenesis, with only 297 genes commonly induced. Among the genes uniquely induced in SGBS cells, we focused on GALNT15, which encodes polypeptide N-acetylgalactosaminyltransferase-15. Its expression increased transiently during adipogenesis in SGBS cells but remained low in 3T3-L1 cells. Overexpression of GALNT15 increased mRNA levels of CCAAT-enhancer binding protein (C/EBPα) and leptin but had no significant impact on adipogenesis in SGBS cells. Conversely, knockdown of GALNT15 suppressed mRNA expression of adipocyte marker genes, reduced lipid accumulation, and decreased the percentage of cells with oil droplets. The induction of C/EBPα and peroxisome proliferator-activated receptor γ during adipogenesis was promoted or suppressed in SGBS cells subjected to overexpression or knockdown of GALNT15, respectively. These data suggest that polypeptide N-acetylgalactosaminyltransferase-15 is a novel regulatory molecule that enhances adipogenesis in SGBS cells.
Assuntos
Células 3T3-L1 , Adipogenia , N-Acetilgalactosaminiltransferases , Polipeptídeo N-Acetilgalactosaminiltransferase , Adipogenia/genética , Humanos , N-Acetilgalactosaminiltransferases/metabolismo , N-Acetilgalactosaminiltransferases/genética , Camundongos , Animais , Adipócitos/metabolismo , Adipócitos/citologia , PPAR gama/metabolismo , PPAR gama/genética , Linhagem Celular , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Leptina/metabolismo , Leptina/genéticaRESUMO
INTRODUCTION: Acute myeloid leukemia with normal cytogenetics (CN-AML) represents a heterogeneous group having diverse genetic mutations. Understanding the significance of each of these mutations is necessary. In this study, we evaluated the prognostic role of MN1 expression in adult CN-AML patients. METHOD: One hundred and sixty-three de-novo adult AML patients were evaluated for MN1 expression by real-time PCR. MN1 expression was correlated with the clinical characteristics of the patients and their outcomes. RESULTS: Higher MN1 expression was associated with NPM1 wild-type (p<0.0001), CD34 positivity (p=0.006), and lower clinical remission rate (p=0.027). FLT3-ITD and CEBPA mutations had no association with MN1 expression. On survival analysis, a high MN1 expression was associated with poor event-free survival (Hazard Ratio 2.47, 95% Confidence Interval: 1.42-4.3; p<0.0001) and overall survival (Hazard Ratio 4.18, 95% Confidence Interval: 2.17-8.08; p<0.0001). On multivariate analysis, the MN1 copy number emerged as an independent predictor of EFS (p<0.0001) and OS (p<0.0001). CONCLUSION: MN1 expression is an independent predictor of outcome in CN-AML.
Assuntos
Biomarcadores Tumorais , Leucemia Mieloide Aguda , Nucleofosmina , Transativadores , Proteínas Supressoras de Tumor , Humanos , Masculino , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Feminino , Adulto , Pessoa de Meia-Idade , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Prognóstico , Adulto Jovem , Transativadores/genética , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Taxa de Sobrevida , Seguimentos , Adolescente , Mutação , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Medição de Risco , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Idoso de 80 Anos ou maisRESUMO
Fibrosis in the lung is thought to be driven by epithelial cell dysfunction and aberrant cell-cell interactions. Unveiling the molecular mechanisms of cellular plasticity and cell-cell interactions is imperative to elucidating lung regenerative capacity and aberrant repair in pulmonary fibrosis. By mining publicly available RNA-Seq data sets, we identified loss of CCAAT enhancer-binding protein alpha (CEBPA) as a candidate contributor to idiopathic pulmonary fibrosis (IPF). We used conditional KO mice, scRNA-Seq, lung organoids, small-molecule inhibition, and potentially novel gene manipulation methods to investigate the role of CEBPA in lung fibrosis and repair. Long-term (6 months or more) of Cebpa loss in AT2 cells caused spontaneous fibrosis and increased susceptibility to bleomycin-induced fibrosis. Cebpa knockout (KO) in these mice significantly decreased AT2 cell numbers in the lung and reduced expression of surfactant homeostasis genes, while increasing inflammatory cell recruitment as well as upregulating S100a8/a9 in AT2 cells. In vivo treatment with an S100A8/A9 inhibitor alleviated experimental lung fibrosis. Restoring CEBPA expression in lung organoids ex vivo and during experimental lung fibrosis in vivo rescued CEBPA deficiency-mediated phenotypes. Our study establishes a direct mechanistic link between CEBPA repression, impaired AT2 cell identity, disrupted tissue homeostasis, and lung fibrosis.
Assuntos
Bleomicina , Proteínas Estimuladoras de Ligação a CCAAT , Homeostase , Camundongos Knockout , Animais , Camundongos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Bleomicina/toxicidade , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/induzido quimicamente , Humanos , Modelos Animais de Doenças , Pulmão/patologia , Pulmão/metabolismo , Organoides/metabolismo , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , MasculinoRESUMO
Biallelic mutations of the CEBPA gene (CEBPAbi) are generally associated with favorable prognosis in patients with acute myeloid leukemia (AML). Monoallelic mutations of the CEBPA gene in carboxy-terminal DNA-binding region (CEBPAsmbZIP) and amino-terminal transactivation domains (CEBPAsmTAD) indicate distinct clinical characteristics and therapeutic outcomes. However, further investigation is required to fully understand these differences. In this retrospective study, we enrolled 77 AML patients with CEBPA mutations, including 53 with CEBPAbi, 12 with CEBPAsmbZIP and 12 with CEBPAsmTAD. The clinical characteristics of the three CEBPAmut groups presented significant differences in age, FAB classification, hemoglobin level and platelet count at diagnosis. The CEBPAsmTAD group exhibited shorter 2-year overall survival (OS) and relapse-free survival (RFS) compared to the CEBPAbi group and CEBPAsmbZIP group in AML patients. The most common co-mutations observed in CEBPAmut AML patients were TET2 and GATA2, which had no effect on prognosis. 2-year RFS of 27 CEBPAmut AML patients who underwent allo-HSCT was better than those who did not. MRD3 positive was identified as an influencing factor for 2-year OS and RFS. Allo-HSCT was found to improve the prognosis of CEPBAmut AML patients with positive MRD3 and adverse co-mutations.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Leucemia Mieloide Aguda , Mutação , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/diagnóstico , Proteínas Estimuladoras de Ligação a CCAAT/genética , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Estudos Retrospectivos , Idoso , Transplante de Células-Tronco Hematopoéticas , Resultado do Tratamento , Adulto Jovem , Adolescente , Intervalo Livre de Doença , Prognóstico , Fator de Transcrição GATA2/genética , Taxa de SobrevidaRESUMO
BACKGROUND: Previous studies have shown that peptides encoded by noncoding RNAs (ncRNAs) can be used as peptide drugs to alleviate diseases. We found that microRNA-31 (miR-31) is involved in the regulation of hypertension and that the peptide miPEP31, which is encoded by the primary transcript of miR-31 (pri-miR-31), can inhibit miR-31 expression. However, the role and mechanism of miPEP31 in hypertension have not been elucidated. METHODS: miPEP31 expression was determined by western blot analysis. miPEP31-deficient mice (miPEP31-/-) were used, and synthetic miPEP31 was injected into Ang II-induced hypertensive mice. Blood pressure was monitored through the tail-cuff method. Histological staining was used to evaluate renal damage. Regulatory T (Treg) cells were assessed by flow cytometry. Differentially expressed genes were analysed through RNA sequencing. The transcription factors were predicted by JASPAR. Luciferase reporter and electrophoretic mobility shift assays (EMSAs) were used to determine the effect of pri-miR-31 on the promoter activity of miPEP31. Images were taken to track the entry of miPEP31 into the cell. RESULTS: miPEP31 is endogenously expressed in target organs and cells related to hypertension. miPEP31 deficiency exacerbated but exogenous miPEP31 administration mitigated the Ang II-induced systolic blood pressure (SBP) elevation, renal impairment and Treg cell decreases in the kidney. Moreover, miPEP31 deletion increased the expression of genes related to Ang II-induced renal fibrosis. miPEP31 inhibited the transcription of miR-31 and promoted Treg differentiation by occupying the Cebpα binding site. The minimal functional domain of miPEP31 was identified and shown to regulate miR-31. CONCLUSION: miPEP31 was identified as a potential therapeutic peptide for treating hypertension by promoting Treg cell differentiation in vivo. Mechanistically, we found that miPEP31 acted as a transcriptional repressor to specifically inhibit miR-31 transcription by competitively occupying the Cebpα binding site in the pri-miR-31 promoter. Our study highlights the significant therapeutic effect of miPEP31 on hypertension and provides novel insight into the role and mechanism of miPEPs.
Assuntos
Angiotensina II , Pressão Sanguínea , Modelos Animais de Doenças , Hipertensão , Rim , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs , Regiões Promotoras Genéticas , Linfócitos T Reguladores , Animais , MicroRNAs/metabolismo , MicroRNAs/genética , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Hipertensão/genética , Sítios de Ligação , Pressão Sanguínea/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/imunologia , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Regulação da Expressão Gênica , Transdução de Sinais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Anti-Hipertensivos/farmacologia , HumanosRESUMO
Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias.
Assuntos
Ilhas de CpG , Metilação de DNA , Epigênese Genética , Redes Reguladoras de Genes , Humanos , Metilação de DNA/genética , Ilhas de CpG/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Regulação Leucêmica da Expressão Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cromatina/metabolismo , Cromatina/genética , Masculino , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Feminino , Hematopoese/genética , Criança , Transcriptoma , Proteínas Proto-Oncogênicas , TransativadoresRESUMO
Pancreatic cancer, predominantly pancreatic ductal adenocarcinoma (PDAC), remains a highly lethal malignancy with limited therapeutic options and a dismal prognosis. By targeting the underlying molecular abnormalities responsible for PDAC development and progression, gene therapy offers a promising strategy to overcome the challenges posed by conventional radiotherapy and chemotherapy. This study sought to explore the therapeutic potential of small activating RNAs (saRNAs) specifically targeting the CCAAT/enhancer-binding protein alpha (CEBPA) gene in PDAC. To overcome the challenges associated with saRNA delivery, tetrahedral framework nucleic acids (tFNAs) were rationally engineered as nanocarriers. These tFNAs were further functionalized with a truncated transferrin receptor aptamer (tTR14) to enhance targeting specificity for PDAC cells. The constructed tFNA-based saRNA formulation demonstrated exceptional stability, efficient saRNA release ability, substantial cellular uptake, biocompatibility, and nontoxicity. In vitro experiments revealed successful intracellular delivery of CEBPA-saRNA utilizing tTR14-decorated tFNA nanocarriers, resulting in significant activation of tumor suppressor genes, namely, CEBPA and its downstream effector P21, leading to notable inhibition of PDAC cell proliferation. Moreover, in a mouse model of PDAC, the tTR14-decorated tFNA-mediated delivery of CEBPA-saRNA effectively upregulated the expression of the CEBPA and P21 genes, consequently suppressing tumor growth. These compelling findings highlight the potential utility of saRNA delivered via a designed tFNA nanocarrier to induce the activation of tumor suppressor genes as an innovative therapeutic approach for PDAC.
Assuntos
Aptâmeros de Nucleotídeos , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Receptores da Transferrina , Animais , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Carcinoma Ductal Pancreático/tratamento farmacológico , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Receptores da Transferrina/metabolismo , Camundongos , Linhagem Celular Tumoral , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proliferação de Células/efeitos dos fármacos , Terapia Genética/métodos , RNA Interferente Pequeno/farmacologia , Camundongos NusRESUMO
Hematologic neoplasms with germline predisposition have been increasingly recognized as a distinct category of tumors over the last few years. As such, this category was added to the World Health Organization (WHO) 4th edition as well as maintained in the WHO 5th edition and International Consensus Classification (ICC) 2022 classification systems. In practice, these tumors require a high index of suspicion and confirmation by molecular testing. Flow cytometry is a cost-effective diagnostic tool that is routinely performed on peripheral blood and bone marrow samples. In this review, we sought to summarize the current body of research correlating flow cytometric immunophenotype to assess its utility in diagnosis of and clinical decision making in germline hematologic neoplasms. We also illustrate these findings using cases mostly from our own institution. We review some of the more commonly mutated genes, including CEBPA, DDX41, RUNX1, ANKRD26, GATA2, Fanconi anemia, Noonan syndrome, and Down syndrome. We highlight that flow cytometry may have a role in the diagnosis (GATA2, Down syndrome) and screening (CEBPA) of some germline predisposition syndromes, although appears to show nonspecific findings in others (DDX41, RUNX1). In many of the others, such as ANKRD26, Fanconi anemia, and Noonan syndrome, further studies are needed to better understand whether specific flow cytometric patterns are observed. Ultimately, we conclude that further studies such as large case series and organized data pipelines are needed in most germline settings to better understand the flow cytometric immunophenotype of these neoplasms.
Assuntos
Citometria de Fluxo , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Neoplasias Hematológicas , Imunofenotipagem , Humanos , Citometria de Fluxo/métodos , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/patologia , Imunofenotipagem/métodos , Criança , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas Estimuladoras de Ligação a CCAAT/genéticaRESUMO
We have recently discovered that the so-called subcortical maternal complex (SCMC) proteins composing of cytoplasmic lattices are destabilized in Uhrf1 knockout murine fully grown oocytes (FGOs). Here we report that human UHRF1 interacts with human NLRP5 and OOEP, which are core components of the SCMC. Moreover, NLRP5 and OOEP interact with DPPA3, which is an essential factor for exporting UHRF1 from the nucleus to the cytoplasm in oocytes. We identify that NLRP5, not OOEP, stabilizes UHRF1 protein in the cytoplasm utilizing specifically engineered cell lines mimicking UHRF1 status in oocytes and preimplantation embryos. Further, UHRF1 is destabilized both in the cytoplasm and nucleus of Nlrp5 knockout murine FGOs. Since pathogenic variants of the SCMC components frequently cause multilocus imprinting disturbance and UHRF1 is essential for maintaining CpG methylation of imprinting control regions during preimplantation development, our results suggest possible pathogenesis behind the disease, which has been a long-standing mystery.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Citoplasma , Metilação de DNA , Impressão Genômica , Camundongos Knockout , Oócitos , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Humanos , Camundongos , Animais , Citoplasma/metabolismo , Oócitos/metabolismo , Feminino , Núcleo Celular/metabolismo , Núcleo Celular/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Blastocisto/metabolismo , Autoantígenos , Proteínas Nucleares , Proteínas MitocondriaisRESUMO
To explore the expression and prognostic value of UHRF1 gene in soft tissue sarcoma (STS) and its related molecular mechanism. The expression data and clinicopathological parameters of STS were downloaded from the Cancer Genome Atlas (TCGA). The expression level of UHRF1 in STS and adjacent tissues and its relationship with clinicopathological characteristics were analyzed. The expression level of UHRF1 in STS tissues was significantly higher than that in paracancerous tissues (Pâ <â .001), and the overall survival (OS) time of patients with high UHRF1 expression was significantly shorter than that of patients with low UHRF1 expression (Pâ =â .002). The expression of UHRF1 was correlated with tumor necrosis, histological type and metastasis, and the differences were statistically significant (Pâ =â .013; Pâ =â .001; Pâ =â .002). The area ratio under receiver operating characteristic (ROC) curve between STS tissue and adjacent tissue of UHRF1 expression was 0.994. Number of tumors (HRâ =â 0.416, 95%CIâ =â 0.260-0.666, Pâ <â .001), depth of tumor (HRâ =â 2.888, 95%CIâ =â 0.910-9.168, Pâ =â .033), metastasis (HRâ =â 2.888, 95% CIâ =â 1.762-4.732, Pâ <â .001), residual tumor (HRâ =â 2.637, 95% CIâ =â 1.721-4.038, Pâ <â .001) and UHRF1 expression (HRâ =â 1.342, 95% CIâ =â 1.105-1.630, Pâ =â .003) were significantly associated with OS, and high expression of UHRF1 (HRâ =â 1.387, 95%CIâ =â 1.008-1.907, Pâ =â .044) was an independent risk factor for the prognosis of STS patients. The results of the nomogram exhibited that UHRF1 expression level had a significant effect on the total score value. GSEA enrichment analysis suggested that UHRF1 was involved in 14 signaling pathways regulating mRNA spliceosome, cell cycle, P53 signaling pathway were identified. Single sample gene set enrichment analysis (ssGSEA) exhibited that the expression of UHRF1 in STS was positively correlated with the level of Th2 cell infiltration, and negatively correlated with plasmacytoid dendritic cells (pDC), natural killer cells (NK), Eosinophils, Mast cells, etc. UHRF1 expression is involved in the immune microenvironment of HCC and affects the occurrence and development of HCC. UHRF1 is highly expressed in STS tissues. It is involved in the regulation of multiple tumor-related signaling pathways and immune cell microenvironment, suggesting that UHRF1 may be a potential molecular marker for prognosis prediction and targeted therapy of STS patients.