RESUMO
How tumor cells genetically lose antigenicity and evade immune checkpoints remains largely elusive. We report that tissue-specific expression of the human long noncoding RNA LINK-A in mouse mammary glands initiates metastatic mammary gland tumors, which phenotypically resemble human triple-negative breast cancer (TNBC). LINK-A expression facilitated crosstalk between phosphatidylinositol-(3,4,5)-trisphosphate and inhibitory G-protein-coupled receptor (GPCR) pathways, attenuating protein kinase A-mediated phosphorylation of the E3 ubiquitin ligase TRIM71. Consequently, LINK-A expression enhanced K48-polyubiquitination-mediated degradation of the antigen peptide-loading complex (PLC) and intrinsic tumor suppressors Rb and p53. Treatment with LINK-A locked nucleic acids or GPCR antagonists stabilized the PLC components, Rb and p53, and sensitized mammary gland tumors to immune checkpoint blockers. Patients with programmed ccll death protein-1(PD-1) blockade-resistant TNBC exhibited elevated LINK-A levels and downregulated PLC components. Hence we demonstrate lncRNA-dependent downregulation of antigenicity and intrinsic tumor suppression, which provides the basis for developing combinational immunotherapy treatment regimens and early TNBC prevention.
Assuntos
Apresentação de Antígeno/imunologia , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Neoplasias/imunologia , Oncogenes , RNA Longo não Codificante/genética , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Adenoma/genética , Adenoma/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Humanos , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
According to the established model of murine innate lymphoid cell (ILC) development, helper ILCs develop separately from natural killer (NK) cells. However, it is unclear how helper ILCs and NK cells develop in humans. Here we elucidated key steps of NK cell, ILC2, and ILC3 development within human tonsils using ex vivo molecular and functional profiling and lineage differentiation assays. We demonstrated that while tonsillar NK cells, ILC2s, and ILC3s originated from a common CD34-CD117+ ILC precursor pool, final steps of ILC2 development deviated independently and became mutually exclusive from those of NK cells and ILC3s, whose developmental pathways overlapped. Moreover, we identified a CD34-CD117+ ILC precursor population that expressed CD56 and gave rise to NK cells and ILC3s but not to ILC2s. These data support a model of human ILC development distinct from the mouse, whereby human NK cells and ILC3s share a common developmental pathway separate from ILC2s.
Assuntos
Células Matadoras Naturais/imunologia , Linfócitos/imunologia , Tonsila Palatina/imunologia , Animais , Antígenos CD34/metabolismo , Antígeno CD56/metabolismo , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Imunidade Inata , Ativação Linfocitária , Camundongos , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
Skeletal muscle regeneration involves a signaling network that regulates the proliferation, differentiation, and fusion of muscle precursor cells to injured myofibers. IRE1α, one of the arms of the unfolded protein response, regulates cellular proteostasis in response to ER stress. Here, we demonstrate that inducible deletion of IRE1α in satellite cells of mice impairs skeletal muscle regeneration through inhibiting myoblast fusion. Knockdown of IRE1α or its downstream target, X-box protein 1 (XBP1), also inhibits myoblast fusion during myogenesis. Transcriptome analysis revealed that knockdown of IRE1α or XBP1 dysregulates the gene expression of molecules involved in myoblast fusion. The IRE1α-XBP1 axis mediates the gene expression of multiple profusion molecules, including myomaker (Mymk). Spliced XBP1 (sXBP1) transcription factor binds to the promoter of Mymk gene during myogenesis. Overexpression of myomaker in IRE1α-knockdown cultures rescues fusion defects. Inducible deletion of IRE1α in satellite cells also inhibits myoblast fusion and myofiber hypertrophy in response to functional overload. Collectively, our study demonstrates that IRE1α promotes myoblast fusion through sXBP1-mediated up-regulation of the gene expression of multiple profusion molecules, including myomaker.
Assuntos
Fusão Celular , Endorribonucleases , Desenvolvimento Muscular , Músculo Esquelético , Mioblastos , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Proteína 1 de Ligação a X-Box , Animais , Proteína 1 de Ligação a X-Box/metabolismo , Proteína 1 de Ligação a X-Box/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Camundongos , Mioblastos/metabolismo , Mioblastos/citologia , Músculo Esquelético/metabolismo , Músculo Esquelético/citologia , Desenvolvimento Muscular/genética , Endorribonucleases/metabolismo , Endorribonucleases/genética , Células Satélites de Músculo Esquelético/metabolismo , Regeneração/genética , Diferenciação Celular/genética , Regulação da Expressão Gênica , Proteínas de Membrana , Proteínas MuscularesRESUMO
Ischemic heart disease, a leading cause of death worldwide, manifests clinically as myocardial infarction. Contemporary therapies using mesenchymal stromal cells (MSCs) and their derivative (exosomes, EXOs) were developed to decrease the progression of cell damage during ischemic injury. Laminin alpha 2 (LAMA2) is an important extracellular matrix protein of the heart. Here, we generated MSC-derived exosomes cultivated under LAMA2 coating to enhance human-induced pluripotent stem cell (hiPSC)-cardiomyocyte recognition of LAMA2-EXOs, thus, increasing cell protection during ischemia reoxygenation. We mapped the mRNA content of LAMA2 and gelatin-EXOs and identified 798 genes that were differentially expressed, including genes associated with cardiac muscle development and extracellular matrix organization. Cells were treated with LAMA2-EXOs 2 h before a 4 h ischemia period (1% O2, 5% CO2, glucose-free media). LAMA2-EXOs had a two-fold protective effect compared to non-treatment on plasma membrane integrity and the apoptosis activation pathway; after a 1.5 h recovery period (20% O2, 5% CO2, cardiomyocyte-enriched media), cardiomyocytes treated with LAMA2-EXOs showed faster recovery than did the control group. Although EXOs had a protective effect on endothelial cells, there was no LAMA2-enhanced protection on these cells. This is the first report of LAMA2-EXOs used to treat cardiomyocytes that underwent ischemia-reoxygenation injury. Overall, we showed that membrane-specific EXOs may help improve cardiomyocyte survival in treating ischemic cardiovascular disease.
Assuntos
Exossomos , Células-Tronco Pluripotentes Induzidas , Laminina , Humanos , Miócitos Cardíacos , Dióxido de Carbono , Células Endoteliais , IsquemiaRESUMO
TP53 is commonly altered in human cancer, and Tp53 reactivation suppresses tumours in vivo in mice (TP53 and Tp53 are also known as p53). This strategy has proven difficult to implement therapeutically, and here we examine an alternative strategy by manipulating the p53 family members, Tp63 and Tp73 (also known as p63 and p73, respectively). The acidic transactivation-domain-bearing (TA) isoforms of p63 and p73 structurally and functionally resemble p53, whereas the ΔN isoforms (lacking the acidic transactivation domain) of p63 and p73 are frequently overexpressed in cancer and act primarily in a dominant-negative fashion against p53, TAp63 and TAp73 to inhibit their tumour-suppressive functions. The p53 family interacts extensively in cellular processes that promote tumour suppression, such as apoptosis and autophagy, thus a clear understanding of this interplay in cancer is needed to treat tumours with alterations in the p53 pathway. Here we show that deletion of the ΔN isoforms of p63 or p73 leads to metabolic reprogramming and regression of p53-deficient tumours through upregulation of IAPP, the gene that encodes amylin, a 37-amino-acid peptide co-secreted with insulin by the ß cells of the pancreas. We found that IAPP is causally involved in this tumour regression and that amylin functions through the calcitonin receptor (CalcR) and receptor activity modifying protein 3 (RAMP3) to inhibit glycolysis and induce reactive oxygen species and apoptosis. Pramlintide, a synthetic analogue of amylin that is currently used to treat type 1 and type 2 diabetes, caused rapid tumour regression in p53-deficient thymic lymphomas, representing a novel strategy to target p53-deficient cancers.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Linfoma/metabolismo , Linfoma/patologia , Proteína Supressora de Tumor p53/deficiência , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Genes Supressores de Tumor , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/uso terapêutico , Linfoma/tratamento farmacológico , Linfoma/genética , Masculino , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteína 3 Modificadora da Atividade de Receptores/metabolismo , Receptores da Calcitonina/metabolismo , Timo/metabolismo , Timo/patologia , Transativadores/genética , Transativadores/metabolismo , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
We used a screening strategy to test for reprogramming factors for the conversion of human cardiac progenitor cells (CPCs) into Pacemaker-like cells. Human transcription factors SHOX2, TBX3, TBX5, TBX18, and the channel protein HCN2, were transiently induced as single factors and in trio combinations into CPCs, first transduced with the connexin 30.2 (CX30.2) mCherry reporter. Following screens for reporter CX30.2 mCherry gene activation and FACS enrichment, we observed the definitive expression of many pacemaker specific genes; including, CX30.2, KCNN4, HCN4, HCN3, HCN1, and SCN3b. These findings suggest that the SHOX2, HCN2, and TBX5 (SHT5) combination of transcription factors is a much better candidate in driving the CPCs into Pacemaker-like cells than other combinations and single transcription factors. Additionally, single-cell RNA sequencing of SHT5 mCherry+ cells revealed cellular enrichment of pacemaker specific genes including TBX3, KCNN4, CX30.2, and BMP2, as well as pacemaker specific potassium and calcium channels (KCND2, KCNK2, and CACNB1). In addition, similar to human and mouse sinoatrial node (SAN) studies, we also observed the down-regulation of NKX2.5. Patch-clamp recordings of the converted Pacemaker-like cells exhibited HCN currents demonstrated the functional characteristic of pacemaker cells. These studies will facilitate the development of an optimal Pacemaker-like cell-based therapy within failing hearts through the recovery of SAN dysfunction.
Assuntos
Relógios Biológicos , Diferenciação Celular , Miocárdio/citologia , Células-Tronco/citologia , Conexinas/metabolismo , Fenômenos Eletrofisiológicos , Regulação da Expressão Gênica , Células HEK293 , Humanos , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
BACKGROUND: Over 96% of high-grade ovarian carcinomas and 50% of all cancers are characterized by alterations in the p53 gene. Therapeutic strategies to restore and/or reactivate the p53 pathway have been challenging. By contrast, p63, which shares many of the downstream targets and functions of p53, is rarely mutated in cancer. METHODS: A novel strategy is presented for circumventing alterations in p53 by inducing the tumor-suppressor isoform TAp63 (transactivation domain of tumor protein p63) through its direct downstream target, microRNA-130b (miR-130b), which is epigenetically silenced and/or downregulated in chemoresistant ovarian cancer. RESULTS: Treatment with miR-130b resulted in: 1) decreased migration/invasion in HEYA8 cells (p53 wild-type) and disruption of multicellular spheroids in OVCAR8 cells (p53-mutant) in vitro, 2) sensitization of HEYA8 and OVCAR8 cells to cisplatin (CDDP) in vitro and in vivo, and 3) transcriptional activation of TAp63 and the B-cell lymphoma (Bcl)-inhibitor B-cell lymphoma 2-like protein 11 (BIM). Overexpression of TAp63 was sufficient to decrease cell viability, suggesting that it is a critical downstream effector of miR-130b. In vivo, combined miR-130b plus CDDP exhibited greater therapeutic efficacy than miR-130b or CDDP alone. Mice that carried OVCAR8 xenograft tumors and were injected with miR-130b in 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) liposomes had a significant decrease in tumor burden at rates similar to those observed in CDDP-treated mice, and 20% of DOPC-miR-130b plus CDDP-treated mice were living tumor free. Systemic injections of scL-miR-130b plus CDDP in a clinically tested, tumor-targeted nanocomplex (scL) improved survival in 60% and complete remissions in 40% of mice that carried HEYA8 xenografts. CONCLUSIONS: The miR-130b/TAp63 axis is proposed as a new druggable pathway that has the potential to uncover broad-spectrum therapeutic options for the majority of p53-altered cancers.
Assuntos
MicroRNAs/uso terapêutico , Mutação de Sentido Incorreto , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Lipossomos , Camundongos , Camundongos Nus , MicroRNAs/administração & dosagem , MicroRNAs/genética , MicroRNAs/metabolismo , Invasividade Neoplásica/prevenção & controle , Isoformas de Proteínas/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Transfecção , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Understanding the mechanisms of early cardiac fate determination may lead to better approaches in promoting heart regeneration. We used a mesoderm posterior 1 (Mesp1)-Cre/Rosa26-EYFP reporter system to identify microRNAs (miRNAs) enriched in early cardiac progenitor cells. Most of these miRNA genes bear MESP1-binding sites and active histone signatures. In a calcium transient-based screening assay, we identified miRNAs that may promote the cardiomyocyte program. An X-chromosome miRNA cluster, miR-322/-503, is the most enriched in the Mesp1 lineage and is the most potent in the screening assay. It is specifically expressed in the looping heart. Ectopic miR-322/-503 mimicking the endogenous temporal patterns specifically drives a cardiomyocyte program while inhibiting neural lineages, likely by targeting the RNA-binding protein CUG-binding protein Elav-like family member 1 (Celf1). Thus, early miRNAs in lineage-committed cells may play powerful roles in cell-fate determination by cross-suppressing other lineages. miRNAs identified in this study, especially miR-322/-503, are potent regulators of early cardiac fate.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas CELF1/genética , Proteínas CELF1/metabolismo , Diferenciação Celular , Linhagem da Célula/genética , Embrião de Mamíferos , Perfilação da Expressão Gênica , Genes Reporter , Integrases/genética , Integrases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mesoderma/citologia , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , MicroRNAs/metabolismo , Morfogênese/genética , Células-Tronco Embrionárias Murinas/citologia , Miócitos Cardíacos/citologia , Cultura Primária de Células , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Transdução de SinaisRESUMO
Self-renewing embryonic stem (ES) cells have an exceptional need for timely biomass production, yet the transcriptional control mechanisms responsible for meeting this requirement are largely unknown. We report here that Ronin (Thap11), which is essential for the self-renewal of ES cells, binds with its transcriptional coregulator, Hcf-1, to a highly conserved enhancer element that previously lacked a recognized binding factor. The subset of genes bound by Ronin/Hcf-1 function primarily in transcription initiation, mRNA splicing, and cell metabolism; genes involved in cell signaling and cell development are conspicuously underrepresented in this target gene repertoire. Although Ronin/Hcf-1 represses the expression of some target genes, its activity at promoter sites more often leads to the up-regulation of genes essential to protein biosynthesis and energy production. We propose that Ronin/Hcf-1 controls a genetic program that contributes to the unimpeded growth of ES cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Fator C1 de Célula Hospedeira/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Metabolismo Energético , Camundongos , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas Repressoras , Transcrição GênicaRESUMO
MicroRNAs (miRNAs) are known to play diverse roles in the regulation of vertebrate development. To investigate miRNA-target mRNA relationships in embryonic development, we have carried out small-RNA sequencing to identify miRNAs expressed in the early gastrula of Xenopus laevis. We identify a total of 180 miRNAs, and we have identified the locations of the miRNA precursor sequences in the X. laevis genome. Of these miRNAs, 141 represent miRs previously identified in Xenopus tropicalis. Alignment to human miRNAs led to the identification of 39 miRNAs that have not previously been described for Xenopus. We have also used a biochemical approach to isolate mRNAs that are associated with the RNA-Induced Silencing Complex (RISC) in early gastrulae and thus candidate targets of miRNA-dependent regulation. Interrogation of this RISC-associated mRNA pool by RT-PCR indicates that a number of genes essential for early patterning and specification may be under regulation by miRNAs. Smad1 transcripts are associated with the RISC; target prediction algorithms identify a single miRNA-binding site for miR-26, which is common to the 3'UTRs of Smad1a and Smad1b. Disruption of the interaction between miR-26 and the Smad1 3'UTR via a Target Protector Morpholino Oligonucleotide (TPMO) leads to a 2-fold increase in Smad1 protein accumulation, moderate increases in the expression of BMP4/Smad1 target genes, and a reduction in organizer gene expression, as well as a partially ventralized phenotype in approximately 25% of embryos. Overexpression of miR-26 resulted in moderately decreased expression of Smad1-dependent genes and an expansion of the region expressing the Organizer gene not1. Our findings indicate that interactions between miR-26 and the Smad1 3'UTR modulate Smad1 function in the establishment of axial patterning; they also establish a foundation for the functional analysis of miRNAs and their regulatory interactions during gastrulation.
Assuntos
Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Proteína Smad1/genética , Proteínas de Xenopus/genética , Xenopus/embriologia , Xenopus/genética , Regiões 3' não Traduzidas/genética , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sequência de Bases , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Gástrula/embriologia , Imunoprecipitação , MicroRNAs/genética , Dados de Sequência Molecular , Fenótipo , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Proteína Smad1/metabolismo , Proteínas de Xenopus/metabolismoRESUMO
Highly differentiated sex chromosomes create a lethal imbalance in gene expression in one sex. To accommodate hemizygosity of the X chromosome in male fruit flies, expression of X-linked genes increases twofold. This is achieved by the male- specific lethal (MSL) complex, which modifies chromatin to increase expression. Mutations that disrupt the X localization of this complex decrease the expression of X-linked genes and reduce male survival. The mechanism that restricts the MSL complex to X chromatin is not understood. We recently reported that the siRNA pathway contributes to localization of the MSL complex, raising questions about the source of the siRNAs involved. The X-linked 1.688 g/cm(3) satellite related repeats (1.688(X) repeats) are restricted to the X chromosome and produce small RNA, making them an attractive candidate. We tested RNA from these repeats for a role in dosage compensation and found that ectopic expression of single-stranded RNAs from 1.688(X) repeats enhanced the male lethality of mutants with defective X recognition. In contrast, expression of double-stranded hairpin RNA from a 1.688(X) repeat generated abundant siRNA and dramatically increased male survival. Consistent with improved survival, X localization of the MSL complex was largely restored in these males. The striking distribution of 1.688(X) repeats, which are nearly exclusive to the X chromosome, suggests that these are cis-acting elements contributing to identification of X chromatin.
Assuntos
Drosophila melanogaster/genética , RNA Interferente Pequeno/fisiologia , Sequências Repetitivas de Ácido Nucleico , Cromossomo X/genética , Animais , Animais Geneticamente Modificados , Pareamento de Bases , Sequência de Bases , Mapeamento Cromossômico , DNA Satélite/genética , Proteínas de Ligação a DNA/análise , Mecanismo Genético de Compensação de Dose , Drosophila/classificação , Drosophila/genética , Proteínas de Drosophila/análise , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/ultraestrutura , Eucromatina/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Letais , Larva , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/análise , Interferência de RNA , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Sequências de Repetição em Tandem , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , TransgenesRESUMO
The roles of microRNAs (miRNAs) and the miRNA processing machinery in the regulation of stem cell biology are not well understood. Here, we show that the p53 family member and p63 isoform, ΔNp63, is a transcriptional activator of a cofactor critical for miRNA processing (DGCR8). This regulation gives rise to a unique miRNA signature resulting in reprogramming cells to multipotency. Strikingly, ΔNp63(-/-) epidermal cells display profound defects in terminal differentiation and express a subset of markers and miRNAs present in embryonic stem cells and fibroblasts induced to pluripotency using Yamanaka factors. Moreover, ΔNp63(-/-) epidermal cells transduced with an inducible DGCR8 plasmid can differentiate into multiple cell fates in vitro and in vivo. We found that human primary keratinocytes depleted of ΔNp63 or DGCR8 can be reprogrammed in 6 d and express a unique miRNA and gene expression signature that is similar but not identical to human induced pluripotent stem cells. Our data reveal a role for ΔNp63 in the transcriptional regulation of DGCR8 to reprogram adult somatic cells into multipotent stem cells.
Assuntos
Regulação para Baixo/genética , Queratinócitos/metabolismo , Células-Tronco Multipotentes/citologia , Fosfoproteínas/genética , Proteínas/genética , Proteínas de Ligação a RNA/genética , Transativadores/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Adulto , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Quimera , Embrião de Mamíferos/citologia , Células Epidérmicas , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Queratinócitos/citologia , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Multipotentes/metabolismo , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/metabolismo , Fosfoproteínas/deficiência , Fosfoproteínas/metabolismo , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transativadores/deficiência , Transativadores/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/metabolismoRESUMO
Next-generation sequencing experiments have shown that microRNAs (miRNAs) are expressed in many different isoforms (isomiRs), whose biological relevance is often unclear. We found that mature miR-21, the most widely researched miRNA because of its importance in human disease, is produced in two prevalent isomiR forms that differ by 1 nt at their 3' end, and moreover that the 3' end of miR-21 is posttranscriptionally adenylated by the noncanonical poly(A) polymerase PAPD5. PAPD5 knockdown caused an increase in the miR-21 expression level, suggesting that PAPD5-mediated adenylation of miR-21 leads to its degradation. Exoribonuclease knockdown experiments followed by small-RNA sequencing suggested that PARN degrades miR-21 in the 3'-to-5' direction. In accordance with this model, microarray expression profiling demonstrated that PAPD5 knockdown results in a down-regulation of miR-21 target mRNAs. We found that disruption of the miR-21 adenylation and degradation pathway is a general feature in tumors across a wide range of tissues, as evidenced by data from The Cancer Genome Atlas, as well as in the noncancerous proliferative disease psoriasis. We conclude that PAPD5 and PARN mediate degradation of oncogenic miRNA miR-21 through a tailing and trimming process, and that this pathway is disrupted in cancer and other proliferative diseases.
Assuntos
Adenina/metabolismo , MicroRNAs/metabolismo , Neoplasias/genética , RNA Nucleotidiltransferases/metabolismo , Estabilidade de RNA , Sequência de Bases , Citosina/metabolismo , Exorribonucleases/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células MCF-7 , MicroRNAs/química , MicroRNAs/genética , Modelos Biológicos , Dados de Sequência Molecular , Neoplasias/patologia , Conformação de Ácido Nucleico , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ribonuclease III/metabolismoRESUMO
Altered microRNA expression patterns are implicated in the formation of many human diseases, including ovarian cancer. Our laboratory previously created Dicer(fl/fl)/Pten(fl/fl)/Amhr2(cre/+) mice, which developed high-grade serous carcinomas originating from mouse fallopian tubes, while neither Dicer(fl/fl)/Amhr2(cre/+) nor Pten(fl/fl)/Amhr2(cre/+) mice developed tumors. To explore miRNAs involved in the tumorigenesis in the double-knockout (DKO) mice, tumor cell lines were established from mouse primary tumors, and the most abundant miRNAs present in mouse normal fallopian tubes, let-7b and miR-34c, were expressed in these cell lines. We found that miR-34c had a more dramatic effect on inhibiting tumor cell viability than let-7b. The action of miR-34c induced tumor cell cycle arrest in G1 phase and apoptosis, and was accompanied with the regulation of key genes involved in cell proliferation and cell cycle G1/S transition. miR-34c suppressed the expression of Ezh2 and Mybl2, which may transcriptionally and functionally activate Cdkn1c. Furthermore, miR-34c levels are extremely low in human serous adenocarcinomas compared with human normal fallopian tubes. Expression of miR-34c in human ovarian cancer cells phenocopied its effects in DKO mouse tumor cells. However, miR-34b/c(-/-)/Pten(fl/fl)/Amhr2(cre/+) mice failed to develop high-grade serous carcinomas, implicating a combination of miRNAs in the tumorigenesis process. Thus, while miR-34c is a putative tumor suppressor in high-grade serous ovarian carcinoma with potential therapeutic advantages, screening of additional miRNAs for their effects alone and in combination with miR-34c is highly warranted to uncover miRNAs that synergize with miR-34c against cancer.
Assuntos
Cistadenocarcinoma Seroso/genética , Genes Supressores de Tumor , MicroRNAs/fisiologia , Neoplasias Ovarianas/genética , Animais , Linhagem Celular Tumoral , Cistadenocarcinoma Seroso/patologia , RNA Helicases DEAD-box/genética , Tubas Uterinas , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Gradação de Tumores , Neoplasias Ovarianas/patologia , PTEN Fosfo-Hidrolase/genética , Ribonuclease III/genéticaRESUMO
Approximately 15% of colorectal carcinomas (CRCs) exhibit a hypermutated genotype accompanied by high levels of microsatellite instability (MSI-H) and defects in DNA mismatch repair. These tumours, unlike the majority of colorectal carcinomas, are often diploid, exhibit frequent epigenetic silencing of the MLH1 DNA mismatch repair gene, and have a better clinical prognosis. As an adjunct study to The Cancer Genome Atlas consortium that recently analysed 224 colorectal cancers by whole exome sequencing, we compared the 35 CRCs (15.6%) with a hypermutated genotype to those with a non-hypermutated genotype. We found that 22 (63%) of the hypermutated CRCs exhibited transcriptional silencing of the MLH1 gene, a high frequency of BRAF V600E gene mutations, and infrequent APC and KRAS mutations, a mutational pattern significantly different from their non-hypermutated counterparts. However, the remaining 13 (37%) hypermutated CRCs lacked MLH1 silencing, contained tumours with the highest mutation rates ('ultramutated' CRCs), and exhibited higher incidences of APC and KRAS mutations, but infrequent BRAF mutations. These patterns were confirmed in an independent validation set of 250 exome-sequenced CRCs. Analysis of mRNA and microRNA expression signatures revealed that hypermutated CRCs with MLH1 silencing had greatly reduced levels of WNT signalling and increased BRAF signalling relative to non-hypermutated CRCs. Our findings suggest that hypermutated CRCs include one subgroup with fundamentally different pathways to malignancy than the majority of CRCs. Examination of MLH1 expression status and frequencies of APC, KRAS, and BRAF mutation in CRC may provide a useful diagnostic tool that could supplement the standard microsatellite instability assays and influence therapeutic decisions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma/genética , Neoplasias Colorretais/genética , Inativação Gênica , Mutação , Proteínas Nucleares/genética , Proteína da Polipose Adenomatosa do Colo/genética , Carcinoma/enzimologia , Carcinoma/patologia , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Perfilação da Expressão Gênica/métodos , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Frequência do Gene , Predisposição Genética para Doença , Humanos , MicroRNAs/metabolismo , Instabilidade de Microssatélites , Proteína 1 Homóloga a MutL , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Prognóstico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , RNA Mensageiro/metabolismo , Via de Sinalização Wnt/genética , Proteínas ras/genéticaRESUMO
The p53 family member TP63 encodes two sets of N-terminal isoforms, TAp63 and ΔNp63 isoforms. They each regulate diverse biological functions in epidermal morphogenesis and in cancer. In the skin, where their activities have been extensively characterized, TAp63 prevents premature aging by regulating the quiescence and genomic stability of stem cells required for wound healing and hair regeneration, while ΔNp63 controls maintenance and terminal differentiation of epidermal basal cells. This functional diversity is surprising given that these isoforms share a high degree of similarity, including an identical sequence for a DNA-binding domain. To understand the mechanisms of the transcriptional programs regulated by each p63 isoform and leading to diverse biological functions, we performed genome-wide analyses using p63 isoform-specific chromatin immunoprecipitation, RNA sequencing, and metabolomics of TAp63-/- and ΔNp63-/- mouse epidermal cells. Our data indicate that TAp63 and ΔNp63 physically and functionally interact with distinct transcription factors for the downstream regulation of their target genes, thus ultimately leading to the regulation of unique transcriptional programs and biological processes. Our findings unveil novel transcriptomes regulated by the p63 isoforms to control diverse biological functions, including the cooperation between TAp63 and NRF2 in the modulation of metabolic pathways and response to oxidative stress providing a mechanistic explanation for the TAp63 knock out phenotypes. SIGNIFICANCE: The p63 isoforms, TAp63 and ΔNp63, control epithelial morphogenesis and tumorigenesis through the interaction with distinct transcription factors and the subsequent regulation of unique transcriptional programs.
Assuntos
Fator 2 Relacionado a NF-E2 , Neoplasias , Camundongos , Animais , Fator 2 Relacionado a NF-E2/genética , Estudo de Associação Genômica Ampla , Neoplasias/genética , Isoformas de Proteínas/genética , Epiderme/metabolismoRESUMO
This article describes a monolayer-coated gold nanoparticle-based transfection system for the delivery of microRNA (miRNA) into human osteosarcoma (HOS) cells. Two distinct ammonium-terminated adsorbates were used in this study, which provided a platform for ionic bonding of the miRNA onto gold nanoparticles (AuNPs). The custom-designed monolayer-coated gold nanoparticles were characterized by dynamic light scattering, gel mobility shift assay, transmission electron microscopy, ultraviolet-visible spectrometry, zeta potential, and X-ray photoelectron spectroscopy. The miRNA-loaded gold nanoparticles were transfected, and the level of intracellular miRNA delivered and taken up by cells was measured by Taqman qPCR. The overall analysis indicated a successful delivery of miRNA into the HOS cells at an â¼11,000-fold increase compared to nontreated cells.
Assuntos
Nanopartículas Metálicas , MicroRNAs , Humanos , Ouro/química , MicroRNAs/genética , Nanopartículas Metálicas/química , Transfecção , Técnicas de Transferência de GenesRESUMO
Interferon gamma (IFNγ) is a critical cytokine known for its diverse roles in immune regulation, inflammation, and tumor surveillance. However, while IFNγ levels were elevated in sera of most newly diagnosed acute myeloid leukemia (AML) patients, its complex interplay in AML remains insufficiently understood. We aim to characterize these complex interactions through comprehensive bulk and single-cell approaches in bone marrow of newly diagnosed AML patients. We identify monocytic AML as having a unique microenvironment characterized by IFNγ producing T and NK cells, high IFNγ signaling, and immunosuppressive features. IFNγ signaling score strongly correlates with venetoclax resistance in primary AML patient cells. Additionally, IFNγ treatment of primary AML patient cells increased venetoclax resistance. Lastly, a parsimonious 47-gene IFNγ score demonstrates robust prognostic value. In summary, our findings suggest that inhibiting IFNγ is a potential treatment strategy to overcoming venetoclax resistance and immune evasion in AML patients.
Assuntos
Interferon gama , Leucemia Mieloide Aguda , Sulfonamidas , Humanos , Interferon gama/farmacologia , Prognóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/diagnóstico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Microambiente TumoralRESUMO
Skeletal muscle regeneration involves coordinated activation of an array of signaling pathways. Fibroblast growth factor-inducible 14 (Fn14) is a bona fide receptor for the TWEAK cytokine. Levels of Fn14 are increased in the skeletal muscle of mice after injury. However, the cell-autonomous role of Fn14 in muscle regeneration remains unknown. Here, we demonstrate that global deletion of the Fn14 receptor in mice attenuates muscle regeneration. Conditional ablation of Fn14 in myoblasts but not in differentiated myofibers of mice inhibits skeletal muscle regeneration. Fn14 promotes myoblast fusion without affecting the levels of myogenic regulatory factors in the regenerating muscle. Fn14 deletion in myoblasts hastens initial differentiation but impairs their fusion. The overexpression of Fn14 in myoblasts results in the formation of myotubes having an increased diameter after induction of differentiation. Ablation of Fn14 also reduces the levels of various components of canonical Wnt and calcium signaling both in vitro and in vivo. Forced activation of Wnt signaling rescues fusion defects in Fn14-deficient myoblast cultures. Collectively, our results demonstrate that Fn14-mediated signaling positively regulates myoblast fusion and skeletal muscle regeneration.
Assuntos
Comunicação Celular , Mioblastos , Receptor de TWEAK , Animais , Camundongos , Diferenciação Celular , Desenvolvimento Muscular , Mioblastos/metabolismo , Via de Sinalização Wnt , Receptor de TWEAK/metabolismoRESUMO
BACKGROUND: Non-small cell lung cancer (NSCLC) comprises the majority (~85%) of all lung tumors, with lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) being the most frequently diagnosed histological subtypes. Multi-modal omics profiling has been carried out in NSCLC, but no studies have yet reported a unique metabolite-related gene signature and altered metabolic pathways associated with LUAD and LUSC. METHODS: We integrated transcriptomics and metabolomics to analyze 30 human lung tumors and adjacent noncancerous tissues. Differential co-expression was used to identify modules of metabolites that were altered between normal and tumor. RESULTS: We identified unique metabolite-related gene signatures specific for LUAD and LUSC and key pathways aberrantly regulated at both transcriptional and metabolic levels. Differential co-expression analysis revealed that loss of coherence between metabolites in tumors is a major characteristic in both LUAD and LUSC. We identified one metabolic onco-module gained in LUAD, characterized by nine metabolites and 57 metabolic genes. Multi-omics integrative analysis revealed a 28 metabolic gene signature associated with poor survival in LUAD, with six metabolite-related genes as individual prognostic markers. CONCLUSIONS: We demonstrated the clinical utility of this integrated metabolic gene signature in LUAD by using it to guide repurposing of AZD-6482, a PI3Kß inhibitor which significantly inhibited three genes from the 28-gene signature. Overall, we have integrated metabolomics and transcriptomics analyses to show that LUAD and LUSC have distinct profiles, inferred gene signatures with prognostic value for patient survival, and identified therapeutic targets and repurposed drugs for potential use in NSCLC treatment.