RESUMO
Here we describe the synthesis and use of a directed hydroxyl radical probe, tethered to a pre-mRNA substrate, to map the structure of this substrate during the spliceosome assembly process. These studies indicate an early organization and proximation of conserved pre-mRNA sequences during spliceosome assembly. This methodology may be adapted to the synthesis of a wide variety of modified RNAs for use as probes of RNA structure and RNA-protein interaction.
Assuntos
Radical Hidroxila/química , Técnicas de Sonda Molecular , Sondas Moleculares/síntese química , Precursores de RNA/química , Spliceossomos/metabolismo , Cristalografia por Raios X/métodos , RNA Polimerases Dirigidas por DNA/metabolismo , Radical Hidroxila/metabolismo , Oligonucleotídeos/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Spliceossomos/química , Proteínas Virais/metabolismoRESUMO
Although activating mutations in RAS oncogenes are known to result in aberrant signaling through multiple pathways, the role of microRNAs (miRNAs) in the Ras oncogenic program remains poorly characterized. Here we demonstrate that Ras activation leads to repression of the miR-143/145 cluster in cells of human, murine, and zebrafish origin. Loss of miR-143/145 expression is observed frequently in KRAS mutant pancreatic cancers, and restoration of these miRNAs abrogates tumorigenesis. miR-143/145 down-regulation requires the Ras-responsive element-binding protein (RREB1), which represses the miR-143/145 promoter. Additionally, KRAS and RREB1 are targets of miR-143/miR-145, revealing a feed-forward mechanism that potentiates Ras signaling.
Assuntos
Regulação para Baixo/genética , MicroRNAs/genética , Neoplasias Pancreáticas/etiologia , Proteínas ras/fisiologia , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Humanos , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas ras/genéticaRESUMO
miR-143 and miR-145 are co-expressed microRNAs (miRNAs) that have been extensively studied as potential tumor suppressors. These miRNAs are highly expressed in the colon and are consistently reported as being downregulated in colorectal and other cancers. Through regulation of multiple targets, they elicit potent effects on cancer cell growth and tumorigenesis. Importantly, a recent discovery demonstrates that miR-143 and miR-145 are not expressed in colonic epithelial cells; rather, these two miRNAs are highly expressed in mesenchymal cells such as fibroblasts and smooth muscle cells. The expression patterns of miR-143 and miR-145 and other miRNAs were initially determined from tissue level data without consideration that multiple different cell types, each with their own unique miRNA expression patterns, make up each tissue. Herein, we discuss the early reports on the identification of dysregulated miR-143 and miR-145 expression in colorectal cancer and how lack of consideration of cellular composition of normal tissue led to the misconception that these miRNAs are downregulated in cancer. We evaluate mechanistic data from miR-143/145 studies in context of their cell type-restricted expression pattern and the potential of these miRNAs to be considered tumor suppressors. Further, we examine other examples of miRNAs being investigated in inappropriate cell types modulating pathways in a non-biological fashion. Our review highlights the importance of determining the cellular expression pattern of each miRNA, so that downstream studies are conducted in the appropriate cell type.
Assuntos
MicroRNAs/metabolismo , Colo/citologia , Colo/metabolismo , Células Epiteliais/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Neoplasias Epiteliais e Glandulares/terapiaRESUMO
RATIONALE: Mitochondria are semiautonomous cellular organelles with their own genome, which not only supply energy but also participate in cell death pathways. MicroRNAs (miRNAs) are usually 19 to 25 nt long, noncoding RNAs, involved in posttranscriptional gene regulation by binding to the 3'-untranslated regions of target mRNA, which impact on diverse cellular processes. OBJECTIVE: To determine if nuclear miRNAs translocate into the mitochondria and regulate mitochondrial function with possible pathophysiological implications in cardiac myocytes. METHODS AND RESULTS: We find that miR-181c is encoded in the nucleus, assembled in the cytoplasm, and finally translocated into the mitochondria of cardiac myocytes. Immunoprecipitation of Argonaute 2 from the mitochondrial fraction indicates binding of cytochrome c oxidase subunit 1 (mt-COX1) mRNA from the mitochondrial genome with miR-181c. Also, a luciferase reporter construct shows that mi-181c binds to the 3'UTR of mt-COX1. To study whether miR-181c regulates mt-COX1, we overexpressed precursor miR-181c (or a scrambled sequence) in primary cultures of neonatal rat ventricular myocytes. Overexpression of miR-181c did not change mt-COX1 mRNA but significantly decreased mt-COX1 protein, suggesting that miR-181c is primarily a translational regulator of mt-COX1. In addition to altering mt-COX1, overexpression of miR-181c results in increased mt-COX2 mRNA and protein content, with an increase in both mitochondrial respiration and reactive oxygen species generation in neonatal rat ventricular myocytes. Thus, our data show for the first time that miR-181c can enter and target the mitochondrial genome, ultimately causing electron transport chain complex IV remodeling and mitochondrial dysfunction. CONCLUSIONS: Nuclear miR-181c translocates into the mitochondria and regulates mitochondrial genome expression. This unique observation may open a new dimension to our understanding of mitochondrial dynamics and the role of miRNA in mitochondrial dysfunction.
Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , Genoma Mitocondrial/genética , MicroRNAs/fisiologia , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/fisiologia , Animais , Células Cultivadas , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Ovarian cancer remains the most lethal gynecological malignancy worldwide due to lack of effective screening, vague early symptoms, poor description of biomarkers, and absence of effective treatment regimes. Epithelial ovarian carcinoma (EOC) is categorized into five distinct disease subtypes which collectively account for ~90% of ovarian carcinomas. Most women present at advanced stages contributing to a poor overall 5-year survival rate. Standard treatment for EOC is cytoreductive surgery and platinum-based chemotherapy; however, most patients suffer from recurrence and platinum-resistant disease, which highlights an urgent need for targeted therapy. The high frequency of molecular alterations affecting gain-of-function signaling through the RAS mitogen-activated protein kinase (MAPK) pathway in EOC has prompted pre-clinical and clinical efforts toward research into the effectiveness of MAPK pathway inhibition as a second-line treatment. The RAS/MAPK pathway is a highly conserved signal transduction cascade, often disrupted in cancer, that regulates tumorigenic phenotypes including cellular proliferation, survival, migration, apoptosis, and differentiation. Herein, the role of the MAPK pathway in EOC with emphasis on targetability of the pathway is described. Pre-clinical and clinical efforts to target MAPK signaling in EOC have identified several MAPK pathway inhibitors that offer efficacious potential for monotherapy and in combination with other compounds. Thus, inhibition of the RAS/MAPK pathway is emerging as a tractable strategy for treatment of ovarian cancer that may permit development of personalized therapy and improved prognosis for women challenged by this disease.
RESUMO
MicroRNAs (miRNAs) regulate gene expression in a variety of biological pathways such as development and tumourigenesis. miRNAs are initially expressed as long primary transcripts (pri-miRNAs) that undergo sequential processing by Drosha and then Dicer to yield mature miRNAs. miR-17~92 is a miRNA cluster that encodes 6 miRNAs and while it is essential for development it also has reported oncogenic activity. To date, the role of RNA structure in miRNA biogenesis has only been considered in terms of the secondary structural elements required for processing of pri-miRNAs by Drosha. Here we report that the miR-17~92 cluster has a compact globular tertiary structure where miRNAs internalized within the core of the folded structure are processed less efficiently than miRNAs on the surface of the structure. Increased miR-92 expression resulting from disruption of the compact miR-17~92 structure results in increased repression of integrin α5 mRNA, a known target of miR-92a. In summary, we describe the first example of pri-miRNA structure modulating differential expression of constituent miRNAs.
Assuntos
MicroRNAs/química , Dobramento de RNA , Sequência de Bases , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Integrina alfa5/genética , Dados de Sequência Molecular , Família Multigênica , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
Ovarian cancer (OC) is the most deadly gynecological malignancy, with unmet clinical need for new therapeutic approaches. The relaxin peptide is a pleiotropic hormone with reproductive functions in the ovary. Relaxin induces cell growth in several types of cancer, but the role of relaxin in OC is poorly understood. Here, using cell lines and xenograft models, we demonstrate that relaxin and its associated GPCR RXFP1 form an autocrine signaling loop essential for OC in vivo tumorigenesis, cell proliferation, and viability. We determined that relaxin signaling activates expression of prooncogenic pathways, including RHO, MAPK, Wnt, and Notch. We found that relaxin is detectable in patient-derived OC tumors, ascites, and serum. Further, inflammatory cytokines IL-6 and TNF-α activated transcription of relaxin via recruitment of STAT3 and NF-κB to the proximal promoter, initiating an autocrine feedback loop that potentiated expression. Inhibition of RXFP1 or relaxin increased cisplatin sensitivity of OC cell lines and abrogated in vivo tumor formation. Finally, we demonstrate that a relaxin-neutralizing antibody reduced OC cell viability and sensitized cells to cisplatin. Collectively, these data identify the relaxin/RXFP1 autocrine loop as a therapeutic vulnerability in OC.
Assuntos
Comunicação Autócrina , Carcinogênese/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Relaxina/metabolismo , Via de Sinalização Wnt , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapiaRESUMO
RAS-MAPK signaling mediates processes critical to normal development including cell proliferation, survival, and differentiation. Germline mutation of RAS-MAPK genes lead to the Noonan-spectrum of syndromes. Here, we present a patient affected by a 6p-interstitial microdeletion with unknown underlying molecular etiology. Examination of 6p-interstitial microdeletion cases reveals shared clinical features consistent with Noonan-spectrum disorders including short stature, facial dysmorphia and cardiovascular abnormalities. We find the RAS-responsive element binding protein-1 (RREB1) is the common deleted gene in multiple 6p-interstitial microdeletion cases. Rreb1 hemizygous mice display orbital hypertelorism and cardiac hypertrophy phenocopying the human syndrome. Rreb1 haploinsufficiency leads to sensitization of MAPK signaling. Rreb1 recruits Sin3a and Kdm1a to control H3K4 methylation at MAPK pathway gene promoters. Haploinsufficiency of SIN3A and mutations in KDM1A cause syndromes similar to RREB1 haploinsufficiency suggesting genetic perturbation of the RREB1-SIN3A-KDM1A complex represents a new category of RASopathy-like syndromes arising through epigenetic reprogramming of MAPK pathway genes.
Assuntos
Proteínas de Ligação a DNA/genética , Haploinsuficiência , Sistema de Sinalização das MAP Quinases/genética , Síndrome de Noonan/etiologia , Fatores de Transcrição/genética , Proteínas ras/metabolismo , Anormalidades Múltiplas/genética , Animais , Deleção Cromossômica , Cromossomos Humanos Par 6 , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/metabolismo , Humanos , Masculino , Metilação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo Correpressor Histona Desacetilase e Sin3/genética , Complexo Correpressor Histona Desacetilase e Sin3/metabolismo , Fatores de Transcrição/metabolismo , Proteínas ras/genéticaRESUMO
Oncogenic KRAS engages multiple effector pathways including the MAPK cascade to promote proliferation and survival of pancreatic cancer cells. KRAS-transformed cancer cells exhibit oncogene addiction to sustained activity of RAS for maintenance of malignant phenotypes. Previously, we have shown an essential role for the RHO guanine exchange factor ARHGEF2 for growth and survival of RAS-transformed pancreatic tumors. Here, we have determined that pancreatic cancer cells demonstrating KRAS addiction are significantly dependent on expression of ARHGEF2. Furthermore, enforced expression of ARHGEF2 desensitizes cells to pharmacological MEK inhibition and initiates a positive feedback loop which activates ERK phosphorylation and the downstream ARHGEF2 promoter. Therefore, targeting ARHGEF2 expression may increase the efficacy of MAPK inhibitors for treatment of RAS-dependent pancreatic cancers.
Assuntos
Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismoRESUMO
Early recognition of pre-mRNA during spliceosome assembly in mammals proceeds through the association of U1 small nuclear ribonucleoprotein particle (snRNP) with the 5' splice site as well as the interactions of the branch binding protein SF1 with the branch region and the U2 snRNP auxiliary factor U2AF with the polypyrimidine tract and 3' splice site. These factors, along with members of the SR protein family, direct the ATP-independent formation of the early (E) complex that commits the pre-mRNA to splicing. We report here the observation in U2AF-depleted HeLa nuclear extract of a distinct, ATP-independent complex designated E' which can be chased into E complex and itself commits a pre-mRNA to the splicing pathway. The E' complex is characterized by a U1 snRNA-5' splice site base pairing, which follows the actual commitment step, an interaction of SF1 with the branch region, and a close association of the 5' splice site with the branch region. These results demonstrate that both commitment to splicing and the early proximity of conserved sequences within pre-mRNA substrates can occur in a minimal complex lacking U2AF, which may function as a precursor to E complex in spliceosome assembly.
Assuntos
Proteínas Nucleares/fisiologia , Ribonucleoproteínas/fisiologia , Spliceossomos/metabolismo , Trifosfato de Adenosina/química , Processamento Alternativo , Núcleo Celular/metabolismo , Cromatografia em Gel , Eletroforese em Gel de Ágar , Células HeLa , Humanos , Radical Hidroxila , Imunoprecipitação , Modelos Biológicos , Modelos Genéticos , Proteínas Nucleares/química , Ligação Proteica , Pirimidinas/química , Precursores de RNA , Splicing de RNA , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteínas/química , Fator de Processamento U2AF , Raios UltravioletaRESUMO
MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and cardiovascular disease, have been shown to activate tissue and/or cell-specific miRNA expression associated with disease progression. The inhibition of miRNA expression offers the potential for a therapeutic intervention. However, traditional approaches to inhibit miRNAs, employing antagomir oligonucleotides, affect specific miRNA functions upon global delivery. Herein, we present a protocol for the in vivo cardio-specific inhibition of the miR-181 family in a rat model. A miRNA-sponge construct is designed to include 10 repeated anti-miR-181 binding sequences. The cardio-specific α-MHC promoter is cloned into the pEGFP backbone to drive the cardio-specific miR-181 miRNA-sponge expression. To create a stable cell line expressing the miR-181-sponge, myoblast H9c2 cells are transfected with the α-MHC-EGFP-miR-181-sponge construct and sorted by fluorescence-activated cell sorting (FACs) into GFP positive H9c2 cells which are cultured with neomycin (G418). Following stable growth in neomycin, monoclonal cell populations are established by additional FACs and single cell cloning. The resulting myoblast H9c2-miR-181-sponge-GFP cells exhibit a loss of function of miR-181 family members as assessed through the increased expression of miR-181 target proteins and compared to H9c2 cells expressing a scramble non-functional sponge. In addition, we develop a nanovector for the systemic delivery of the miR-181-sponge construct by complexing positively charged liposomal nanoparticles and negatively charged miR-181-sponge plasmids. In vivo imaging of GFP reveals that multiple tail vein injections of a nanovector over a three-week period are able to promote a significant expression of the miR-181-sponge in a cardio-specific manner. Importantly, a loss of miR-181 function is observed in the heart tissue but not in the kidney or the liver. The miRNA-sponge is a powerful method to inhibit tissue-specific miRNA expression. Driving the miRNA-sponge expression from a tissue-specific promoter provides specificity for the miRNA inhibition, which can be confined to a targeted organ or tissue. Furthermore, combining nanovector and miRNA-sponge technologies permits an effective delivery and tissue-specific miRNA inhibition in vivo.
Assuntos
Coração/fisiologia , MicroRNAs/genética , Nanopartículas/metabolismo , Animais , Humanos , Ratos , TransfecçãoRESUMO
Activating mutations of KRAS are nearly ubiquitous in pancreatic adenocarcinomas occurring in greater than 90% of cases. Cellular transformation by oncogenic RAS requires the RHO guanine exchange factor ARHGEF2 (also known as GEF-H1) for tumor growth and survival. Here, we find oncogenic KRAS activates ARHGEF2 through a minimal RAS responsive promoter. We have determined the endogenous ARHGEF2 promoter is positively regulated by the transcription factors ELK1, ETS1, SP1 and SP3 and negatively regulated by the RAS responsive element binding protein (RREB1). We find that the panel of ARHGEF2-regulating transcription factors modulates RAS transformed phenotypes including cellular viability, anchorage-independent growth and invasion-migration of pancreatic cancer cells. RREB1 knockdown activates the amplitude and duration of RHOA via increased ARHGEF2 expression. By relieving the negative regulation of RREB1 on the ARHGEF2 promoter, we determined that ETS1 and SP3 are essential for the normal expression of ARHGEF2 and contribute to the migratory behavior of pancreatic cancer cells. Furthermore, enforced expression of ARHGEF2 rescues loss of SP3 driven invasion-migration and anchorage-independent growth defective phenotypes through restored activation of RHOA. Collectively, our results identify a transcription factor program required for RAS transformation and provide mechanistic insight into the highly metastatic behavior of pancreatic cancer.
Assuntos
Transformação Celular Neoplásica/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Ativação Transcricional , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Transformação Celular Neoplásica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Neoplasias Pancreáticas/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-1/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Fator de Transcrição Sp3/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: MicroRNA (miRNA) is a type of noncoding RNA that can repress the expression of target genes through posttranscriptional regulation. In addition to numerous physiologic roles for miRNAs, they play an important role in pathophysiologic processes affecting cardiovascular health. Previously, we reported that nuclear encoded microRNA (miR-181c) is present in heart mitochondria, and importantly, its overexpression affects mitochondrial function by regulating mitochondrial gene expression. METHODS AND RESULTS: To investigate further how the miR-181 family affects the heart, we suppressed miR-181 using a miR-181-sponge containing 10 repeated complementary miR-181 "seed" sequences and generated a set of H9c2 cells, a cell line derived from rat myoblast, by stably expressing either a scrambled or miR-181-sponge sequence. Sponge-H9c2 cells showed a decrease in reactive oxygen species production and reduced basal mitochondrial respiration and protection against doxorubicin-induced oxidative stress. We also found that miR-181a/b targets phosphatase and tensin homolog (PTEN), and the sponge-expressing stable cells had increased PTEN activity and decreased PI3K signaling. In addition, we have used miR-181a/b-/- and miR-181c/d-/- knockout mice and subjected them to ischemia-reperfusion injury. Our results suggest divergent effects of different miR-181 family members: miR-181a/b targets PTEN in the cytosol, resulting in an increase in infarct size in miR-181a/b-/- mice due to increased PTEN signaling, whereas miR-181c targets mt-COX1 in the mitochondria, resulting in decreased infarct size in miR-181c/d-/- mice. CONCLUSIONS: The miR-181 family alters the myocardial response to oxidative stress, notably with detrimental effects by targeting mt-COX1 (miR-181c) or with protection by targeting PTEN (miR-181a/b).
Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Animais , Ciclo-Oxigenase 1/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mioblastos/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , RatosRESUMO
Bone undergoes continuous remodeling due to balanced bone formation and resorption mediated by osteoblasts and osteoclasts, respectively. Osteoclasts arise from the macrophage lineage, and their differentiation is dependent on RANKL, a member of the TNF family of cytokines. Here, we have provided evidence that RANKL controls the expression of 3BP2, an adapter protein that is required for activation of SRC tyrosine kinase and simultaneously coordinates the attenuation of ß-catenin, both of which are required to execute the osteoclast developmental program. We found that RANKL represses the transcription of the E3 ubiquitin ligase RNF146 through an NF-κB-related inhibitory element in the RNF146 promoter. RANKL-mediated suppression of RNF146 results in the stabilization of its substrates, 3BP2 and AXIN1, which consequently triggers the activation of SRC and attenuates the expression of ß-catenin, respectively. Depletion of RNF146 caused hypersensitivity to LPS-induced TNF-α production in vivo. RNF146 thus acts as an inhibitory switch to control osteoclastogenesis and cytokine production and may be a control point underlying the pathogenesis of chronic inflammatory diseases.
Assuntos
Osteoclastos/metabolismo , Ligante RANK/metabolismo , Elementos de Resposta , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Lipopolissacarídeos/toxicidade , Camundongos , Osteoclastos/citologia , Ligante RANK/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/genética , beta Catenina/genética , beta Catenina/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
UNLABELLED: Activating KRAS mutations are nearly ubiquitous in pancreatic cancer occurring in more than 95% of clinical cases. miRNAs are small noncoding RNAs that regulate gene expression by binding sequences within the 3'UTRs of target mRNAs. An integral role for miRNAs in cancer pathogenesis is well established; however, the role of miRNAs in KRAS-mediated tumorigenesis is poorly characterized. Here it is demonstrated that expression of miR-31 is coupled to the expression of oncogenic KRAS and activity of the MAPK pathway. miR-31 is highly expressed in patient-derived xenografts and a panel of pancreatic and colorectal cancer cells harboring activating KRAS mutations. The miR-31 host gene is a large noncoding RNA that correlates with miR-31 expression and enabled identification of the putative miR-31 promoter. Using luciferase reporters, a minimal RAS-responsive miR-31 promoter was found to drive robust luciferase activity dependent on expression of mutant KRAS and the transcription factor ELK1. Furthermore, ELK1 interacts directly with the endogenous miR-31 promoter in a MAPK-dependent manner. Expression of enforced miR-31 significantly enhanced invasion and migration of multiple pancreatic cancer cells resulting from the activation of RhoA through regulation of the miR-31 target gene RASA1. Importantly, acute knockdown of RASA1 phenocopied enforced miR-31 expression on the migratory behavior of pancreatic cancer cells through increased RhoA activation. IMPLICATIONS: Oncogenic KRAS can activate Rho through the miR-31-mediated regulation of RASA1 indicating miR-31 acts as a KRAS effector to modulate invasion and migration in pancreatic cancer.
Assuntos
Neoplasias Colorretais/genética , MicroRNAs/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína p120 Ativadora de GTPase/genética , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Sistema de Sinalização das MAP Quinases , MicroRNAs/metabolismo , Mutação , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismo , Proteína p120 Ativadora de GTPase/metabolismoRESUMO
Cellular identity in metazoan organisms is frequently established through lineage-specifying transcription factors, which control their own expression through transcriptional positive feedback, while antagonizing the developmental networks of competing lineages. Here, we have uncovered a distinct positive feedback loop that arises from the reciprocal stabilization of the tyrosine kinase ABL and the transcriptional coactivator TAZ. Moreover, we determined that this loop is required for osteoblast differentiation and embryonic skeletal formation. ABL potentiated the assembly and activation of the RUNX2-TAZ master transcription factor complex that is required for osteoblastogenesis, while antagonizing PPARγ-mediated adipogenesis. ABL also enhanced TAZ nuclear localization and the formation of the TAZ-TEAD complex that is required for osteoblast expansion. Last, we have provided genetic data showing that regulation of the ABL-TAZ amplification loop lies downstream of the adaptor protein 3BP2, which is mutated in the craniofacial dysmorphia syndrome cherubism. Our study demonstrates an interplay between ABL and TAZ that controls the mesenchymal maturation program toward the osteoblast lineage and is mechanistically distinct from the established model of lineage-specific maturation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Núcleo Celular/genética , Querubismo/genética , Querubismo/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , PPAR gama/genética , PPAR gama/metabolismo , Proteínas Proto-Oncogênicas c-abl/genética , TransativadoresRESUMO
Cellular transformation by oncogenic RAS engages the MAPK pathway under strict regulation by the scaffold protein KSR-1. Here, we report that the guanine nucleotide exchange factor GEF-H1 plays a critical role in a positive feedback loop for the RAS/MAPK pathway independent of its RhoGEF activity. GEF-H1 acts as an adaptor protein linking the PP2A B' subunits to KSR-1, thereby mediating the dephosphorylation of KSR-1 S392 and activation of MAPK signaling. GEF-H1 is important for the growth and survival of HRAS(V12)-transformed cells and pancreatic tumor xenografts. GEF-H1 expression is induced by oncogenic RAS and is correlated with pancreatic neoplastic progression. Our results, therefore, identify GEF-H1 as an amplifier of MAPK signaling and provide mechanistic insight into the progression of RAS mutant tumors.
Assuntos
Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/patologia , Proteínas Quinases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas ras/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Camundongos , Células NIH 3T3 , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Quinases/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Transdução de Sinais , Células Tumorais Cultivadas , Proteínas ras/genéticaAssuntos
Proteínas/química , RNA/química , Ácido Edético/química , Ensaio de Desvio de Mobilidade Eletroforética , Estrutura Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas/metabolismo , RNA/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Fator de Processamento U2AFRESUMO
BACKGROUND: MicroRNAs are ~22-nt long regulatory RNAs that serve as critical modulators of post-transcriptional gene regulation. The diversity of miRNAs in endothelial cells (ECs) and the relationship of this diversity to epithelial and hematologic cells is unknown. We investigated the baseline miRNA signature of human ECs cultured from the aorta (HAEC), coronary artery (HCEC), umbilical vein (HUVEC), pulmonary artery (HPAEC), pulmonary microvasculature (HPMVEC), dermal microvasculature (HDMVEC), and brain microvasculature (HBMVEC) to understand the diversity of miRNA expression in ECs. RESULTS: We identified 166 expressed miRNAs, of which 3 miRNAs (miR-99b, miR-20b and let-7b) differed significantly between EC types and predicted EC clustering. We confirmed the significance of these miRNAs by RT-PCR analysis and in a second data set by Sylamer analysis. We found wide diversity of miRNAs between endothelial, epithelial and hematologic cells with 99 miRNAs shared across cell types and 31 miRNAs unique to ECs. We show polycistronic miRNA chromosomal clusters have common expression levels within a given cell type. CONCLUSIONS: EC miRNA expression levels are generally consistent across EC types. Three microRNAs were variable within the dataset indicating potential regulatory changes that could impact on EC phenotypic differences. MiRNA expression in endothelial, epithelial and hematologic cells differentiate these cell types. This data establishes a valuable resource characterizing the diverse miRNA signature of ECs.