RESUMO
The molecular mechanisms underlying atrial fibrillation, the most common sustained cardiac arrhythmia, remain poorly understood. Genome-wide association studies uncovered a major atrial fibrillation susceptibility locus on human chromosome 4q25 in close proximity to the paired-like homeodomain transcription factor 2 (Pitx2) homeobox gene. Pitx2, a target of the left-sided Nodal signaling pathway that initiates early in development, represses the sinoatrial node program and pacemaker activity on the left side. To address the mechanisms underlying this repressive activity, we hypothesized that Pitx2 regulates microRNAs (miRs) to repress the sinoatrial node genetic program. MiRs are small noncoding RNAs that regulate gene expression posttranscriptionally. Using an integrated genomic approach, we discovered that Pitx2 positively regulates miR-17-92 and miR-106b-25. Intracardiac electrical stimulation revealed that both miR-17-92 and miR-106b-25 deficient mice exhibit pacing-induced atrial fibrillation. Furthermore electrocardiogram telemetry revealed that mice with miR-17-92 cardiac-specific inactivation develop prolonged PR intervals whereas mice with miR-17-92 cardiac-specific inactivation and miR-106b-25 heterozygosity develop sinoatrial node dysfunction. Both arrhythmias are risk factors for atrial fibrillation in humans. Importantly, miR-17-92 and miR-106b-25 directly repress genes, such as Shox2 and Tbx3, that are required for sinoatrial node development. Together, to our knowledge, these findings provide the first genetic evidence for an miR loss-of-function that increases atrial fibrillation susceptibility.
Assuntos
Fibrilação Atrial/metabolismo , Predisposição Genética para Doença , Proteínas de Homeodomínio/metabolismo , MicroRNAs/metabolismo , Nó Sinoatrial/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fibrilação Atrial/genética , Fibrilação Atrial/patologia , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Knockout , MicroRNAs/genética , Nó Sinoatrial/patologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Proteína Homeobox PITX2RESUMO
Among the most common human congenital anomalies, cleft lip and palate (CL/P) affects up to 1 in 700 live births. MicroRNA (miR)s are small, non-coding RNAs that repress gene expression post-transcriptionally. The miR-17-92 cluster encodes six miRs that have been implicated in human cancers and heart development. We discovered that miR-17-92 mutant embryos had severe craniofacial phenotypes, including incompletely penetrant CL/P and mandibular hypoplasia. Embryos that were compound mutant for miR-17-92 and the related miR-106b-25 cluster had completely penetrant CL/P. Expression of Tbx1 and Tbx3, the DiGeorge/velo-cardio-facial (DGS) and Ulnar-mammary syndrome (UMS) disease genes, was expanded in miR-17-92 mutant craniofacial structures. Both Tbx1 and Tbx3 had functional miR seed sequences that mediated gene repression. Analysis of miR-17-92 regulatory regions uncovered conserved and functional AP-2α recognition elements that directed miR-17-92 expression. Together, our data indicate that miR-17-92 modulates expression of critical T-box transcriptional regulators during midface development and is itself a target of Bmp-signaling and the craniofacial pioneer factor AP-2α. Our data are the first genetic evidence that an individual miR or miR cluster is functionally important in mammalian CL/P.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , MicroRNAs/genética , Fator de Transcrição AP-2/genética , Animais , Sequência de Bases , Fenda Labial/patologia , Fissura Palatina/patologia , Modelos Animais de Doenças , Embrião de Mamíferos/patologia , Regulação da Expressão Gênica , Humanos , Sequências Reguladoras de Ácido Nucleico/genética , Transdução de Sinais , Proteínas com Domínio T/genética , Fator de Transcrição AP-2/metabolismoRESUMO
The claudin-low subtype is a recently identified rare molecular subtype of human breast cancer that expresses low levels of tight and adherens junction genes and shows high expression of epithelial-to-mesenchymal transition (EMT) genes. These tumors are enriched in gene expression signatures derived from human tumor-initiating cells (TICs) and human mammary stem cells. Through cross-species analysis, we discovered mouse mammary tumors that have similar gene expression characteristics as human claudin-low tumors and were also enriched for the human TIC signature. Such claudin-low tumors were similarly rare but came from a number of distinct mouse models, including the p53 null transplant model. Here we present a molecular characterization of 50 p53 null mammary tumors compared with other mouse models and human breast tumor subtypes. Similar to human tumors, the murine p53 null tumors fell into multiple molecular subtypes, including two basal-like, a luminal, a claudin-low, and a subtype unique to this model. The claudin-low tumors also showed high gene expression of EMT inducers, low expression of the miR-200 family, and low to absent expression of both claudin 3 and E-cadherin. These murine subtypes also contained distinct genomic DNA copy number changes, some of which are similarly altered in their cognate human subtype counterpart. Finally, limiting dilution transplantation revealed that p53 null claudin-low tumors are highly enriched for TICs compared with the more common adenocarcinomas arising in the same model, thus providing a unique preclinical mouse model to investigate the therapeutic response of TICs.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Genômica/métodos , Neoplasias Mamárias Animais/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Adenocarcinoma/classificação , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Neoplasias da Mama/classificação , Claudinas/metabolismo , Análise por Conglomerados , Variações do Número de Cópias de DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos/genética , Genoma Humano/genética , Humanos , Neoplasias Mamárias Animais/classificação , Neoplasias Mamárias Animais/patologia , Camundongos , MicroRNAs/metabolismo , Transplante de Neoplasias , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismoRESUMO
In an effort to understand the potential role of microRNAs (miRNAs) in mammary-gland stem or progenitor cells, miRNA microarrays were performed on subpopulations of the mouse mammary epithelial cell (MEC) line COMMA-DbetaGeo. This cell line contains a heterogeneous subpopulation of progenitors characterized by the expression of stem cell antigen 1 (Sca-1; encoded by Ly6a). Microarray analysis indicated that the Sca-1 subpopulations have distinct miRNA expression profiles. Functional studies were performed on miR-205, which was highly expressed in the Sca-1-positive (Sca-1(+)) cells. When miR-205 was overexpressed in vitro, the COMMA-DbetaGeo cells underwent several significant morphological and molecular changes. miR-205 overexpression led to an expansion of the progenitor-cell population, decreased cell size and increased cellular proliferation. In addition, the colony-forming potential of the two Sca-1 subpopulations was increased. Target prediction for miR-205 indicated that it might regulate the expression of the tumor-suppressor protein PTEN. Overexpression studies using reporter constructs confirmed that PTEN expression is regulated by miR-205. In addition to PTEN, several other putative and previously validated miR-205 targets were identified by microarray analysis, including the previously reported miR-205 targets ZEB1 and ZEB2. Additionally, in normal mouse MECs, high expression of miR-205 was observed in stem-cell-enriched cell populations isolated by FACS using established cell-surface markers.
Assuntos
Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Antígenos Ly/metabolismo , Sequência de Bases , Diferenciação Celular , Proliferação de Células , Tamanho Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Células Epiteliais/classificação , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Proteínas de Membrana/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Células-Tronco/classificaçãoRESUMO
Congenital heart malformations are the most common of all congenital human birth anomalies. During the past decade, research with zebrafish, chick, and mouse models have elucidated many fundamental genetic pathways that govern early cardiac patterning and differentiation. This review highlights the roles of the bone morphogenetic protein (BMP) signaling pathway in cardiogenesis and how defective BMP signals can disrupt the intricate steps of cardiac formation and cause congenital heart defects.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Cardiopatias Congênitas/metabolismo , Transdução de Sinais , Animais , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/genética , HumanosRESUMO
Recent studies have demonstrated that miR-205 has a role in both normal development and cancer, however conflicting reports on its function illustrate the complexity of its regulation and targets. Additionally, miR-205 was found to be highly expressed in stem cell-enriched populations from the mouse mammary gland, and thus may play a function in normal mammary stem cell maintenance. The role that miR-205 plays in tumor formation and metastasis is likely context-dependent as reports have indicated that it may function as either a tumor suppressor or an oncogene. The role that miR-205 plays in directing stem cell fate is still unknown.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Glândulas Mamárias Humanas/embriologia , MicroRNAs/genética , MicroRNAs/fisiologia , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma/metabolismo , Carcinoma/patologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Glândulas Mamárias Humanas/metabolismo , Camundongos , MicroRNAs/metabolismo , Modelos Biológicos , Organogênese/genéticaRESUMO
The sarcoplasmic reticulum (SR) plays a critical role in excitation-contraction coupling by regulating the cytoplasmic calcium concentration of striated muscle. The histidine-rich calcium-binding protein (HRCBP) is expressed in the junctional SR, the site of calcium release from the SR. HRCBP is expressed exclusively in muscle tissues and binds calcium with low affinity and high capacity. In addition, HRCBP interacts with triadin, a protein associated with the ryanodine receptor and thought to be involved in calcium release. Its calcium binding properties, localization to the SR, and interaction with triadin suggest that HRCBP is involved in calcium handling by the SR. To determine the function of HRCBP in vivo, we inactivated HRC, the gene encoding HRCBP, in mice. HRC knockout mice exhibited impaired weight gain beginning at 11 months of age, which was marked by reduced skeletal muscle and fat mass, and triadin protein expression was upregulated in the heart of HRC knockout mice. In addition, HRC null mice displayed a significantly exaggerated response to the induction of cardiac hypertrophy by isoproterenol compared to their wild-type littermates. The exaggerated response of HRC knockout mice to the induction of cardiac hypertrophy is consistent with a regulatory role for HRCBP in calcium handling in vivo and suggests that mutations in HRC, in combination with other genetic or environmental factors, might contribute to pathological hypertrophy and heart failure.
Assuntos
Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Cardiomegalia/genética , Cardiomegalia/fisiopatologia , Predisposição Genética para Doença , Isoproterenol , Proteínas Musculares/deficiência , Aumento de Peso/genética , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Músculo Esquelético/metabolismoRESUMO
The HRC gene encodes the histidine-rich calcium-binding protein, which is found in the lumen of the junctional sarcoplasmic reticulum (SR) of cardiac and skeletal muscle and within calciosomes of arterial smooth muscle. The expression of HRC in cardiac, skeletal, and smooth muscle raises the possibility of a common transcriptional mechanism governing its expression in all three muscle cell types. In this study, we identified a transcriptional enhancer from the HRC gene that is sufficient to direct the expression of lacZ in the expression pattern of endogenous HRC in transgenic mice. The HRC enhancer contains a small, highly conserved sequence that is required for expression in all three muscle lineages. Within this conserved region is a consensus site for myocyte enhancer factor 2 (MEF2) proteins that we show is bound efficiently by MEF2 and is required for transgene expression in all three muscle lineages in vivo. Furthermore, the entire HRC enhancer sequence lacks any discernible CArG motifs, the binding site for serum response factor (SRF), and we show that the enhancer is not activated by SRF. Thus, these studies identify the HRC enhancer as the first MEF2-dependent, CArG-independent transcriptional target in smooth muscle and represent the first analysis of the transcriptional regulation of an SR gene in vivo.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Músculo Esquelético/embriologia , Músculo Liso Vascular/embriologia , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Genes Reporter , Coração/fisiologia , Humanos , Fatores de Transcrição MEF2 , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Músculo Liso Vascular/fisiologia , Fatores de Regulação Miogênica , Regiões Promotoras Genéticas , Alinhamento de Sequência , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Background: Lung cancer treatment has become increasingly dependent upon invasive biopsies to profile tumors for personalized therapy. Recently, tumor expression of programmed death-ligand 1 (PD-L1) has gained interest as a potential predictor of response to immunotherapy. Circulating biomarkers present an opportunity for tumor profiling without the risks of invasive procedures. We characterized PD-L1 expression within populations of nucleated cells in the peripheral blood of lung cancer patients in hopes of expanding the role of liquid biopsy in this setting.Methods: Peripheral blood samples from a multi-institutional prospective study of patients with clinical diagnosis of lung cancer were subjected to cytomorphometric and immunohistochemical evaluation using single-cell, automated slide-based, digital pathology. PD-L1 expression was determined by immunofluorescence.Results: PD-L1 expression was detected within peripheral circulating cells associated with malignancy (CCAM) in 26 of 112 (23%) non-small cell lung cancer patients. Two distinct populations of nucleated, nonhematolymphoid, PD-L1-expressing cells were identified; cytokeratin positive (CK+, PD-L1+, CD45-) and cytokeratin negative (CK-, PD-L1+, CD45-) cells, both with cytomorphometric features (size, nuclear-to-cytoplasm ratio) consistent with tumor cells. Patients with >1.1 PD-L1(+) cell/mL (n = 14/112) experienced worse overall survival than patients with ≤1.1 PD-L1(+) cell/mL (2-year OS: 31.2% vs. 78.8%, P = 0.00159). In a Cox model adjusting for stage, high PD-L1(+) cell burden remained a significant predictor of mortality (HR = 3.85; 95% confidence interval, 1.64-9.09; P = 0.002).Conclusions: PD-L1 expression is detectable in two distinct cell populations in the peripheral blood of lung cancer patients and is associated with worse survival.Impact: These findings could represent a step forward in the development of minimally invasive liquid biopsies for the profiling of tumors. Cancer Epidemiol Biomarkers Prev; 26(7); 1139-45. ©2017 AACR.
Assuntos
Antígeno B7-H1/análise , Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Idoso , Células Sanguíneas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/sangue , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Biópsia Líquida , Neoplasias Pulmonares/sangue , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Estudos ProspectivosRESUMO
Genomic instability is a hallmark of cancer often associated with poor patient outcome and resistance to targeted therapy. Assessment of genomic instability in bulk tumor or biopsy can be complicated due to sample availability, surrounding tissue contamination, or tumor heterogeneity. The Epic Sciences circulating tumor cell (CTC) platform utilizes a non-enrichment based approach for the detection and characterization of rare tumor cells in clinical blood samples. Genomic profiling of individual CTCs could provide a portrait of cancer heterogeneity, identify clonal and sub-clonal drivers, and monitor disease progression. To that end, we developed a single cell Copy Number Variation (CNV) Assay to evaluate genomic instability and CNVs in patient CTCs. For proof of concept, prostate cancer cell lines, LNCaP, PC3 and VCaP, were spiked into healthy donor blood to create mock patient-like samples for downstream single cell genomic analysis. In addition, samples from seven metastatic castration resistant prostate cancer (mCRPC) patients were included to evaluate clinical feasibility. CTCs were enumerated and characterized using the Epic Sciences CTC Platform. Identified single CTCs were recovered, whole genome amplified, and sequenced using an Illumina NextSeq 500. CTCs were then analyzed for genome-wide copy number variations, followed by genomic instability analyses. Large-scale state transitions (LSTs) were measured as surrogates of genomic instability. Genomic instability scores were determined reproducibly for LNCaP, PC3, and VCaP, and were higher than white blood cell (WBC) controls from healthy donors. A wide range of LST scores were observed within and among the seven mCRPC patient samples. On the gene level, loss of the PTEN tumor suppressor was observed in PC3 and 5/7 (71%) patients. Amplification of the androgen receptor (AR) gene was observed in VCaP cells and 5/7 (71%) mCRPC patients. Using an in silico down-sampling approach, we determined that DNA copy number and genomic instability can be detected with as few as 350K sequencing reads. The data shown here demonstrate the feasibility of detecting genomic instabilities at the single cell level using the Epic Sciences CTC Platform. Understanding CTC heterogeneity has great potential for patient stratification prior to treatment with targeted therapies and for monitoring disease evolution during treatment.
Assuntos
Instabilidade Cromossômica/genética , Variações do Número de Cópias de DNA/genética , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Biblioteca Gênica , Instabilidade Genômica , Genômica , Humanos , Hibridização in Situ Fluorescente , Masculino , Células Neoplásicas Circulantes/patologia , PTEN Fosfo-Hidrolase/genética , Receptores Androgênicos/genética , Reprodutibilidade dos TestesRESUMO
IMPORTANCE: A critical decision in the management of metastatic castration-resistant prostate cancer (mCRPC) is when to administer an androgen receptor signaling (ARS) inhibitor or a taxane. OBJECTIVE: To determine if pretherapy nuclear androgen-receptor splice variant 7 (AR-V7) protein expression and localization on circulating tumor cells (CTCs) is a treatment-specific marker for response and outcomes between ARS inhibitors and taxanes. DESIGN, SETTING, AND PARTICIPANTS: For this cross-sectional cohort study at Memorial Sloan Kettering Cancer Center, 265 men with progressive mCRPC undergoing a change in treatment were considered; 86 were excluded because they were not initiating ARS or taxane therapy; and 18 were excluded for processing time constraints, leaving 161 patients for analysis. Between December 2012 and March 2015, blood was collected and processed from patients with progressive mCRPC immediately prior to new line of systemic therapy. Patients were followed up to 3 years. MAIN OUTCOMES AND MEASURES: Prostate-specific antigen (PSA) response, time receiving therapy, radiographic progression-free survival (rPFS), and overall survival (OS). RESULTS: Overall, of 193 prospectively collected blood samples from 161 men with mCRPC, 191 were evaluable (128 pre-ARS inhibitor and 63 pretaxane). AR-V7-positive CTCs were found in 34 samples (18%), including 3% of first-line, 18% of second-line, and 31% of third- or greater line samples. Patients whose samples had AR-V7-positive CTCs before ARS inhibition had resistant posttherapy PSA changes (PTPC), shorter rPFS, shorter time on therapy, and shorter OS than those without AR-V7-positive CTCs. Overall, resistant PTPC were seen in 65 of 112 samples (58%) without detectable AR-V7-positive CTCs prior to ARS inhibition. There were statistically significant differences in OS but not in PTPC, time on therapy, or rPFS for patients with or without pretherapy AR-V7-positive CTCs treated with a taxane. A multivariable model adjusting for baseline factors associated with survival showed superior OS with taxanes relative to ARS inhibitors when AR-V7-positive CTCs were detected pretherapy (hazard ratio, 0.24; 95% CI, 0.10-0.57; P = .035). CONCLUSIONS AND RELEVANCE: The results validate CTC nuclear expression of AR-V7 protein in men with mCRPC as a treatment-specific biomarker that is associated with superior survival on taxane therapy over ARS-directed therapy in a clinical practice setting. Continued examination of this biomarker in prospective studies will further aid clinical utility.
Assuntos
Células Neoplásicas Circulantes/metabolismo , Neoplasias de Próstata Resistentes à Castração/sangue , Receptores Androgênicos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antagonistas de Receptores de Andrógenos/farmacologia , Antagonistas de Receptores de Andrógenos/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Estudos Transversais , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Humanos , Calicreínas/sangue , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Antígeno Prostático Específico/sangue , Neoplasias de Próstata Resistentes à Castração/diagnóstico , Neoplasias de Próstata Resistentes à Castração/mortalidade , Neoplasias de Próstata Resistentes à Castração/terapia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Androgênicos/genética , Sensibilidade e Especificidade , Taxoides/farmacologia , Taxoides/uso terapêutico , Resultado do TratamentoRESUMO
The Epic Platform was developed for the unbiased detection and molecular characterization of circulating tumour cells (CTCs). Here, we report assay performance data, including accuracy, linearity, specificity and intra/inter-assay precision of CTC enumeration in healthy donor (HD) blood samples spiked with varying concentrations of cancer cell line controls (CLCs). Additionally, we demonstrate clinical feasibility for CTC detection in a small cohort of metastatic castrate-resistant prostate cancer (mCRPC) patients. The Epic Platform demonstrated accuracy, linearity and sensitivity for the enumeration of all CLC concentrations tested. Furthermore, we established the precision between multiple operators and slide staining batches and assay specificity showing zero CTCs detected in 18 healthy donor samples. In a clinical feasibility study, at least one traditional CTC/mL (CK+, CD45-, and intact nuclei) was detected in 89 % of 44 mCRPC samples, whereas 100 % of samples had CTCs enumerated if additional CTC subpopulations (CK-/CD45- and CK+ apoptotic CTCs) were included in the analysis. In addition to presenting Epic Platform's performance with respect to CTC enumeration, we provide examples of its integrated downstream capabilities, including protein biomarker expression and downstream genomic analyses at single cell resolution.
RESUMO
As modulators of gene expression, microRNAs (miRNAs) are essential for normal development. Not surprisingly, aberrant expression of miRNAs is associated with many diseases, including cancer. Studies of various breast cancer subtypes have demonstrated that, like gene expression profiles and pathological differences, miRNA profiles can distinguish various tumor subtypes. Over the last few years, roles for miRNAs during many stages of breast cancer progression have been established. This includes potential breast cancer associated polymorphisms in miRNA target sites or miRNAs themselves, miRNAs that can act as tumor suppressors or oncogenes, and miRNAs that can modulate metastatic spread. Recent studies have also suggested key roles for miRNAs in regulating cancer stem cells. Thus, miRNAs have now become important therapeutic targets. This can be achieved by replacing miRNA expression where it has been lost or decreased, or conversely by inhibiting miRNA expression where it has been amplified or overexpressed in cancers. Ultimately, miRNAs should provide both important prognostic biomarkers as well as new targetable molecules for the treatment of breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , MicroRNAs/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes p53 , Humanos , MicroRNAs/genética , Terapia de Alvo Molecular , Metástase Neoplásica , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/fisiologia , Oncogenes , PrognósticoRESUMO
MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression posttranscriptionally. We investigated the hypothesis that bone morphogenetic protein (Bmp) signaling regulates miRNAs in cardiac progenitor cells. Bmp2 and Bmp4 regulate OFT myocardial differentiation via regulation of the miRNA-17-92 cluster. In Bmp mutant embryos, myocardial differentiation was delayed, and multiple miRNAs encoded by miRNA-17-92 were reduced. We uncovered functional miRNA-17-92 seed sequences within the 3' UTR of cardiac progenitor genes such as Isl1 and Tbx1. In both Bmp and miRNA-17-92 mutant embryos, Isl1 and Tbx1 expression failed to be correctly downregulated. Transfection experiments indicated that miRNA-17 and miRNA-20a directly repressed Isl1 and Tbx1. Genetic interaction studies uncovered a synergistic interaction between miRNA-17-92 cluster and Bmp4, providing direct in vivo evidence for the Bmp-miRNA-17-92 regulatory pathway. Our findings indicate that Bmp signaling directly regulates a miRNA-mediated effector mechanism that downregulates cardiac progenitor genes and enhances myocardial differentiation.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Mioblastos Cardíacos/citologia , Mioblastos Cardíacos/metabolismo , Animais , Sequência de Bases , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Gravidez , Transdução de Sinais , Proteínas com Domínio T/genética , Fatores de TranscriçãoRESUMO
Members of the basic helix-loop-helix (bHLH) transcription factor family play an essential role in multiple developmental processes. During neurogenesis, positive and negative regulation by bHLH proteins is essential for proper development. Here we report the identification and initial characterization of the bHLH gene, Neuronal twist (N-twist), named for its neural expression pattern and high sequence homology and physical linkage to the mesodermal inhibitor, M-twist. N-twist is expressed in the developing mouse central nervous system in the midbrain, hindbrain, and neural tube. This neural expression is conserved in invertebrates, as expression of the Drosophila ortholog of N-twist is also restricted to the central nervous system. Like other bHLH family members, N-Twist heterodimerizes with E protein and binds DNA at a consensus bHLH-binding site, the E box. We show that N-Twist inhibits MASH1-dependent transcriptional activation by sequestering E protein in a dominant negative fashion. Thus, these studies support the notion that N-Twist represents a novel negative regulator of neurogenesis.