RESUMO
Traumatic Brain Injury (TBI) is a global driver of disability, and we currently lack effective therapies to promote neural repair and recovery. TBI is characterized by an initial insult, followed by a secondary injury cascade, including inflammation, excitotoxicity, and glial cellular response. This cascade incorporates molecular mechanisms that represent potential targets of therapeutic intervention. In this study, we investigate the response to focal impact injury to the optic tectum of Xenopus laevis tadpoles. This injury disrupts the blood-brain barrier, causing edema, and produces deficits in visually-driven behaviors which are resolved within one week. Within 3 h, injured brains show a dramatic transcriptional activation of inflammatory cytokines, upregulation of genes associated with inflammation, and recruitment of microglia to the injury site and surrounding tissue. Shortly afterward, astrocytes undergo morphological alterations and accumulate near the injury site, and these changes persist for at least 48 h following injury. Genes associated with astrocyte reactivity and neuroprotective functions also show elevated levels of expression following injury. Since our results demonstrate that the response to focal impact injury in Xenopus resembles the cellular alterations observed in rodents and other mammalian models, the Xenopus tadpole offers a new, scalable vertebrate model for TBI.
Assuntos
Astrócitos , Lesões Encefálicas Traumáticas , Animais , Astrócitos/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Modelos Animais de Doenças , Inflamação/metabolismo , Larva , Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Doenças Neuroinflamatórias , Xenopus laevisRESUMO
To investigate microRNA (miR) functions in early eye development, we asked whether eye field transcription factors (EFTFs) are targets of miR-dependent regulation in Xenopus embryos. Argonaute (AGO) ribonucleoprotein complexes, including miRs and targeted mRNAs, were coimmunoprecipitated from transgenic embryos expressing myc-tagged AGO under the control of the rax1 promoter; mRNAs for all EFTFs coimmunoprecipitated with Ago in late neurulae. Computational predictions of miR binding sites within EFTF 3'UTRs identified miR-199a-3p ("miR-199") as a candidate regulator of EFTFs, and miR-199 was shown to regulate rax1 in vivo. Targeted overexpression of miR-199 led to small eyes, a reduction in EFTF expression, and reduced cell proliferation. Inhibition of interactions between mir-199 and the rax1 3'UTR reversed the small eye phenotype. Although targeted knockdown of miR-199 left the eye field intact, it reduced optic cup outgrowth and disrupted eye formation. Computational identification of candidate miR-199 targets within the Xenopus transcriptome led to the identification of ptk7 as a candidate regulator. Targeted overexpression of ptk7 resulted in abnormal optic cup formation and a reduction or loss of eye development, recapitulating the range of eye phenotypes seen following miR-199 knockdown. Our results indicate that miR-199 plays both positive and negative regulatory roles in eye development.
Assuntos
Olho/embriologia , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Interferência de RNA , Xenopus laevis/embriologia , Xenopus laevis/genética , Animais , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Estudos de Associação Genética , Mutação com Perda de Função , Organogênese/genética , Fenótipo , Ligação Proteica , Receptores Proteína Tirosina Quinases/genética , Reprodutibilidade dos Testes , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
The establishment of cell lineages occurs via a dynamic progression of gene regulatory networks (GRNs) that underlie developmental commitment and differentiation. To investigate how microRNAs (miRs) function in this process, we compared miRs and miR targets at the initiation of the two major ectodermal lineages in Xenopus. We used next-generation sequencing to identify over 170 miRs expressed in midgastrula ectoderm expressing either noggin or a constitutively active BMP receptor, reflecting anterior neural or epidermal ectoderm, respectively; 125 had not previously been identified in Xenopus. We identified the locations of the pre-miR sequences in the X. laevis genome. Neural and epidermal ectoderm express broadly similar sets of miRs. To identify targets of miR-dependent translational control, we co-immunoprecipitated Argonaute-Ribonucleoprotein (Ago-RNP) complexes from early neural and epidermal ectoderm and sequenced the associated RNA. The Ago-RNP RNAs from these tissues represent overlapping, yet distinct, subsets of genes. Moreover, the profile of Ago-RNP associated genes differs substantially from the profile of total RNAs in these tissues. We generated target predictions for the "high confidence" Ago-RNP RNAs using the identified ectodermal miRs; These RNAs generally had target sites for multiple miRs. Oct4 orthologues, as well as many of their previously identified transcriptional targets, are represented in the Ago-RNP pool in both tissues, suggesting that miR-dependent regulation contributes to the downregulation of the oct4 gene regulatory network and the reduction in ectodermal pluripotency.
Assuntos
Ectoderma/metabolismo , Epiderme/embriologia , MicroRNAs/genética , Placa Neural/metabolismo , RNA Mensageiro/genética , Xenopus laevis/embriologia , Animais , Diferenciação Celular , Embrião não Mamífero/metabolismo , Epiderme/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , MicroRNAs/biossíntese , Microinjeções , Fenótipo , RNA Mensageiro/administração & dosagem , RNA Mensageiro/biossíntese , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Conditional overexpression of histone reader Tripartite motif containing protein 24 (TRIM24) in mouse mammary epithelia (Trim24COE) drives spontaneous development of mammary carcinosarcoma tumors, lacking ER, PR and HER2. Human carcinosarcomas or metaplastic breast cancers (MpBC) are a rare, chemorefractory subclass of triple-negative breast cancers (TNBC). Comparison of Trim24COE metaplastic carcinosarcoma morphology, TRIM24 protein levels and a derived Trim24COE gene signature reveals strong correlation with human MpBC tumors and MpBC patient-derived xenograft (PDX) models. Global and single-cell tumor profiling reveal Met as a direct oncogenic target of TRIM24, leading to aberrant PI3K/mTOR activation. Here, we find that pharmacological inhibition of these pathways in primary Trim24COE tumor cells and TRIM24-PROTAC treatment of MpBC TNBC PDX tumorspheres decreased cellular viability, suggesting potential in therapeutically targeting TRIM24 and its regulated pathways in TRIM24-expressing TNBC.
Assuntos
Carcinossarcoma/genética , Proteínas de Transporte/genética , Neoplasias Mamárias Experimentais/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Neoplasias de Mama Triplo Negativas/genética , Animais , Mama/patologia , Carcinossarcoma/patologia , Proteínas de Transporte/metabolismo , Ensaios Clínicos como Assunto , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-met/genética , RNA-Seq , Análise de Célula Única , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Sequenciamento Completo do Genoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Small RNAs from early neural (i.e., Noggin-expressing, or NOG) and epidermal (expressing a constitutively active BMP4 receptor, CABR) ectoderm in Xenopus laevis were sequenced to identify microRNAs (miRs) expressed in each tissue. Argonaute-associated mRNAs were isolated and sequenced to identify genes that are regulated by microRNAs in these tissues. Interactions between these ectodermal miRs and selected miR-regulated mRNAs were predicted using the PITA algorithm; PITA predictions for over 600 mRNAs are presented. All sequencing data are available at NCBI (NCBI Bioproject Accession number: PRJNA325834). This article accompanies the manuscript "MicroRNAs and ectodermal specification I. Identification of miRs and miR-targeted mRNAs in early anterior neural and epidermal ectoderm" (V.V. Shah, B. Soibam, R.A. Ritter, A. Benham, J. Oomen, A.K. Sater, 2016) [1].