RESUMO
Hypoxia-inducible factor 1α (HIF1α) is a transcription factor that regulates angiogenesis under hypoxic conditions. To investigate the posttranscriptional regulatory mechanism of HIF1α, we performed a cell-based screening to reveal potential cis-elements and the regulatory RNA-binding proteins that act as trans-factors. We found that LIN28A promoted HIF1α protein expression independently of the downregulation of microRNA let-7, which is also directly mediated by LIN28A. Transcriptome analysis and evaluation of RNA stability using RNA-seq and SLAM-seq analyses, respectively, revealed that LIN28A upregulates HIF1A expression via mRNA stabilization. To investigate the physical association of LIN28A with HIF1A mRNA, we performed enhanced crosslinking immunoprecipitation in 293FT cells and integrally analyzed the transcriptome. We observed that LIN28A associates with HIF1A mRNA via its cis-element motif "UGAU". The "UGAU" motifs are recognized by the cold shock domain of LIN28A, and the introduction of a loss-of-function mutation to the cold shock domain diminished the upregulatory activities performed by LIN28A. Finally, the microvessel density assay showed that the expression of LIN28A promoted angiogenesis in vivo. In conclusion, our study elucidated the role of LIN28A in enhancing the HIF1α axis at the posttranscription layer.
Assuntos
Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia , Estabilidade de RNA , Proteínas de Ligação a RNA , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
4-Nonylphenol (NP) has weak estrogen-like activity, and can therefore act as an endocrine disruptor. This study examined the effects of perinatal exposure to low-dose NP on learning and memory, general activity, and emotionality in male rat offspring. Dams were orally administered 1 or 10 mg/kg/day of NP or vehicle from gestational day 10 to postnatal day 14. The male offspring were evaluated using a battery of behavioral tests, including an appetite-motivated maze test (MAZE test) used to assess spatial learning and memory. In the MAZE test, times to reach goal (food) for both groups treated with NP were significantly shorter than those for the control group. In other behavioral tests (the open-field, elevated plus-maze, and step-through passive avoidance tests), NP did not affect any of each behavioral parameter. Thus, this study indicates perinatal exposure to low-dose NP specifically improves spatial learning and memory in male rat offspring.
Assuntos
Feto/efeitos dos fármacos , Memória/efeitos dos fármacos , Fenóis/farmacologia , Aprendizagem Espacial/efeitos dos fármacos , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Donor organ shortages for transplantation remain a serious global concern, and alternative treatment is in high demand. Fetal cells and tissues have considerable therapeutic potential as, for example, organoid technology that uses human induced pluripotent stem cells (hiPSCs) to generate unlimited human fetal-like cells and tissues. We previously reported the in vivo vascularization of early fetal liver-like hiPSC-derived liver buds (LBs) and subsquent improved survival of recipient mice with subacute liver failure. Here, we show hiPSC-liver organoids (LOs) that recapitulate midgestational fetal liver promote de novo liver generation when grafted onto the surface of host livers in chemical fibrosis models, thereby recovering liver function. We found that fetal liver, a hematopoietic tissue, highly expressed macrophage-recruiting factors and antifibrotic M2 macrophage polarization factors compared with the adult liver, resulting in fibrosis reduction because of CD163+ M2-macrophage polarization. Next, we created midgestational fetal liver-like hiPSC-LOs by fusion of hiPSC-LBs to induce static cell-cell interactions and found that these contained complex structures such as hepatocytes, vasculature, and bile ducts after transplantation. This fusion allowed the generation of a large human tissue suitable for transplantation into immunodeficient rodent models of liver fibrosis. hiPSC-LOs showed superior liver function compared with hiPSC-LBs and improved survival and liver function upon transplantation. In addition, hiPSC-LO transplantation ameliorated chemically induced liver fibrosis, a symptom of liver cirrhosis that leads to organ dysfunction, through immunomodulatory effects, particularly on CD163+ phagocytic M2-macrophage polarization. Together, our results suggest hiPSC-LO transplantation as a promising therapeutic option for liver fibrosis.
Assuntos
Imunomodulação , Células-Tronco Pluripotentes Induzidas , Cirrose Hepática , Fígado , Organoides , Humanos , Cirrose Hepática/patologia , Cirrose Hepática/terapia , Animais , Fígado/patologia , Macrófagos , Transplante de Fígado , CamundongosRESUMO
The heterogeneity of cancer stem cells (CSCs) within tumors presents a challenge in therapeutic targeting. To decipher the cellular plasticity that fuels phenotypic heterogeneity, we undertook single-cell transcriptomics analysis in triple-negative breast cancer (TNBC) to identify subpopulations in CSCs. We found a subpopulation of CSCs with ancestral features that is marked by FXYD domain-containing ion transport regulator 3 (FXYD3), a component of the Na+/K+ pump. Accordingly, FXYD3+ CSCs evolve and proliferate, while displaying traits of alveolar progenitors that are normally induced during pregnancy. Clinically, FXYD3+ CSCs were persistent during neoadjuvant chemotherapy, hence linking them to drug-tolerant persisters (DTPs) and identifying them as crucial therapeutic targets. Importantly, FXYD3+ CSCs were sensitive to senolytic Na+/K+ pump inhibitors, such as cardiac glycosides. Together, our data indicate that FXYD3+ CSCs with ancestral features are drivers of plasticity and chemoresistance in TNBC. Targeting the Na+/K+ pump could be an effective strategy to eliminate CSCs with ancestral and DTP features that could improve TNBC prognosis.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Células-Tronco Neoplásicas/patologia , Linhagem Celular Tumoral , Proteínas de Membrana , Proteínas de Neoplasias/genéticaRESUMO
Programmed death-ligand 1 (PD-L1) is a co-inhibitory molecule expressed on tumor cells. Immune checkpoint inhibitors focusing on the PD-L1 mechanism are now being studied for the treatment of various cancer types. However, the regulatory mechanism of PD-L1 is yet to be fully clarified, and a high-throughput system for comparing the abilities of small compounds in regulating PD-L1 has not yet been established. Therefore, we created a HiBiT-tagged lung adenocarcinoma cell line, PC9-KI, for easier and faster detection of changes in PD-L1 protein expression. Using PC9-KI cells, we screened 1280 chemical compounds from the Library of Pharmacologically Active Compounds and identified microtubule polymerization inhibitors and thapsigargin as PD-L1 upregulators and a p97 inhibitor as a PD-L1 downregulator.
Assuntos
Antineoplásicos/farmacologia , Antígeno B7-H1/genética , Proteínas Recombinantes de Fusão/genética , Mucosa Respiratória/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Moduladores de Tubulina/farmacologia , Antígeno B7-H1/agonistas , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Efeito Fundador , Expressão Gênica , Genes Reporter , Ensaios de Triagem em Larga Escala , Humanos , Medições Luminescentes , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Engenharia de Proteínas/métodos , Quinazolinas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Tapsigargina/farmacologia , Proteína com Valosina/antagonistas & inibidores , Proteína com Valosina/genética , Proteína com Valosina/metabolismoRESUMO
BACKGROUNDS: Sevoflurane is a most frequently used volatile anesthetics, but its molecular mechanisms of action remain unclear. We hypothesized that specific genes play regulatory roles in brain exposed to sevoflurane. Thus, we aimed to evaluate the effects of sevoflurane inhalation and identify potential regulatory genes by RNA-seq analysis. METHODS: Eight-week old mice were exposed to sevoflurane. RNA from medial prefrontal cortex, striatum, hypothalamus, and hippocampus were analysed using RNA-seq. Differently expressed genes were extracted and their gene ontology terms were analysed using Metascape. These our anesthetized mouse data and the transcriptome array data of the cerebral cortex of sleeping mice were compared. Finally, the activities of transcription factors were evaluated using a weighted parametric gene set analysis (wPGSA). JASPAR was used to confirm the existence of binding motifs in the upstream sequences of the differently expressed genes. RESULTS: The gene ontology term enrichment analysis result suggests that sevoflurane inhalation upregulated angiogenesis and downregulated neural differentiation in each region of brain. The comparison with the brains of sleeping mice showed that the gene expression changes were specific to anesthetized mice. Focusing on individual genes, sevoflurane induced Klf4 upregulation in all sampled parts of brain. wPGSA supported the function of KLF4 as a transcription factor, and KLF4-binding motifs were present in many regulatory regions of the differentially expressed genes. CONCLUSIONS: Klf4 was upregulated by sevoflurane inhalation in the mouse brain. The roles of KLF4 might be key to elucidating the mechanisms of sevoflurane induced functional modification in the brain.
Assuntos
Encéfalo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Sevoflurano/farmacologia , Transcriptoma/efeitos dos fármacos , Animais , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Ontologia Genética , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Fatores de Transcrição/genética , Transcriptoma/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
In human genome, there are approximately 1,500 RNA-binding proteins (RBPs). They can regulate mRNA stability or translational efficiency via ribosomes and these processes are known as 'post-transcriptional regulation'. Accumulating evidences indicate that post-transcriptional regulation is the determinant of the accurate levels of cytokines mRNAs. While transcriptional regulation of cytokines mRNAs has been well studied and found to be important for the rapid induction of mRNA and regulation of the acute phase of inflammation, post-transcriptional regulation by RBPs is essential for resolving inflammation in the later phase, and their dysfunction may lead to severe autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus. For post-transcriptional regulation, RBPs recognize and directly bind to cis-regulatory elements in 3' untranslated region of mRNAs such as AU-rich or constitutive decay elements and play various roles. In this review, we summarize the recent findings regarding the role of RBPs in the regulation of inflammation.
Assuntos
Inflamação/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica/genética , Animais , Humanos , Inflamação/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologiaRESUMO
Nonylphenol ethoxylate (NPE) is a non-ionic surfactant, that is degraded to short-chain NPE and 4-nonylphenol (NP) by bacteria in the environment. NP, one of the most common environmental endocrine disruptors, exhibits weak estrogen-like activity. In this study, we investigated whether oral administration of NP (at 0.5 and 5 mg/kg doses) affects spatial learning and memory, general activity, emotionality, and fear-motivated learning and memory in male and female Sprague-Dawley (SD) rats. SD rats of both sexes were evaluated using a battery of behavioral tests, including an appetite-motivated maze test (MAZE test) that was used to assess spatial learning and memory. In the MAZE test, the time required to reach the reward in male rats treated with 0.5 mg/kg NP group and female rats administered 5 mg/kg NP was significantly longer than that for control animals of the corresponding sex. In other behavioral tests, no significant differences were observed between the control group and either of the NP-treated groups of male rats. In female rats, inner and ambulation values for animals administered 0.5 mg/kg NP were significantly higher than those measured in control animals in open-field test, while the latency in the group treated with 5 mg/kg NP was significantly shorter compared to the control group in step-through passive avoidance test. This study indicates that oral administration of a low-dose of NP slightly impairs spatial learning and memory performance in male and female rats, and alters emotionality and fear-motivated learning and memory in female rats only.
Assuntos
Comportamento Animal/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Memória/efeitos dos fármacos , Fenóis/administração & dosagem , Fenóis/toxicidade , Desempenho Psicomotor/efeitos dos fármacos , Aprendizagem Espacial/efeitos dos fármacos , Tensoativos/administração & dosagem , Tensoativos/toxicidade , Administração Oral , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Emoções/efeitos dos fármacos , Disruptores Endócrinos/administração & dosagem , Medo/efeitos dos fármacos , Feminino , Masculino , Ratos Sprague-Dawley , CaminhadaRESUMO
Cognitive dysfunction due to higher blood glucose level has been reported previously. Genistein (GEN) is a phytoestrogen that we hypothesized might lead to improved memory, despite elevated blood glucose levels at the time of memory consolidation. To investigate this hypothesis, we compared the effects of orally administered GEN on the central nervous system in normal versus glucose-loaded adult male rats. A battery of behavioral assessments was carried out. In the MAZE test, which measured spatial learning and memory, the time of normal rats was shortened by GEN treatment compared to the vehicle group, but only in the early stages of testing. In the glucose-loaded group, GEN treatment improved performance as mazes were advanced. In the open-field test, GEN treatment delayed habituation to the new environment in normal rats, and increased the exploratory behaviors of glucose-loaded rats. There were no significant differences observed for emotionality or fear-motivated learning and memory. Together, these results indicate that GEN treatment improved spatial learning and memory only in the early stages of testing in the normal state, but improved spatial learning and memory when glucose levels increased during memory consolidation.