RESUMEN
Adverse early life experiences are well-established risk factors for neurological disorders later in life. However, the molecular mechanisms underlying the impact of adverse experiences on neurophysiological systems throughout life remain incompletely understood. Previous studies suggest that social attachment to parents in early development are indispensable for infants to grow into healthy adults. In situations where multiple offspring are born in a single birth in common marmosets, human hand-rearing is employed to ensure the survival of the offspring in captivity. However, hand-reared marmosets often exhibit behavioral abnormalities, including abnormal vocalizations, excessive attachment to the caretaker, and aggressive behavior. In this study, comprehensive transcriptome analyses were conducted on hippocampus tissues, a neuroanatomical region sensitive to social attachment, obtained from human hand-reared (N = 6) and parent-reared male marmosets (N = 5) at distinct developmental stages. Our analyses revealed consistent alterations in a subset of genes, including those related to neurodevelopmental diseases, across different developmental stages, indicating their continuous susceptibility to the effects of early parental deprivation. These findings highlight the dynamic nature of gene expression in response to early life experiences and suggest that the impact of early parental deprivation on gene expression may vary across different stages of development.
Asunto(s)
Callithrix , Padres , Animales , Adulto , Humanos , Masculino , Callithrix/fisiología , Relaciones Familiares , Encéfalo , Expresión GénicaRESUMEN
The chromatin-associated AAA+ ATPases Tip48 and Tip49 are the core components of various complexes implicated in diverse nuclear events such as DNA repair and gene regulation. Although they are frequently overexpressed in many human cancers, their functional significance remains unclear. Here, we show that loss of Tip49 triggered p53-dependent apoptosis and inhibited leukemia development in vivo. To examine the impact of chemical inhibition of this complex on leukemia, we have developed the novel compound DS-4950, which interferes with the ATPase activity of the Tip48/49. Administration of DS-4950 was well-tolerated in healthy mice, and the drug effectively reduced tumor burden and improved survival. We also provide evidence that the dependency on Tip48/49 is widely conserved in non-hematologic malignancies with wild type p53. These results demonstrated that the Tip48/49 ATPases are functionally necessary and therapeutically targetable for the treatment of human cancers.
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ADN Helicasas , Leucemia Mieloide Aguda , Humanos , Ratones , Animales , ADN Helicasas/genética , Proteínas Portadoras/genética , Proteína p53 Supresora de Tumor/genética , Adenosina Trifosfatasas/genética , ATPasas Asociadas con Actividades Celulares Diversas/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genéticaRESUMEN
Disease-risk stratification and development of intensified chemotherapy protocols have substantially improved the outcome of acute lymphoblastic leukemia (ALL). However, outcomes of relapsed or refractory cases remain poor. Previous studies have discussed the oncogenic role of enhancer of zeste homolog 1 and 2 (EZH1/2), and the efficacy of dual inhibition of EZH1/2 as a treatment for hematological malignancy. Here, we investigated whether an EZH1/2 dual inhibitor, DS-3201 (valemetostat), has antitumor effects on B cell ALL (B-ALL). DS-3201 inhibited growth of B-ALL cell lines more significantly and strongly than the EZH2-specific inhibitor EPZ-6438, and induced cell cycle arrest and apoptosis in vitro. RNA-seq analysis to determine the effect of DS-3201 on cell cycle arrest-related genes expressed by B-ALL cell lines showed that DS-3201 upregulated CDKN1C and TP53INP1. CRIPSR/Cas9 knockout confirmed that CDKN1C and TP53INP1 are direct targets of EZH1/2 and are responsible for the antitumor effects of DS-3201 against B-ALL. Furthermore, a patient-derived xenograft (PDX) mouse model showed that DS-3201 inhibited the growth of B-ALL harboring MLL-AF4 significantly. Thus, DS-3201 provides another option for treatment of B-ALL.
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Linfoma de Burkitt , Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Ratones , Animales , Complejo Represivo Polycomb 2 , Regulación hacia Arriba , Proteína Potenciadora del Homólogo Zeste 2 , Inhibidores Enzimáticos/farmacología , Puntos de Control del Ciclo Celular/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas Portadoras/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismoRESUMEN
Malignant rhabdoid tumors (MRTs) are rare and highly aggressive pediatric cancers with no standard of care. MRTs are characterized by loss of SMARCB1, which results in upregulated expression of enhancer of zeste homolog 2 (EZH2), which is responsible for the methylation of lysine 27 of histone H3 (H3K27me3), leading to the repression of gene expression. Although previous reports suggest EZH2 as an effective therapeutic target, the functions of EZH1, the other homolog of EZH, in MRT remain unknown. Here, we show that EZH1, as well as EZH2, contributes to MRT cell growth and H3K27 methylation. Depletion or selective inhibition of EZH2 led to a compensatory increase in EZH1 expression, and depletion of EZH1 enhanced the effect of EZH2 inhibition. EZH1/2 dual inhibitors suppressed MRT cell growth markedly, reflecting the reduction of H3K27me3 accumulation at one of the EZH1/2 targets, the CDKN2A locus. Dual inhibition of EZH1/2 in vivo suppressed tumor growth completely, with no significant adverse effects. These findings indicate that both EZH1 and EZH2 are potential targets for MRT therapy, and that EZH1/2 dual inhibitors may be promising therapeutic strategies for MRT.
RESUMEN
Alzheimer's disease (AD) is the leading cause of dementia which afflicts tens of millions of people worldwide. Despite many scientific progresses to dissect the AD's molecular basis from studies on various mouse models, it has been suffered from evolutionary species differences. Here, we report generation of a non-human primate (NHP), common marmoset model ubiquitously expressing Amyloid-beta precursor protein (APP) transgenes with the Swedish (KM670/671NL) and Indiana (V717F) mutations. The transgene integration of generated two transgenic marmosets (TG1&TG2) was thoroughly investigated by genomic PCR, whole-genome sequencing, and fluorescence in situ hybridization. By reprogramming, we confirmed the validity of transgene expression in induced neurons in vitro. Moreover, we discovered structural changes in specific brain regions of transgenic marmosets by magnetic resonance imaging analysis, including in the entorhinal cortex and hippocampus. In immunohistochemistry, we detected increased Aß plaque-like structures in TG1 brain at 7 years old, although evident neuronal loss or glial inflammation was not observed. Thus, this study summarizes our attempt to establish an NHP AD model. Although the transgenesis approach alone seemed not sufficient to fully recapitulate AD in NHPs, it may be beneficial for drug development and further disease modeling by combination with other genetically engineered models and disease-inducing approaches.
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Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Animales , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Callithrix/genética , Modelos Animales de Enfermedad , Hibridación Fluorescente in Situ , Ratones Transgénicos , TransgenesRESUMEN
Common marmosets are non-human primate models used in biomedical research and genome editing technology. This study aimed to establish cell lines from common marmosets and evaluate their characteristics. We obtained normal fibroblasts derived from muscle tissues of two common marmosets and immortalized them with the introduction of a mutat form of cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomere reverse transcriptase (TERT) using the piggyBac transposon. Compared to parental cells, the immortalized cell lines (named K4DT cells) showed telomerase activity and an accelerated cell proliferation rate. To our knowledge, this is the first study describing the successful establishment of immortalized common marmoset-derived fibroblasts using piggyBac transposition of CDK4R24C, Cyclin D1, and TERT. Our generated cell lines might be a beneficial tool for future studies on disease modeling and targeted gene therapies.
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Callithrix , Telomerasa , Animales , Callithrix/metabolismo , Ciclo Celular/genética , Línea Celular , Ciclina D1/metabolismo , Fibroblastos/metabolismo , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
In veterinary medicine, blood transfusion is commonly performed on companion animals. The common marmoset is a small nonhuman primate with increasing popularity as an animal model in biomedical research. Because of its small whole blood volume, the marmoset is at high risk of exsanguination, and blood transfusion is required to care for life-threatening bleeding. However, few clinical evaluations exist on transfusions for marmosets. This study performed whole blood transfusion with cross-matching on nine marmosets and surveyed the therapeutic effects. Recipients included clinical cases with persistent bleeding, anemia, and coma, as well as animals subjected to postoperative bleeding prophylaxis. Donors were selected from healthy marmosets, including littermates. Cross-match assay before transfusion were all negative, and recipients showed no visible signs of transfusion-related adverse reactions. Whole blood transfusions caused hemostasis and successful recovery in bleeding marmosets, including long-term improvement of anemia cases. Our results indicated that blood transfusion is effective for marmosets with severe anemia and persistent hemorrhage from both non-experimental and surgical causes. Furthermore, DNA sequencing for blood-group classification revealed that all subject marmosets were type A, suggesting that the risk of blood type mismatch may be low in this species.
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Anemia , Callithrix , Anemia/terapia , Anemia/veterinaria , Animales , Transfusión Sanguínea/veterinariaRESUMEN
Pluripotent stem cells (PSCs), embryonic stem cells (ESCs), and induced PSCs (iPSCs) are excellent tools for studying embryonic development in organisms and classified into naïve and primed states. ESC-derived germline chimera individuals can be produced by injecting naïve ESCs/iPSCs into preimplantation embryos, and conversion of primed human ESCs/iPSCs into a naïve state provides insights into epiblast cell features. Non-human ESCs/iPSCs are alternatives to human naïve ESCs/iPSCs, which elicit ethical issues. In this study, we used the common marmoset (Callithrix jacchus) as an animal model. Since 1996, 16 marmoset ESC lines have been established. Because most of these ESC lines are female and were derived >10 years ago, new ESCs, particularly male marmoset ESC lines, are needed. Here, we successfully established 17 novel marmoset ESC lines, including six male ESC lines from in vitro-fertilized (IVF) embryos and 12 ESC lines under feeder-free conditions. This report is the first to establish ESC lines using feeder-free conditions and IVF preimplantation blastocysts in marmosets, and these novel ESC lines could potentially facilitate future non-human primate ESC studies.
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Callithrix , Técnicas de Cultivo de Célula , Animales , Diferenciación Celular , Línea Celular , Células Madre Embrionarias , Femenino , Masculino , EmbarazoRESUMEN
The neocortex has expanded during mammalian evolution. Overexpression studies in developing mouse and ferret neocortex have implicated the human-specific gene ARHGAP11B in neocortical expansion, but the relevance for primate evolution has been unclear. Here, we provide functional evidence that ARHGAP11B causes expansion of the primate neocortex. ARHGAP11B expressed in fetal neocortex of the common marmoset under control of the gene's own (human) promoter increased the numbers of basal radial glia progenitors in the marmoset outer subventricular zone, increased the numbers of upper-layer neurons, enlarged the neocortex, and induced its folding. Thus, the human-specific ARHGAP11B drives changes in development in the nonhuman primate marmoset that reflect the changes in evolution that characterize human neocortical development.
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Proteínas Activadoras de GTPasa/metabolismo , Neocórtex/embriología , Animales , Animales Modificados Genéticamente , Callithrix , Feto , Proteínas Activadoras de GTPasa/genética , Humanos , Ventrículos Laterales/embriología , Ventrículos Laterales/metabolismo , Neocórtex/anatomía & histología , Neocórtex/metabolismo , Células-Madre Neurales/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Tamaño de los Órganos , Regiones Promotoras GenéticasRESUMEN
Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine and the treatment of various diseases. Before proceeding to clinical trials, it is important to test the efficacy and safety of iPS cell-based treatments using experimental animals. The common marmoset is a new world monkey widely used in biomedical studies. However, efficient methods that could generate iPS cells from a variety of cells have not been established. Here, we report that marmoset cells are efficiently reprogrammed into iPS cells by combining RNA transfection and chemical compounds. Using this novel combination, we generate transgene integration-free marmoset iPS cells from a variety of cells that are difficult to reprogram using conventional RNA transfection method. Furthermore, we show this is similarly effective for human and cynomolgus monkey iPS cell generation. Thus, the addition of chemical compounds during RNA transfection greatly facilitates reprogramming and efficient generation of completely integration-free safe iPS cells in primates, particularly from difficult-to-reprogram cells.
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Reprogramación Celular , Fibroblastos/citología , Células Madre Pluripotentes Inducidas/citología , Preparaciones Farmacéuticas/administración & dosificación , ARN/administración & dosificación , Transfección/métodos , Anciano , Animales , Diferenciación Celular , Células Cultivadas , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/fisiología , PlatirrinosRESUMEN
It has been well established that microRNA (miR)-143 is downregulated in human bladder cancer (BC). Recent precision medicine has shown that mutations in BC are frequently observed in FGFR3, RAS and PIK3CA genes, all of which correlate with RAS signaling networks. We have previously shown that miR-143 suppresses cell growth by inhibiting RAS signaling networks in several cancers including BC. In the present study, we showed that synthetic miR-143 negatively regulated the RNA-binding protein Musashi-2 (MSI2) in BC cell lines. MSI2 is an RNA-binding protein that regulates the stability of certain mRNAs and their translation by binding to the target sequences of the mRNAs. Of note, the present study clarified that MSI2 positively regulated KRAS expression through directly binding to the target sequence of KRAS mRNA and promoting its translation, thus contributing to the maintenance of KRAS expression. Thus, miR-143 silenced KRAS and MSI2, which further downregulated KRAS expression through perturbation of the MSI2/KRAS cascade.
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MicroARNs/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas de Unión al ARN/genética , Neoplasias de la Vejiga Urinaria/patología , Animales , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas de Unión al ARN/metabolismo , Regulación hacia Arriba , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
Gastric cancer (GC) is one of the most common cancers worldwide. In the clinical setting, the identification of HER2 overexpression in GC was a significant finding, as trastuzumab, an anti-HER2 drug, provides a survival advantage to HER2-positive GC patients. In HER2-postive GC, the dysregulation of PI3K/AKT and MAPK/ERK signaling pathways has been reported, and inhibition of these pathways is an important therapeutic strategy. MiR-143 is known to act as a tumor suppressor in several cancers, such as bladder cancer, breast cancer, colorectal cancer, and gastric cancer. In the current study, we developed a novel chemically-modified miR-143 and explored the functions of this synthetic miR-143 (syn-miR-143) in HER2-positive gastric cancer. The expression level of miR-143 was down-regulated in GC cell lines, including HER2-positive GC cell lines, MKN7, and KATO-III. The ectopic expression of miR-143 in those cell lines suppressed cell growth through systemic silencing of KRAS and its effector signaling molecules, AKT and ERK. Furthermore, syn-miR-143 indirectly down-regulated the expression of HER2, an upstream molecule of KRAS, through silencing DEAD/H-box RNA helicase 6 (DDX6), RNA helicase, which enhanced HER2 protein expression at the translational step in HER2-positive GC cells. These findings suggested that syn-miR-143 acted as a tumor suppressor through the impairment of KRAS networks including the DDX6.
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ARN Helicasas DEAD-box/metabolismo , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Animales , Antagomirs/metabolismo , Apoptosis/genética , Secuencia de Bases , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones Desnudos , MicroARNs/genética , Modelos Biológicos , Transducción de Señal , Regulación hacia Arriba/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Therapy based on targeted inhibition of BCR-ABL tyrosine kinase has greatly improved the prognosis for patients with Philadelphia chromosome (Ph)-positive leukemia and tyrosine kinase inhibitors (TKI) have become standard therapy. However, some patients acquire resistance to TKI that is frequently associated with point mutations in BCR-ABL. We previously reported that a medium-chain fatty-acid derivative AIC-47 induced transcriptional suppression of BCR-ABL and perturbation of the Warburg effect, leading to growth inhibition in Ph-positive leukemia cells. Herein, we showed that AIC-47 had anti-leukemic effects in either wild type (WT)- or mutated-BCR-ABL-harboring cells. AIC-47 suppressed transcription of BCR-ABL gene regardless of the mutation through downregulation of transcriptional activator, c-Myc. Reprogramming of the metabolic pathway has been reported to be associated with resistance to anti-cancer drugs; however, we found that a point mutation of BCR-ABL was independent of the profile of pyruvate kinase muscle (PKM) isoform expression. Even in T315I-mutated cells, AIC-47 induced switching of the expression profile of PKM isoforms from PKM2 to PKM1, suggesting that AIC-47 disrupted the Warburg effect. In a leukemic mouse model, AIC-47 greatly suppressed the increase in BCR-ABL mRNA level and improved hepatosplenomegaly regardless of the BCR-ABL mutation. Notably, the improvement of splenomegaly by AIC-47 was remarkable and might be equal to or greater than that of TKI. These findings suggest that AIC-47 might be a promising agent for overcoming the resistance of Ph-positive leukemia to therapy.
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Proliferación Celular/efectos de los fármacos , Ácidos Grasos/farmacología , Proteínas de Fusión bcr-abl/genética , Compuestos Heterocíclicos con 1 Anillo/farmacología , Cetonas/farmacología , Leucemia/tratamiento farmacológico , Mutación Puntual/genética , Inhibidores de Proteínas Quinasas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Humanos , Leucemia/genética , Ratones , Ratones Endogámicos BALB C , Ratones DesnudosAsunto(s)
Autofagia , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Fusión bcr-abl/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis , Animales , Antineoplásicos/farmacología , Células Cultivadas , Proteínas de Fusión bcr-abl/genética , Compuestos Heterocíclicos con 1 Anillo/farmacología , Humanos , Cetonas/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Mutación , Células Precursoras de Linfocitos B/efectos de los fármacos , Células Precursoras de Linfocitos B/metabolismo , Células Precursoras de Linfocitos B/patologíaRESUMEN
Serine and arginine rich splicing factor 3 (SRSF3), an SR-rich family protein, has an oncogenic function in various kinds of cancer. However, the detailed mechanism of the function had not been previously clarified. Here, we showed that the SRSF3 splicer regulated the expression profile of the pyruvate kinase, which is one of the rate-limiting enzymes in glycolysis. Most cancer cells express pyruvate kinase muscle 2 (PKM2) dominantly to maintain a glycolysis-dominant energy metabolism. Overexpression of SRSF3, as well as that of another splicer, polypyrimidine tract binding protein 1 (PTBP1) and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), in clinical cancer samples supported the notion that these proteins decreased the Pyruvate kinase muscle 1 (PKM1)/PKM2 ratio, which positively contributed to a glycolysis-dominant metabolism. The silencing of SRSF3 in human colon cancer cells induced a marked growth inhibition in both in vitro and in vivo experiments and caused an increase in the PKM1/PKM2 ratio, thus resulting in a metabolic shift from glycolysis to oxidative phosphorylation. At the same time, the silenced cells were induced to undergo autophagy. SRSF3 contributed to PKM mRNA splicing by co-operating with PTBP1 and hnRNPA1, which was validated by the results of RNP immunoprecipitation (RIP) and immunoprecipitation (IP) experiments. These findings altogether indicated that SRSF3 as a PKM splicer played a positive role in cancer-specific energy metabolism.
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Neoplasias del Colon/metabolismo , Metabolismo Energético , Piruvato Quinasa/genética , Empalme del ARN/genética , Factores de Empalme Serina-Arginina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Autofagia , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular , Neoplasias del Colon/patología , Neoplasias del Colon/ultraestructura , Femenino , Silenciador del Gen , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones Desnudos , Persona de Mediana Edad , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Piruvato Quinasa/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKI) targeted against BCR-ABL. We previously reported the investigation of residual CML diseases during TKI treatment using FACS-sorting and quantitative RT-PCR of BCR-ABL among each population; total mononuclear cells, hematopoietic stem cells, and myeloid progenitors. The observations also implied that the second-generation of ABL-tyrosine kinase inhibitors (2nd TKIs), dasatinib or nilotinib therapy can be more promising approach for efficient reduction of the CML stem cells. Moreover, we need to develop the evaluation method of the residual CML diseases to establish rational therapy-cessation strategies in CML.
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Dasatinib/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Fusión bcr-abl , Leucemia Mielógena Crónica BCR-ABL Positiva , Células Progenitoras Mieloides , Células Madre Neoplásicas , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucocitos Mononucleares/enzimología , Leucocitos Mononucleares/patología , Células Progenitoras Mieloides/enzimología , Células Progenitoras Mieloides/patología , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patologíaRESUMEN
Pyruvate kinase is known as the glycolytic enzyme catalyzing the final step in glycolysis. In mammals, two different forms of it exist, i.e., pyruvate kinase M1/2 (PKM) and pyruvate kinase L/R (PKLR). Also, PKM has two isoforms, i.e., PKM1 and PKM2. These genes have tissue-specific distribution. Namely, PKM1 is distributed in high-energy-demanding organs, such as brain and muscle. Also, PKM2 is distributed in various other organs, such as the colon. On the other hand, PKLR is distributed in liver and red blood cells (RBCs). Interestingly, PKM2 has been recognized as one of the essential genes for the cancer-specific energy metabolism termed the “Warburg effect”. However, the mechanism(s) underlying this fact have remained largely unclear. Recently, we found that some organ-specific microRNAs (miRNAs, MIR) regulate PKM isoform expression through direct targeting of polypyrimidine tract binding protein 1 (PTBP1), which is the splicer responsible for PKM2-dominant expression. In this study, we examined whether this machinery was conserved in the case of other PTBP1- and PKM-targeting miRNAs. We focused on the MIRs 122, 137, and 206, and investigated the expression profiles of each of these miRNAs in tissues from mouse and human organs. Also, we examined the regulatory mechanisms of PKM isoform expression by testing each of these miRNAs in human cancer cell lines. Presently, we found that brain-specific MIR137 and muscle-specific MIR206 predominantly induced PKM1 expression through direct targeting of PTBP1. Also, liver-specific MIR122 suppressed the expression of both PKM1 and PKM2, which action occurred through direct targeting of PKM to enable the expression of PKLR. Moreover, the expression levels of these miRNAs were downregulated in cancer cells that had originated from these tissues, resulting in PKM2 dominance. Our results suggest that the organ-specific distribution of miRNAs is one of the principal means by which miRNA establishes characteristics of a tissue and that dysregulation of these miRNAs results in cancer development through a change in the ratio of PKM isoform expression. Also, our results contribute to cancer diagnosis and will be useful for cancer-specific therapy for the Warburg effect in the near future.
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Proteínas Portadoras/genética , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/genética , MicroARNs/genética , Hormonas Tiroideas/genética , Regiones no Traducidas 3' , Animales , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Modelos Biológicos , Especificidad de Órganos/genética , Interferencia de ARN , ARN Mensajero/genética , Hormonas Tiroideas/metabolismo , Proteínas de Unión a Hormona TiroideRESUMEN
Despite considerable research on K-Ras inhibitors, none had been established until now. We synthesized nuclease-resistant synthetic miR-143 (miR-143#12), which strongly silenced K-Ras, its effector signal molecules AKT and ERK, and the K-Ras activator Sos1. We examined the anti-proliferative effect of miR-143#12 and the mechanism in human colon cancer DLD-1 cell (G13D) and other cell types harboring K-Ras mutations. Cell growth was markedly suppressed in a concentration-dependent manner by miR-143#12 (IC50 : 1.32 nmol L-1 ) with a decrease in the K-Ras mRNA level. Interestingly, this mRNA level was also downregulated by either a PI3K/AKT or MEK inhibitor, which indicates a positive circuit of K-Ras mRNA expression. MiR-143#12 silenced cytoplasmic K-Ras mRNA expression and impaired the positive circuit by directly targeting AKT and ERK mRNA. Combination treatment with miR-143#12 and a low-dose EGFR inhibitor induced a synergistic inhibition of growth with a marked inactivation of both PI3K/AKT and MAPK/ERK signaling pathways. However, silencing K-Ras by siR-KRas instead of miR-143#12 did not induce this synergism through the combined treatment with the EGFR inhibitor. Thus, miR-143#12 perturbed the K-Ras expression system and K-Ras activation by silencing Sos1 and, resultantly, restored the efficacy of the EGFR inhibitors. The in vivo results also supported those of the in vitro experiments. The extremely potent miR-143#12 enabled us to understand K-Ras signaling networks and shut them down by combination treatment with this miRNA and EGFR inhibitor in K-Ras-driven colon cancer cell lines.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , MicroARNs/administración & dosificación , MicroARNs/síntesis química , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Bencimidazoles/administración & dosificación , Bencimidazoles/farmacología , Benzotiazoles/administración & dosificación , Benzotiazoles/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/genética , Sinergismo Farmacológico , Flavonoides/administración & dosificación , Flavonoides/farmacología , Células HT29 , Humanos , Ratones , MicroARNs/antagonistas & inhibidores , MicroARNs/química , MicroARNs/farmacología , Mutación , Trasplante de Neoplasias , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Human DEAD-box RNA helicase gene DDX6 was cloned from B-cell lymphoma cell line RC-K8. Previously, we reported that DDX6 acts as oncogene in several cancers such as colorectal cancer and hepatocellular carcinoma. However, the detailed mechanism of DDX6 action in carcinogenesis is largely unknown. In this study, we examined the functions of DDX6 in clinical gastric cancer (GC) samples and GC cells. DDX6 protein expression levels of cancer samples were higher than those of the adjacent normal tissues in 25 clinical GC samples (median value: 1.4 times higher). Also, the results of an RNA immunoprecipitation-assay (RIP-assay) showed that DDX6 associated with c-Myc mRNA. Moreover, enforced overexpression of DDX6 promoted both mRNA and protein expression of c-Myc in GC cells. On the other hand, the gene silencing of DDX6 induced growth suppression through down-regulation of c-Myc in GC cells grown in either two or three dimensions. Furthermore, c-Myc mRNA expression levels of cancer samples were higher than those of the adjacent normal tissues in DDX6 up-regulated-GC clinical samples. Our findings in this study suggested that DDX6 acted as oncogene in GC cells through promotion of c-Myc expression by association with the mRNA of c-Myc.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas/metabolismo , Neoplasias Gástricas/metabolismo , Regulación hacia Arriba , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Proliferación Celular , ARN Helicasas DEAD-box/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Neoplasias Gástricas/genéticaRESUMEN
Morphometry studies of human brain development have revealed characteristics of some growth patterns, such as gray matter (GM) and white matter (WM), but the features that make human neurodevelopment distinct from that in other species remain unclear. Studies of the common marmoset (Callithrix jacchus), a small New World primate, can provide insights into unique features such as cooperative behaviors complementary to those from comparative analyses using mouse and rhesus monkey. In the present study, we analyzed developmental patterns of GM, WM, and cortical regions with volume measurements using longitudinal sample (23 marmosets; 11 male, 12 female) between the ages of one and 30months. Regional analysis using a total of 164 magnetic resonance imaging datasets revealed that GM volume increased before puberty (5.4months), but subsequently declined until adulthood, whereas WM volume increased rapidly before stabilizing around puberty (9.9months). Cortical regions showed similar patterns of increase and decrease, patterns with global GM but differed in the timing of volume peak and degree of decline across regions. The progressive-regressive pattern detected in both global and cortical GM was well correlated to phases of synaptogenesis and synaptic pruning reported in previous marmoset studies. A rapid increase in WM in early development may represent a distinctive aspect of human neurodevelopment. These findings suggest that studies of marmoset brain development can provide valuable comparative information that will facilitate a deeper understanding of human brain growth and neurodevelopmental disorders.