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Reactive astrogliosis is a hallmark of Alzheimer's disease (AD). However, a clinically validated neuroimaging probe to visualize the reactive astrogliosis is yet to be discovered. Here, we show that PET imaging with 11C-acetate and 18F-fluorodeoxyglucose (18F-FDG) functionally visualizes the reactive astrocyte-mediated neuronal hypometabolism in the brains with neuroinflammation and AD. To investigate the alterations of acetate and glucose metabolism in the diseased brains and their impact on the AD pathology, we adopted multifaceted approaches including microPET imaging, autoradiography, immunohistochemistry, metabolomics, and electrophysiology. Two AD rodent models, APP/PS1 and 5xFAD transgenic mice, one adenovirus-induced rat model of reactive astrogliosis, and post-mortem human brain tissues were used in this study. We further curated a proof-of-concept human study that included 11C-acetate and 18F-FDG PET imaging analyses along with neuropsychological assessments from 11 AD patients and 10 healthy control subjects. We demonstrate that reactive astrocytes excessively absorb acetate through elevated monocarboxylate transporter-1 (MCT1) in rodent models of both reactive astrogliosis and AD. The elevated acetate uptake is associated with reactive astrogliosis and boosts the aberrant astrocytic GABA synthesis when amyloid-ß is present. The excessive astrocytic GABA subsequently suppresses neuronal activity, which could lead to glucose uptake through decreased glucose transporter-3 in the diseased brains. We further demonstrate that 11C-acetate uptake was significantly increased in the entorhinal cortex, hippocampus and temporo-parietal neocortex of the AD patients compared to the healthy controls, while 18F-FDG uptake was significantly reduced in the same regions. Additionally, we discover a strong correlation between the patients' cognitive function and the PET signals of both 11C-acetate and 18F-FDG. We demonstrate the potential value of PET imaging with 11C-acetate and 18F-FDG by visualizing reactive astrogliosis and the associated neuronal glucose hypometablosim for AD patients. Our findings further suggest that the acetate-boosted reactive astrocyte-neuron interaction could contribute to the cognitive decline in AD.
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Doença de Alzheimer , Camundongos , Humanos , Ratos , Animais , Doença de Alzheimer/metabolismo , Fluordesoxiglucose F18/metabolismo , Astrócitos/metabolismo , Radioisótopos de Carbono/metabolismo , Gliose/diagnóstico por imagem , Encéfalo/patologia , Tomografia por Emissão de Pósitrons/métodos , Ácido gama-Aminobutírico/metabolismoRESUMO
Several radiolabeled prostate-specific membrane antigen (PSMA)-targeted agents have been developed for detecting prostate cancer, using positron emission tomography imaging and targeted radionuclide therapy. Among them, [18F]PSMA-1007 has several advantages, including a comparatively long half-life, delayed renal excretion, and compatible structure with α-/ß-particle emitter-labeled therapeutics. This study aimed to characterize the preclinical pharmacokinetics and internal radiation dosimetry of [18F]PSMA-1007, as well as its repeatability and specificity for target binding using prostate tumor-bearing mice. In PSMA-positive tumor-bearing mice, the kidney showed the greatest accumulation of [18F]PSMA-1007. The distribution in the tumor attained its peak concentration of 2.8%ID/g at 112 min after intravenous injection. The absorbed doses in the tumor and salivary glands were 0.079 ± 0.010 Gy/MBq and 0.036 ± 0.006 Gy/MBq, respectively. The variance of the net influx (Ki) of [18F]PSMA-1007 to the tumor was minimal between scans performed in the same animals (within-subject coefficient of variation = 7.57%). [18F]PSMA-1007 uptake in the tumor was specifically decreased by 32% in Ki after treatment with a PSMA inhibitor 2-(phosphonomethyl)-pentanedioic acid (2-PMPA). In the present study, we investigated the in vivo preclinical characteristics of [18F]PSMA-1007. Our data from [18F]PSMA-1007 PET/computed tomography (CT) studies in a subcutaneous prostate cancer xenograft mouse model supports clinical therapeutic strategies that use paired therapeutic radiopharmaceuticals (such as [177Lu]Lu-PSMA-617), especially strategies with a quantitative radiation dose estimate for target lesions while minimizing radiation-induced toxicity to off-target tissues.
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Neoplasias da Próstata , Compostos Radiofarmacêuticos , Masculino , Humanos , Animais , Camundongos , Compostos Radiofarmacêuticos/farmacocinética , Xenoenxertos , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/radioterapia , Neoplasias da Próstata/tratamento farmacológico , Oligopeptídeos , Glutamato Carboxipeptidase II/metabolismo , Antígenos de Superfície/metabolismo , Linhagem Celular TumoralRESUMO
AIMS: To investigate the effect of dapagliflozin, a sodium-glucose co-transporter-2 (SGLT2) inhibitor, on renal gluconeogenesis in vitro, ex vivo and in vivo. MATERIALS AND METHODS: We treated HK-2 cells (human renal proximal tubule cells) and mouse primary renal proximal tubule cells with dapagliflozin, and evaluated the process of renal gluconeogenesis. We also examined the effect of dapagliflozin on renal gluconeogenesis in normoglycaemic and hyperglycaemic mice. RESULTS: Dapagliflozin enhanced renal gluconeogenesis in vitro, ex vivo and in vivo. It increased phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), peroxisome proliferative activated receptor-gamma co-activator 1α (PGC-1α) and phosphorylated cyclic-AMP response element binding protein (CREB) expression and decreased phosphorylated Forkhead Box O1 (FOXO1) expression in HK-2 cells, mouse primary renal proximal tubule cells, and the mouse renal cortex. Glutamine enhanced the gluconeogenic effect of dapagliflozin in HK-2 cells. Also, dapagliflozin increased 14 C-glutamine utilization in HK-2 cells. Glucagon did not affect dapagliflozin-induced enhancement in renal gluconeogenesis in HK-2 cells. SGLT2 gene knockdown with siRNA resulted in an increase of gluconeogenic gene expression and associated transcription factors in HK-2 cells. Dapagliflozin reduced fasting plasma glucose levels and improved oral glucose tolerance and insulin tolerance in high-fat diet-fed hyperglycaemic mice, although renal gluconeogenesis was enhanced. CONCLUSIONS: Dapagliflozin increased levels of gluconeogenic enzyme in the renal cortex and consequently increased renal gluconeogenesis, which is mediated by SGLT2 inhibition.
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Gluconeogênese , Inibidores do Transportador 2 de Sódio-Glicose , Compostos Benzidrílicos , Glucose/metabolismo , Glucosídeos , Túbulos Renais Proximais/metabolismo , Fígado/metabolismo , Sódio/metabolismo , Transportador 2 de Glucose-Sódio/metabolismoRESUMO
OBJECTIVES: A special vector system was developed to monitor the in vitro and in vivo endogenous level of a primary transcript of miR124a during neuronal differentiation RESULTS: The upstream regions of miR124a were fused with luciferase (Gluc) and their activity was measured. During neurogenesis of P19 cells, the primary transcript level of miR124a was increased 1.5-times compared to the undifferentiated P19 cells. P19 cells grafted to nude mice exhibited the same pattern of luciferase activity in vivo as they did in vitro. CONCLUSION: The expression of primary miR124a during neurogenesis was successfully imaged by in vitro and in vivo luciferase reporter gene-based method.
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MicroRNAs/genética , MicroRNAs/metabolismo , Imagem Molecular/métodos , Neurogênese/genética , Neurogênese/fisiologia , Animais , Sequência de Bases , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/química , Dados de Sequência Molecular , Regiões Promotoras GenéticasRESUMO
Glioblastoma (GBM) is a representative malignant brain tumor characterized by a dismal prognosis, with survival rates of less than 2 years and high recurrence rates. Despite surgical resection and several alternative treatments, GBM remains a refractory disease due to its aggressive invasiveness and resistance to anticancer therapy. In this report, we explore the role of fibronectin type III domain containing 3B (FNDC3B) and its potential as a prognostic and therapeutic biomarker in GBM. GBM exhibited a significantly higher cancer-to-normal ratio compared to other organs, and patients with high FNDC3B expression had a poor prognosis (p < 0.01). In vitro studies revealed that silencing FNDC3B significantly reduced the expression of Survivin, an apoptosis inhibitor, and also reduced cell migration, invasion, extracellular matrix adhesion ability, and stem cell properties in GBM cells. Furthermore, we identified that FNDC3B regulates PTEN/PI3K/Akt signaling in GBM cells using MetaCore integrated pathway bioinformatics analysis and a proteome profiler phospho-kinase array with sequential western blot analysis. Collectively, our findings suggest FNDC3B as a potential biomarker for predicting GBM patient survival and for the development of treatment strategies for GBM.
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BACKGROUND: Glial scar formation is a reactive glial response confining injured regions in a central nervous system. However, it remains challenging to identify key factors formulating glial scar in response to glioblastoma (GBM) due to complex glia-GBM crosstalk. METHODS: Here, we constructed an astrocytic scar enclosing GBM in a human assembloid and a mouse xenograft model. GBM spheroids were preformed and then co-cultured with microglia and astrocytes in 3D Matrigel. For the xenograft model, U87-MG cells were subcutaneously injected to the Balb/C nude female mice. RESULTS: Additional glutamate was released from GBM-microglia assembloid by 3.2-folds compared to GBM alone. The glutamate upregulated astrocytic monoamine oxidase-B (MAO-B) activity and chondroitin sulfate proteoglycans (CSPGs) deposition, forming the astrocytic scar and restricting GBM growth. Attenuating scar formation by the glutamate-MAO-B inhibition increased drug penetration into GBM assembloid, while reducing GBM confinement. CONCLUSIONS: Taken together, our study suggests that astrocytic scar could be a critical modulator in GBM therapeutics.
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BACKGROUND: Reactive astrogliosis is a hallmark of various brain pathologies, including neurodegenerative diseases and glioblastomas. However, the specific intermediate metabolites contributing to reactive astrogliosis remain unknown. This study investigated how glioblastomas induce reactive astrogliosis in the neighboring microenvironment and explores 11C-acetate PET as an imaging technique for detecting reactive astrogliosis. METHODS: Through in vitro, mouse models, and human tissue experiments, we examined the association between elevated 11C-acetate uptake and reactive astrogliosis in gliomas. We explored acetate from glioblastoma cells, which triggers reactive astrogliosis in neighboring astrocytes by upregulating MAO-B and MCT1 expression. We evaluated the presence of cancer stem cells in the reactive astrogliosis region of glioblastomas and assessed the correlation between the volume of 11C-acetate uptake beyond MRI and prognosis. RESULTS: Elevated 11C-acetate uptake is associated with reactive astrogliosis and astrocytic MCT1 in the periphery of glioblastomas in human tissues and mouse models. Glioblastoma cells exhibit increased acetate production as a result of glucose metabolism, with subsequent secretion of acetate. Acetate derived from glioblastoma cells induces reactive astrogliosis in neighboring astrocytes by increasing the expression of MAO-B and MCT1. We found cancer stem cells within the reactive astrogliosis at the tumor periphery. Consequently, a larger volume of 11C-acetate uptake beyond contrast-enhanced MRI was associated with worse prognosis. CONCLUSION: Our results highlight the role of acetate derived from glioblastoma cells in inducing reactive astrogliosis and underscore the potential value of 11C-acetate PET as an imaging technique for detecting reactive astrogliosis, offering important implications for the diagnosis and treatment of glioblastomas.
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HCC is well known for low glycolysis in the tumors, whereas hypoxia induces glycolytic phenotype and tumor progression. This study was conducted to evaluate the expression of SLCs in human HCCs and investigated whether extracellular nutrient administration related to SLCs in low-glycolytic HCC can prevent hypoxic tumor progression. SLCs expression was screened according to the level of glycolysis in HCCs. Then, whether extracellular nutrient treatment can affect hypoxic tumor progression, as well as the mechanisms, were evaluated in an in vitro cell line and an in vivo animal model. Low-glycolytic HCCs showed high SLC13A5/NaCT and SLC16A1/MCT1 but low SLC2A1/GLUT1 and HIF1α/HIF1α expression. Especially, high SLC13A5 expression was significantly associated with good overall survival in the Cancer Genome Atlas (TCGA) database. In HepG2 cells with the highest NaCT expression, extracellular citrate treatment upon hypoxia induced HIF1α degradation, which led to reduced glycolysis and cellular proliferation. Finally, in HepG2-animal models, the citrate-treated group showed smaller tumor with less hypoxic areas than the vehicle-treated group. In patients with HCC, SLC13A5/NaCT is an important SLC, which is associated with low glycolysis and good prognosis. Extracellular citrate treatment induced the failure of metabolic adaptation to hypoxia and tumor growth inhibition, which can be a potential therapeutic strategy in HCCs.
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PURPOSE: 11 C-acetate ( 11 C-ACE) uptake on PET/CT was recently discovered to represent reactive astrocytes in the tumor microenvironment. This study aimed at evaluating the role of 11 C-ACE PET/CT as an imaging biomarker of reactive astrogliosis in characterizing different types of gliomas. METHODS: In this prospective study, a total of 182 patients underwent 11 C-ACE PET/CT before surgery. The ratio of SUV max of a glioma to the SUV mean of the contralateral choroid plexus ( 11 C-ACE TCR) on PET/CT was calculated. 11 C-ACE TCRs were compared with the World Health Organization grades and isocitrate dehydrogenase 1 ( IDH1 ) mutation status. Grade 2 was considered low-grade tumor, and grades 3 and 4 were considered high-grade tumors. RESULTS: The median 11 C-ACE TCR was significantly higher in IDH1 wild-type (wt) tumors (n = 91) than in IDH1 -mutant (mt) tumors (n = 91) (2.38 vs 1.30, P < 0.001). Of the 91 IDH1 -mt tumors, there were no differences in the median 11 C-ACE TCRs between oligodendrogliomas (ODs) and astrocytic tumors (1.40 vs 1.20, P > 0.05). In grading low- versus high-grade gliomas, the receiver operating characteristic curve analyses showed a higher area under the curve (0.951) in IDH1 -wt tumors than in IDH1 -mt tumors (0.783, P = 0.002). Grade 2 ODs were well differentiated from high-grade gliomas. The 11 C-ACE TCR of grade 3 ODs was significantly lower than that of IDH1 -wt glioblastomas. CONCLUSIONS: High 11 C-ACE uptake is associated with high-grade IDH1 -wt tumors, thus facilitating differentiation from high-grade IDH1-mt and low-grade gliomas. In particular, low 11 C-ACE uptake in ODs is advantageous in overcoming the limitation of radiolabeled amino acid tracers.
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Neoplasias Encefálicas , Glioma , Acetatos , Neoplasias Encefálicas/metabolismo , Glioma/patologia , Gliose , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Mutação , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Estudos Prospectivos , Microambiente TumoralRESUMO
BACKGROUND: Currently, several antibody (Ab)-based therapies have shown excellent therapeutic effects in the clinic. Nonetheless, Ab penetration into tumor tissues is limited due to abnormal vasculature, tumor interstitial pressure, and excessive extracellular matrix (ECM) accumulation, thus demanding novel strategies to overcome these barriers. METHODS: The intratumoral distribution of therapeutic Abs were detected by fluorescence microscopy or positron emission tomography in both human gastric xenograft and syngeneic pancreatic hamster tumor models. The antitumor efficacy by combination of oncolytic adenovirus (Ad), which coexpresses relaxin (RLX), interleukin (IL)-12, and granulocyte macrophage colony-stimulating factor (GM-CSF) (oAd/IL12/GM-RLX) and antibody against the programmed cell death protein 1 (αPD-1) was examined in hamster subcutaneous and orthotopic pancreatic tumor models. The immunological aspects of these combination therapy regimen were assessed by flow cytometry or immunohistochemistry in subcutaneous hamster tumor models. RESULTS: Relaxin-expressing oncolytic Ad effectively degraded tumor ECM and enhanced the tumor penetration of trastuzumab in comparison with trastuzumab monotherapy. Based on these results, an oAd/IL12/GM-RLX was used to enhance the potency of immune checkpoint blockade. The combination of the oAd/IL12/GM-RLX and αPD-1 promoted a concomitant degradation of the tumor ECM and amelioration of the immunosuppressive tumor niches, ultimately enhanced intratumoral infiltration of both αPD-1 and activated T cells. Of note, the combination therapy was able to elicit a potent and durable antitumor immune response against cold tumors that were refractory to immune checkpoint inhibitor monotherapy. CONCLUSIONS: Our findings are the first to demonstrate that expression of four genes (IL-12p35, IL-12p40, GM-CSF, and RLX) mediated by a single oncolytic Ad vector can promote remodeling of both physical and immunological aspects of the tumor niches to overcome the major limitations of Ab-based therapies that have emerged in recent clinical trials.
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Adenoviridae/genética , Terapia Viral Oncolítica/métodos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Relaxina/uso terapêutico , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Relaxina/farmacologiaRESUMO
This study aimed to assess how to enhance the value of 18F-Fluorodeoxyglucose (FDG) PET/CTs for glioma grading and better delineation of the tumor boundary by glucose loading. In mouse models of brain tumor using U87MG cells, 18F-FDG-PET images were obtained after fasting and after glucose loading. There was a significant difference in the tumor-to-normal cortex-uptake ratio (TNR) between the fasting and glucose-loading scans. 14C-2-Deoxy-D-glucose (14C-DG) uptake was measured in vitro using U87MG, U373MG and primary neurons cultured with different concentrations of glucose. The tumor-to-neuron ratio of 14C-DG uptake increased with up to 10 mM of glucose. Finally, 10 low-grade and 17 high-grade glioma patients underwent fasting and glucose loading 18F-FDG PET/CT and the TNR was compared between scans. The effect of glucose loading was significant in high-grade but not in low-grade gliomas. The receiver operating characteristic curve analyses with a cut-off TNR of 0.81 showed a higher area under the curve after glucose loading than fasting for differentiating low-grade versus high-grade gliomas. In addition, the glucose loading PET/CT was more useful than the fasting PET/CT for the discrimination of oligodendrogliomas from IDH-wildtype glioblastomas. Glucose loading resulted in a greater reduction in 18F-FDG uptake in the normal cortex than in tumors, which increases the usefulness of 18F-FDG PET/CT for grading.
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BACKGROUNDS: Radioimmunotherapy (RIT) serves as a targeted therapy for non-Hodgkin lymphomas (NHL). Although HIF(Hypoxia-inducible factors)-1α is an important biomarker during radiation therapy, its role in NHL is unclear. Atorvastatin (ATV) is used as a combination drug for chemotherapy. METHODS: We investigated whether ATV downregulated tumor radio-resistance and enhanced the anticancer effect of 131I-RTX (rituximab) in Raji xenograft mouse models. First, the increased uptake and enhanced therapeutic effect of 131I-RTX by ATV was confirmed using molecular imaging in Raji xenograft subcutaneous model and orthotropic model with SPECT and IVIS images. Second, we examined the profile of differentially expressed miRNAs using miRNA array. RESULTS: We found that miR-346 inhibited HIF-1α/VEGF (Vascular endothelial growth factor) during ATV combination therapy with 131I-RTX. The underlying mechanism of ATV involved induction of anti-angiogenesis and radiosensitivity by downregulating HIF-1α in Raji cells. CONCLUSION: Our findings suggested that combination therapy with ATV and 131I-RTX is a promising strategy for enhancing the potency of 131I-RTX therapy in poorly responding patients and those with radio-resistance.
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Glucose hypometabolism in cortical structures after functional disconnection is frequently reported in patients with white matter diseases such as subcortical stroke. However, the molecular and cellular mechanisms have been poorly elucidated. Here we show, in an animal model of internal capsular infarct, that GABA-synthesizing reactive astrocytes in distant cortical areas cause glucose hypometabolism via tonic inhibition of neighboring neurons. We find that reversal of aberrant astrocytic GABA synthesis, by pharmacological inhibition and astrocyte-specific gene silencing of MAO-B, reverses the reduction in cortical glucose metabolism. Moreover, induction of aberrant astrocytic GABA synthesis by cortical injection of putrescine or adenovirus recapitulates cortical hypometabolism. Furthermore, MAO-B inhibition causes a remarkable recovery from post-stroke motor deficits when combined with a rehabilitation regimen. Collectively, our data indicate that cortical glucose hypometabolism in subcortical stroke is caused by aberrant astrocytic GABA and MAO-B inhibition and that attenuating cortical hypometabolism can be a therapeutic approach in subcortical stroke.
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Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiopatologia , Recuperação de Função Fisiológica , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/fisiopatologia , Ácido gama-Aminobutírico/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Córtex Cerebral/ultraestrutura , Glucose/metabolismo , Masculino , Modelos Biológicos , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Atividade Motora/efeitos dos fármacos , Células Piramidais/metabolismo , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacosRESUMO
Semiconductor quantum dots (QDs), which have broad absorption with narrow emission spectra, are useful for multiplex imaging. Here, fluorescence derby imaging using dual color QDs conjugated by the AS1411 aptamer (targeting nucleolin) and the arginine-glycine-aspartic acid (targeting the integrin alpha(v)beta(3)) in cancer cells is reported. Simultaneous fluorescence imaging of cellular distribution of nucleolin and integrin alpha(v)beta(3) using QDs enables easy monitoring of separate targets in the cancer cells and the normal healthy cells. These results suggest the feasibility of a concurrent visualization of QD-based multiple cancer biomarkers using small molecules such as aptamer or peptide ligands.
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Biomarcadores Tumorais/metabolismo , Imageamento Tridimensional/métodos , Pontos Quânticos , Aptâmeros de Nucleotídeos , Linhagem Celular Tumoral , Fluorescência , Humanos , Integrina alfaVbeta3/metabolismo , Microscopia Confocal , Oligodesoxirribonucleotídeos/metabolismo , Oligopeptídeos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , NucleolinaRESUMO
The combination of nanotechnology with molecular imaging has great potential for the development of diagnostics and therapeutics, and multimodal imaging enables versatile applications from cell tracking in animals to clinical applications. Herein, we report a multimodal nanoparticle imaging system that is capable of concurrent fluorescence, bioluminescence, bioluminescence resonance energy transfer (BRET), positron emission tomography (PET) and magnetic resonance (MR) imaging in vivo. A cobalt-ferrite nanoparticle surrounded by rhodamine (MF) was conjugated with luciferase (MFB) and p-SCN-bn-NOTA (2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclonane-1,4,7-triacetic acid) followed by (68)GaCl(3) (magnetic-fluorescent-bioluminescent-radioisotopic particle, MFBR). Confocal microscopy revealed good transfection efficiency of MFB into cells and BRET was also observed in MFB. A good correlation among rhodamine, luciferase, and (68)GaCl(3) was found in MFBR, and the activities of each imaging modality increased dose-dependently with the amount of MFBR in the C6 cells. In vivo optical images were acquired from the thighs of mice after intramuscular and subcutaneous injections of MFBR-laden cells. MicroPET and MR images showed intense radioactivity and ferromagnetic intensities with MFBR-laden cells. The multimodal imaging strategy could be used as potential imaging tools to track cells.
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Diagnóstico por Imagem/métodos , Nanopartículas Metálicas/química , Animais , Linhagem Celular Tumoral , Cobalto/química , Compostos Férricos/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Radioisótopos de Gálio/química , Magnetismo , Camundongos , Camundongos Nus , Tomografia por Emissão de Pósitrons , Ratos , Rodaminas/química , Transplante HeterólogoRESUMO
Generally, the 3'-end of the duplex microRNA (miR) precursor (pre-miR) is known to be stable in vivo and serve as a mature form of miR. However, both the 3'-end (miR9) and 5'-end (miR9*) of a brain-specific miR9 have been shown to function biologically in brain development. In this study, real-time PCR analysis and in vitro/in vivo bioluminescent imaging demonstrated that the upstream region of a primary miR9-1 (pri-miR9-1) can be used to monitor the highly expressed pattern of endogenous pri-miR9-1 during neurogenesis, and that the Luciferase reporter gene can image the unequal expression patterns of miR9 and miR9* seen during the neuronal differentiation of P19 cells. This demonstrates that our bioimaging system can be used to study the participation of miRs in the regulation of neuronal differentiation.
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Encéfalo , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Neurônios/fisiologia , Animais , Sequência de Bases , Encéfalo/citologia , Encéfalo/embriologia , Linhagem Celular Tumoral , Genes Reporter , Camundongos , Camundongos Nus , MicroRNAs/genética , Dados de Sequência Molecular , Neurônios/citologiaRESUMO
The VisuFect-conjugated molecular beacon was developed for non-invasive visualization of microRNA34c in a living single mouse sperm.
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Carbocianinas/análise , Carbocianinas/química , MicroRNAs/análise , Espermatozoides/metabolismo , Animais , Sobrevivência Celular , Fluorescência , Células HEK293 , Humanos , Masculino , Camundongos , MicroRNAs/química , Microscopia de Fluorescência , Análise de Célula ÚnicaRESUMO
A dual fluorophore-based color-tunable molecular beacon visualized the microRNA-294 expression-dependent color change in cells.
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Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Regulação da Expressão Gênica , MicroRNAs/genética , MicroRNAs/metabolismo , Imagem Molecular , Sondas de Oligonucleotídeos/química , Animais , Células CHO , Diferenciação Celular , Cor , Cricetinae , Cricetulus , Camundongos , Microscopia Confocal , Neurônios/citologiaRESUMO
Magnetic resonance imaging (MRI) offers great spatial resolution for viewing deep tissues and anatomy. We developed a self-assembling signal-on magnetic fluorescence nanoparticle to visualize intracellular microRNAs (miRNAs or miRs) during neurogenesis using MRI. The self-assembling nanoparticle (miR124a MR beacon) was aggregated by the incubation of three different oligonucleotides: a 3' adaptor, a 5' adaptor, and a linker containing miR124a-binding sequences. The T2-weighted magnetic resonance (MR) signal of the self-assembled nanoparticle was quenched when miR124a was absent from test tubes or was minimally expressed in cells and tissues. When miR124a was present in test tubes or highly expressed in vitro and in vivo during P19 cell neurogenesis, it hybridized with the miR124a MR beacon, causing the linker to detach, resulting in increased signal-on MRI intensity. This MR beacon can be used as a new imaging probe to monitor the miRNA-mediated regulation of cellular processes.
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Espaço Intracelular/metabolismo , Imageamento por Ressonância Magnética , MicroRNAs/metabolismo , Sondas Moleculares , Neurogênese , Diferenciação Celular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Nanopartículas/ultraestrutura , Neurogênese/genéticaRESUMO
Current methods for delivering foreign genetic materials into mammalian cells are highly successful. However, these methods cannot be applied in oocyte or embryo systems due to their toxicity and low efficiency. Moreover, no satisfactory methods exist for delivering foreign genetic material without inducing physical damage to membranes. Here we developed an organic compound (VisuFect)-mediated small interfering RNA (siRNA) delivery method and evaluated this method in P19 cells and mouse zygotes. Oct4-siRNA conjugated VisuFect (Oct4-siRNA-VF) permeated the zona pellucida effectively and localized inside mouse zygotes without inducing membrane damage. Successful VisuFect-mediated delivery was further demonstrated by strong transcriptional repression of Oct4 expression by the delivered Oct4-siRNA, in addition to repressed embryonic development of mouse zygotes.