RESUMO
BACKGROUND: Radiotherapy is the primary therapeutic option for glioblastoma. Some studies proved that radiotherapy increased the release of exosomes from cells. The mechanism by which these exosomes modify the phenotype of microglia in the tumor microenvironment to further determine the fate of irradiated glioblastoma cells remains to be elucidated. METHODS: We erected the co-culture system of glioblastoma cells and microglia. After radiation, we analyzing the immunophenotype of microglia and the proliferation of radiated glioblastoma cells. By whole transcriptome sequencing, we analyzed of circRNAs in exosomes from glioblastoma cells and microglia. We used some methods, which included RT-PCR, dual-luciferase reporter, et al., to identify how circ_0012381 from radiated glioblastoma cell-derived exosomes regulated the immunophenotype of microglia to further affect the proliferation of radiated glioblastoma cells. RESULTS: Radiated glioblastoma cell-derived exosomes markedly induced M2 microglia polarization. These M2-polarized microglia promoted the proliferation of irradiated glioblastoma cells. Circ_0012381 expression was increased in the irradiated glioblastoma cells, and circ_0012381 entered the microglia via exosomes. Circ_0012381 induced M2 microglia polarization by sponging with miR-340-5p to increase ARG1 expression. M2-polarized microglia suppressed phagocytosis and promoted the growth of the irradiated glioblastoma cells by CCL2/CCR2 axis. Compared with the effects of radiotherapy alone, the inhibition of exosomes significantly inhibited the growth of irradiated glioblastoma cells in a zebrafish model. CONCLUSIONS: Our data suggested that the inhibition of exosome secretion might represent a potential therapeutic strategy to increase the efficacy of radiotherapy in patients with glioblastoma.
Assuntos
Exossomos , Glioblastoma , MicroRNAs , Animais , Exossomos/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/radioterapia , MicroRNAs/genética , MicroRNAs/metabolismo , Microglia/metabolismo , Microambiente Tumoral , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
UNLABELLED: The human immunodeficiency virus type 1 (HIV-1)-encoded virion infectivity factor (Vif) is required to inactivate the host restriction factor APOBEC3 by engaging Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-ß) is a novel regulator of Vif-CRL5 function; as yet, its mechanism of regulation remains unclear. In the present study, we demonstrate that CBF-ß promotion of Vif-CRL5 assembly is independent of its influence on Vif stability and is also a conserved feature of primate lentiviral Vif proteins. Furthermore, CBF-ß is critical for the formation of the Vif-ElonginB/ElonginC-Cul5 core E3 ubiquitin ligase complex in vitro. CBF-ß from diverse vertebrate species supported HIV-1 Vif function, indicating the conserved nature of Vif-CBF-ß interfaces. Considering the importance of the interaction between Vif and CBF-ß in viral CRL5 function, disrupting this interaction represents an attractive pharmacological intervention against HIV-1. IMPORTANCE: HIV-1 encodes virion infectivity factor (Vif) to inactivate its host's antiviral APOBEC3 proteins. Vif triggers APOBEC3 degradation by forming Vif-Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-ß) is a novel regulator of Vif-CRL5 function whose mechanism of regulation remains poorly defined. In the present study, we demonstrate that the promotion of Vif-CRL5 assembly by CBF-ß can be separated from its influence on Vif stability. The promotion of Vif-CRL5 assembly, but not the influence on Vif stability, is conserved among primate lentiviral Vif proteins: we found that CBF-ß from diverse vertebrate species supported HIV-1 Vif function. Considering the importance of the interaction between Vif and CBF-ß in viral CRL5 function and HIV-1 replication, disrupting this interaction is an attractive strategy against HIV-1.
Assuntos
Subunidade beta de Fator de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/metabolismo , Proteínas Culina/metabolismo , Evolução Molecular , Infecções por HIV/metabolismo , HIV-1/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Subunidade beta de Fator de Ligação ao Core/química , Proteínas Culina/genética , Elonguina , Produtos do Gene vif/química , Produtos do Gene vif/genética , Produtos do Gene vif/metabolismo , Infecções por HIV/enzimologia , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/química , HIV-1/genética , Humanos , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de Sequência , Vírus da Imunodeficiência Símia/química , Vírus da Imunodeficiência Símia/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Produtos do Gene vif do Vírus da Imunodeficiência Humana/química , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genéticaRESUMO
Human long interspersed elements 1 (LINE-1 or L1) is the only autonomous non-LTR retroelement in humans and has been associated with genome instability, inherited genetic diseases, and the development of cancer. Certain human APOBEC3 family proteins are known to have LINE-1 restriction activity. The mechanisms by which APOBEC3 affects LINE-1 retrotransposition are not all well characterized; here, we confirm that both A3B and A3DE have a strong ability to inhibit LINE-1 retrotransposition. A3DE interacts with LINE-1 ORF1p to target LINE-1 ribonucleoprotein particles in an RNA-dependent manner. Moreover, A3DE binds to LINE-1 RNA and ORF1 protein in cell culture system. Fluorescence microscopy demonstrated that A3DE co-localizes with ORF1p in cytoplasm. Furthermore, A3DE inhibits LINE-1 reverse transcriptase activity in LINE-1 ribonucleoprotein particles in a cytidine deaminase-independent manner. In contrast, A3B has less inhibitory effects on LINE-1 reverse transcriptase activity despite its strong inhibition of LINE-1 retrotransposition. This study demonstrates that different A3 proteins have been evolved to inhibit LINE-1 activity through distinct mechanisms.
Assuntos
Citidina Desaminase/metabolismo , Elementos Nucleotídeos Longos e Dispersos , Transcrição Reversa , Células HEK293 , Humanos , Fases de Leitura Aberta , RNA/genética , RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMO
Morphological identification is a widespread procedure to assess the presence of apoptosis by visual inspection of the morphological characteristics or the fluorescence images. The procedure is lengthy and results are observer dependent. A quantitative automatic analysis is objective and would greatly help the routine work. We developed an image processing and segmentation method which combined the Otsu thresholding and morphological operators for apoptosis study. An automatic determination method of apoptotic stages of HL-60 cells with fluorescence images was developed. Comparison was made between normal cells, early apoptotic cells and late apoptotic cells about their geometric parameters which were defined to describe the features of cell morphology. The results demonstrated that the parameters we chose are very representative of the morphological characteristics of apoptotic cells. Significant differences exist between the cells in different stages, and automatic quantification of the differences can be achieved.