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1.
Anticancer Res ; 39(9): 4947-4955, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519600

RESUMO

BACKGROUND/AIM: Insulin-like growth factor II mRNA-binding protein 3 (IMP3) plays an important role in the adhesion, invasion, and metastasis of tumor cells. Although emerging evidence suggests that IMP3 promotes tumor progression in several malignancies, the expression of IMP3 and its prognostic implication in adenocarcinoma of the ampulla of Vater (AVAC) has not been clarified to date. MATERIALS AND METHODS: The IMP3 expression status in 87 AVAC tissues was examined using immunostaining, and its association with various clinicopathological features and outcome of patients with AVAC was investigated. RESULTS: The vast majority (87.4%) of AVAC cases displayed at least focal cytoplasmic and membranous IMP3 immunoreactivity in tumor cells, whereas IMP3 expression was consistently absent from normal biliary epithelial cells. Tumor-specific IMP3 expression was associated with submucosal and pancreatic invasion, which were not identified in the corresponding hematoxylin and eosin-stained slides. This finding led to up-staging of the pathological tumor stage in two cases of well-differentiated AVAC. In addition, high IMP3 expression was significantly associated with a poorly differentiated histology (p=0.026). Survival analyses revealed that high IMP3 expression independently predicted shorter recurrence-free (p=0.003) and overall (p=0.029) survival. CONCLUSION: Our study demonstrated tumor-specific IMP3 expression in AVAC, which will be helpful in determining invasion depth and tumor extent in patients with well-differentiated tumors, as well as indicating worse survival of patients with AVAC. Our data highlight IMP3 expression status as a potential diagnostic and prognostic marker for AVAC.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Ampola Hepatopancreática/patologia , Neoplasias do Ducto Colédoco/genética , Neoplasias do Ducto Colédoco/mortalidade , Regulação Neoplásica da Expressão Gênica , Proteínas de Ligação a RNA/metabolismo , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adulto , Idoso , Biomarcadores Tumorais , Neoplasias do Ducto Colédoco/diagnóstico , Neoplasias do Ducto Colédoco/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Proteínas de Ligação a RNA/genética , Carga Tumoral
2.
Zhonghua Zhong Liu Za Zhi ; 41(9): 675-680, 2019 Sep 23.
Artigo em Chinês | MEDLINE | ID: mdl-31550857

RESUMO

Objective: To investigate the inhibitory effect of programmed cell death factor 4 (PDCD4) on arsenic trioxide (As(2)O(3))-induced cell growth and nuclear factor kappa B (NF-κB) signaling pathway in neuroblastoma. Methods: The PDCD4 overexpression vector was transfected into neuroblastoma cells and detected by fluorescence quantitative PCR and Western blot. As(2)O(3) was used to treat PDCD4 overexpressing neuroblastoma cells. MTT assay was used to measure the proliferation. Colony formation assay was used to determine the cell clone forming ability. Apoptosis was measured by flow cytometry. Western blot was used to detect the expression of NF-κB p65 and cleaved caspase-3 protein in cells. Results: The transfection of PDCD4 overexpression vector significantly increased the expression level of PDCD4 in neuroblastoma cells. The cell survival rates of the control group, PDCD4 group, As(2)O(3) group and As(2)O(3)+ PDCD4 group were 100%, (72.14±5.20)%, (62.58±3.14)% and (40.87±2.47)%, respectively. The colony formation rates in these four groups were (91.25±8.36)%, (65.32±7.14)%, (57.23±5.28)% and (37.14±3.64)%, respectively. In addition, the cell apoptotic rates of these four groups were (3.57±0.24)%, (28.64±3.20)%, (36.41±4.58)% and (49.65±5.27)%, respectively. Therefore, overexpression of PDCD4 in the absence or presence of As(2)O(3) inhibited cell proliferation and clone formation ability, while promoted apoptosis. Furthermore, the expression levels of cleaved caspase-3 in the control group, PDCD4 group, As(2)O(3) group and As(2)O(3)+ PDCD4 group were 0.21±0.03, 0.30±0.02, 0.43±0.05 and 0.57±0.06, respectively. And the expression levels of NF-κB p65 protein were 0.68±0.04, 0.52±0.03, 0.43±0.04, and 0.32±0.02, respectively. Compared with the control group, the expression levels of NF-κB p65 protein in PDCD4 group, As(2)O(3) group and As(2)O(3)+ PDCD4 group were significantly decreased (P<0.05), whereas the expression level of cleaved Caspase-3 protein was significantly increased (P<0.05). The changes in As(2)O(3)+ PDCD4 group were more significant than those in PDCD4 group and As(2)O(3) groups (both P<0.05). Conclusion: PDCD4 enhances the inhibitory effect of As(2)O(3) on the growth and NF-κB signaling pathway in neuroblastoma cells.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/efeitos dos fármacos , Trióxido de Arsênio/farmacologia , NF-kappa B/fisiologia , Neuroblastoma/tratamento farmacológico , Proteínas de Ligação a RNA/genética , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos , Proteínas Reguladoras de Apoptose/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Mol Biol (Mosk) ; 53(4): 663-673, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397440

RESUMO

Malignant cutaneous melanoma (CM) is an extremely aggressive cancer characterized by a high level of metastatic activity and unfavorable prognosis due to a high incidence of relapses, as well as resistance to standard chemotherapy. Cutaneous melanoma accounts for 80% of deaths from malignant skin tumors. Nucleolin/C23 and nucleophosmin/B23, which constitute altogether ~70% of the nucleolus volume, are promising targets for molecular therapy of melanoma. These proteins perform many important functions in the cell, so disruption of the NCL and/or NPM gene structure and abnormal expression of the C23 and B23 proteins they encode, can lead to unlimited cell proliferation and progression of a tumor. Therefore, investigation of the structure and expression of these genes is a topical problem, which is important for understanding the mechanisms of CM carcinogenesis and for the development of new therapeutic approaches. This paper describes new NCL and NPM polymorphisms, as well as the levels of C23 and B23 expression in normal tissues, CM and mucosal melanoma.


Assuntos
Melanoma/genética , Melanoma/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Proliferação de Células , Humanos , Melanoma/tratamento farmacológico , Terapia de Alvo Molecular , Proteínas Nucleares/biossíntese , Proteínas Nucleares/química , Fosfoproteínas/biossíntese , Fosfoproteínas/química , Polimorfismo Genético , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/química , Neoplasias Cutâneas/tratamento farmacológico
4.
Cancer Sci ; 110(10): 3204-3214, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31385416

RESUMO

Peritoneal dissemination is the most frequent metastatic route of ovarian cancer. However, due to the high heterogeneity in ovarian cancer, most conventional studies lack parental tumor controls relevant to metastases and, thus, it is difficult to trace the molecular changes of cancer cells along with the selection by the abdominal microenvironment. Here, we established an in vivo mouse peritoneal dissemination scheme that allowed us to select more aggressive sublines from parental ovarian cancer cells, including A2780 and SKOV-3. Microarray and gene profiling analyses indicated that autophagy-related genes were enriched in selected malignant sublines. Detection of LC3-II, p62 and autophagic puncta demonstrated that these malignant variants were more sensitive to autophagic induction when exposed to diverse stress conditions, such as high cell density, starvation and drug treatment. As compared with parental A2780, the selected variant acquired the ability to grow better under high-density stress; however, this effect was reversed by addition of autophagic inhibitors or knockdown of ATG5. When analyzing the clinical profiles of autophagy-related genes identified to be enriched in malignant A2780 variant, 73% of them had prognostic significance for the survival of ovarian cancer patients. Taken together, our findings indicate that an increase in autophagic potency among ovarian cancer cells is crucial for selection of metastatic colonies in the abdominal microenvironment. In addition, the derived autophagic gene profile can not only predict prognosis well but can also be potentially applied to precision medicine for identifying those ovarian cancer patients suitable for taking anti-autophagy cancer drugs.


Assuntos
Proteínas Relacionadas à Autofagia/genética , Perfilação da Expressão Gênica/métodos , Proteínas Associadas aos Microtúbulos/genética , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/secundário , Proteínas de Ligação a RNA/genética , Animais , Autofagia , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Humanos , Camundongos , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/patologia , Medicina de Precisão , Prognóstico , Microambiente Tumoral
5.
Genes Dev ; 33(17-18): 1221-1235, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371437

RESUMO

TRIM71/LIN-41, a phylogenetically conserved regulator of development, controls stem cell fates. Mammalian TRIM71 exhibits both RNA-binding and protein ubiquitylation activities, but the functional contribution of either activity and relevant primary targets remain poorly understood. Here, we demonstrate that TRIM71 shapes the transcriptome of mouse embryonic stem cells (mESCs) predominantly through its RNA-binding activity. We reveal that TRIM71 binds targets through 3' untranslated region (UTR) hairpin motifs and that it acts predominantly by target degradation. TRIM71 mutations implicated in etiogenesis of human congenital hydrocephalus impair target silencing. We identify a set of primary targets consistently regulated in various human and mouse cell lines, including MBNL1 (Muscleblind-like protein 1). MBNL1 promotes cell differentiation through regulation of alternative splicing, and we demonstrate that TRIM71 promotes embryonic splicing patterns through MBNL1 repression. Hence, repression of MBNL1-dependent alternative splicing may contribute to TRIM71's function in regulating stem cell fates.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Proteínas de Ligação a RNA/genética , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Células-Tronco Embrionárias , Humanos , Camundongos , Camundongos Knockout , Mutação , Motivos de Nucleotídeos , Ligação Proteica , Domínios Proteicos/genética , Interferência de RNA , Proteínas de Ligação a RNA/metabolismo
6.
BMC Evol Biol ; 19(1): 149, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31337330

RESUMO

BACKGROUND: Adenosine deaminase enzymes of the ADAR family are conserved in metazoans. They convert adenine into inosine in dsRNAs and thus alter both structural properties and the coding potential of their substrates. Acting on exogenous dsRNAs, ADAR1 exerts a pro- or anti-viral role in vertebrates and Drosophila. RESULTS: We traced 4 ADAR homologs in 14 lophotrochozoan genomes and we classified them into ADAD, ADAR1 or ADAR2, based on phylogenetic and structural analyses of the enzymatic domain. Using RNA-seq and quantitative real time PCR we demonstrated the upregulation of one ADAR1 homolog in the bivalve Crassostrea gigas and in the gastropod Haliotis diversicolor supertexta during Ostreid herpesvirus-1 or Haliotid herpesvirus-1 infection. Accordingly, we demonstrated an extensive ADAR-mediated editing of viral RNAs. Single nucleotide variation (SNV) profiles obtained by pairing RNA- and DNA-seq data from the viral infected individuals resulted to be mostly compatible with ADAR-mediated A-to-I editing (up to 97%). SNVs occurred at low frequency in genomic hotspots, denoted by the overlapping of viral genes encoded on opposite DNA strands. The SNV sites and their upstream neighbor nucleotide indicated the targeting of selected adenosines. The analysis of viral sequences suggested that, under the pressure of the ADAR editing, the two Malacoherpesviridae genomes have evolved to reduce the number of deamination targets. CONCLUSIONS: We report, for the first time, evidence of an extensive editing of Malacoherpesviridae RNAs attributable to host ADAR1 enzymes. The analysis of base neighbor preferences, structural features and expression profiles of molluscan ADAR1 supports the conservation of the enzyme function among metazoans and further suggested that ADAR1 exerts an antiviral role in mollusks.


Assuntos
Antivirais/metabolismo , Vírus de DNA/genética , Moluscos/virologia , Edição de RNA/genética , RNA Viral/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Teorema de Bayes , Vírus de DNA/fisiologia , Regulação da Expressão Gênica , Genoma Viral , Modelos Moleculares , Moluscos/genética , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Transcriptoma/genética
7.
Medicine (Baltimore) ; 98(27): e16009, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31277094

RESUMO

Bladder cancer is one of the most common malignancies of urinary tract. The current study aimed to investigate the role of insulin-like growth factor II messenger RNA binding protein 3 (IMP3) expression in the prognostic evaluation of non-muscle- invasive urothelial carcinoma of the bladder.Immunohistochemistry (IHC) was carried out to examine IMP3 protein expression in specimens from 183 cases of non-muscle-invasive urothelial carcinoma, 20 cases of muscle-invasive urothelial carcinoma and 20 benign tissues adjacent to cancer tissue.The expression of IMP3 was not detected in the adjacent benign tissues. The expression intensity of IMP3 in muscle-invasive samples was significantly higher than that in non-muscle-invasive urothelial carcinoma specimens (P = .008). IMP3 expression was significantly related with advanced tumor stage (P < .001), advanced tumor grade (P = .004), and tumor recurrence (P < .001) in non-muscle-invasive urothelial carcinomas. Kaplan-Meier analysis showed that IMP3-positive patients had much lower disease-free (P < .001), progression-free (P = .002) and metastasis-free (P = .019) survival rates compared with those with IMP3-negative tumors. By multivariable Cox analysis, we also found that IMP3 expression in non-muscle- invasive urothelial carcinomas proved to be an independent unfavorable prognostic factor of disease-free survival (HR: 2.52; 95% CI, 1.39-4.56; P = .002), progression- free survival (HR: 5.19; 95% CI, 1.54-17.46; P = .008) and metastasis-free survival (HR: 4.87; 95% CI, 1.08-22.02; P = .040).Our results demonstrate that the expression of IMP3 in non-muscle- invasive bladder cancer can serve as an independent predictor that will help recognize the subgroup of patients with a high ability to relapse, progress, and metastasize and who might get the maximum benefit from an early and more aggressive treatment strategy.


Assuntos
Carcinoma de Células de Transição/genética , Proteínas de Ligação a RNA/genética , Neoplasias da Bexiga Urinária/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Carcinoma de Células de Transição/mortalidade , Carcinoma de Células de Transição/patologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Modelos de Riscos Proporcionais , Proteínas de Ligação a RNA/metabolismo , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologia , Urotélio/patologia
8.
Gene ; 714: 143994, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31330233

RESUMO

Long non-coding RNA (lncRNA) potentially regulates tumorigenesis. LncRNA small nucleolar RNA host gene 1 (SNHG1) expression remains high in hepatocellular carcinoma cells; however, its biological mechanism in hepatocellular carcinoma remains unknown. In this study, SNHG1 expression in hepatocellular carcinoma cells was detected by qRT-PCR. Proliferative and migratory potentials of hepatocellular carcinoma cells were determined by CCK-8 and Transwell assay, respectively. Then, the nude mice model of xenograft was employed to verify the effect of SNHG1 on tumor formation in vivo. We identified the potential target of SNHG1 through bioinformatics and dual-luciferase reporter gene. Furthermore, Western blot and RIP assay was used for clarifying their interaction and functions in regulating the development of hepatocellular carcinoma. Our results indicated a high expression of SNHG1 in hepatocellular carcinoma cells. Downregulation of SNHG1 inhibited proliferative and migratory potentials of hepatocellular carcinoma cells in vitro and in vivo. Moreover, the expression of programmed cell death 4 (PDCD4) was positively regulated by SNHG1 through competing with miR-195-5p. These results indicated that SNHG1 participated in the development of hepatocellular carcinoma as a ceRNA to competitively bind to miR-195-5p and thus mediate PDCD4 expression.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/genética , Animais , Apoptose/genética , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma Hepatocelular/patologia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus
9.
Nat Commun ; 10(1): 3020, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31289263

RESUMO

Human cytomegalovirus (HCMV) can persistently infect humans, but how HCMV avoids humoral immunity is not clear. The neonatal Fc receptor (FcRn) controls IgG transport from the mother to the fetus and prolongs IgG half-life. Here we show that US11 inhibits the assembly of FcRn with ß2m and retains FcRn in the endoplasmic reticulum (ER), consequently blocking FcRn trafficking to the endosome. Furthermore, US11 recruits the ubiquitin enzymes Derlin-1, TMEM129 and UbE2J2 to engage FcRn, consequently initiating the dislocation of FcRn from the ER to the cytosol and facilitating its degradation. Importantly, US11 inhibits IgG-FcRn binding, resulting in a reduction of IgG transcytosis across intestinal or placental epithelial cells and IgG degradation in endothelial cells. Hence, these results identify the mechanism by which HCMV infection exploits an ER-associated degradation pathway through US11 to disable FcRn functions. These results have implications for vaccine development and immune surveillance.


Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Evasão da Resposta Imune , Imunidade Humoral , Proteínas de Ligação a RNA/metabolismo , Receptores Fc/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/virologia , Degradação Associada com o Retículo Endoplasmático/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Receptores Fc/imunologia , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/genética , Proteínas Virais/imunologia
10.
Cell Prolif ; 52(5): e12615, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31310044

RESUMO

OBJECTIVES: It has been widely reported that long non-coding RNAs (lncRNAs) can participate in multiple biological processes of human cancers. lncRNA HLA complex group 11 (HCG11) has been reported in human cancers as a tumour suppressor. This study focused on investigating the function and mechanism of HCG11 in glioma. MATERIALS AND METHODS: Based on The Cancer Genome Atlas (TCGA) data set and qRT-PCR analysis, the expression pattern of HCG11 was identified in glioma samples. The mechanism associated with HCG11 downregulation was determined by mechanism experiments. Gain-of-function assays were conducted for the identification of HCG11 function in glioma progression. Mechanism investigation based on the luciferase reporter assay, RIP assay and pull-down assay was used to explore the downstream molecular mechanism of HCG11. The role of molecular pathway in the progression of glioma was analysed in accordance with the rescue assays. RESULTS: HCG11 was expressed at low level in glioma samples compared with normal samples. FOXP1 could bind with HCG11 and transcriptionally inactivated HCG11. Overexpression of HCG11 efficiently suppressed cell proliferation, induced cell cycle arrest and promoted cell apoptosis. HCG11 was predominantly enriched in the cytoplasm of glioma cells and acted as a competing endogenous RNAs (ceRNAs) by sponging micro-496 to upregulate cytoplasmic polyadenylation element binding protein 3 (CPEB3). CEPB3 and miR-496 involved in HCG11-mediated glioma progression. CONCLUSIONS: HCG11 inhibited glioma progression by regulating miR-496/CPEB3 axis.


Assuntos
Glioma/patologia , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Antagomirs/metabolismo , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Progressão da Doença , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/metabolismo , Glioma/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Interferência de RNA , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo
11.
Artigo em Inglês | MEDLINE | ID: mdl-31176867

RESUMO

In rice field eel (Monopterus albus), germ cell development in the developing gonad has been revealed in detail. However, it is unclear how primordial germ cells (PGCs) migrate to the somatic part of the gonad (genital ridge). This study visualized PGC migration by injecting a chimeric mRNA containing a fluorescent protein fused to the 3' untranslated region (3'UTR) of three different genes, nanos3 of zebrafish (Danio rerio) and dead end (dnd) and vasa of rice field eel. The mRNAs were injected either alone or in pairs into embryos at the one-cell stage. The results showed that mRNAs containing nanos3 and dnd 3'UTRs labeled PGCs over a wider time frame than those containing vasa 3'UTR, suggesting that nanos3 and dnd 3'UTRs are suitable for visualizing PGCs in rice field eel. Using this direct visualization method, the normal migration route of PGCs was observed from the 50%-epiboly stage to hatching stage for the first time, and the ectopic PGCs were also visualized during this period in rice field eel. These findings extend our knowledge of germ cell development, and lay a foundation for further research on the relationship between PGCs and sex differentiation, and on incubation conditions for embryos in rice field eel.


Assuntos
Células Germinativas/metabolismo , Smegmamorpha/embriologia , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Movimento Celular/genética , Movimento Celular/fisiologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Células Germinativas/citologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Smegmamorpha/genética , Smegmamorpha/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
12.
Genes Dev ; 33(15-16): 1048-1068, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31221665

RESUMO

Fetal hematopoietic stem and progenitor cells (HSPCs) hold promise to cure a wide array of hematological diseases, and we previously found a role for the RNA-binding protein (RBP) Lin28b in respecifying adult HSPCs to resemble their fetal counterparts. Here we show by single-cell RNA sequencing that Lin28b alone was insufficient for complete reprogramming of gene expression from the adult toward the fetal pattern. Using proteomics and in situ analyses, we found that Lin28b (and its closely related paralog, Lin28a) directly interacted with Igf2bp3, another RBP, and their enforced co-expression in adult HSPCs reactivated fetal-like B-cell development in vivo more efficiently than either factor alone. In B-cell progenitors, Lin28b and Igf2bp3 jointly stabilized thousands of mRNAs by binding at the same sites, including those of the B-cell regulators Pax5 and Arid3a as well as Igf2bp3 mRNA itself, forming an autoregulatory loop. Our results suggest that Lin28b and Igf2bp3 are at the center of a gene regulatory network that mediates the fetal-adult hematopoietic switch. A method to efficiently generate induced fetal-like hematopoietic stem cells (ifHSCs) will facilitate basic studies of their biology and possibly pave a path toward their clinical application.


Assuntos
Reprogramação Celular/genética , Proteínas de Ligação a DNA/metabolismo , Redes Reguladoras de Genes , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Ligação a RNA/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Proteínas de Ligação a DNA/genética , Camundongos , MicroRNAs/metabolismo , Modelos Animais , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética
13.
Life Sci ; 232: 116606, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31254586

RESUMO

AIMS: Bupivacaine, a common local anesthetic, can cause neurotoxicity and abnormal neuro-disorders. However, the precise underlying mechanisms have not been fully elucidated. In this study, we investigated the function of lncRNA MALAT1 in the bupivacaine-induced neurotoxicity process. MATERIALS AND METHODS: SH-SY5Y cells and neonatal mouse DRG neurons were cultured in vitro and treated with bupivacaine to establish a neurotoxicity model. Caspase3 activity and cell survival rates were detected to evaluate the function of lncRNA MALAT1. Western blotting was used to detect the expression levels of PDCD4 and cleaved-caspase-3. A dual-luciferase reporter assay was used to explore the potential binding target of lncRNA MALAT1. RESULTS: We found that the expression of lncRNA MALAT1 was upregulated upon exposure to bupivacaine. Knockdown of lncRNA MALAT1 significantly increased the cell death rates, and Caspase3 activity assays revealed that the apoptosis rates were manifestly increased in the MALAT1 downregulation group. In addition, we screened the possible target and found that miR-101-3p is the direct target of MALAT1 using a dual-luciferase reporter assay; these results suggest that lncRNA MALAT1 may function as a decoy to sponge miR-101-3p. Furthermore, we demonstrated that activation of the MALAT1/miR-101-3p/PDCD4 axis protected cells against bupivacaine treatment. CONCLUSION: We elucidated the function and mechanism of MALAT1 in bupivacaine-induced neurotoxicity. Targeting MALAT1 might provide new methods to prevent neurotoxicity.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Bupivacaína/toxicidade , MicroRNAs/metabolismo , Síndromes Neurotóxicas/etiologia , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/metabolismo , Anestésicos Locais/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular Tumoral , Gânglios Espinais/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , MicroRNAs/genética , Síndromes Neurotóxicas/genética , Síndromes Neurotóxicas/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Regulação para Cima/efeitos dos fármacos
14.
Br J Anaesth ; 123(2): e226-e238, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31253357

RESUMO

BACKGROUND: The long-term use of opioid analgesics is limited by the development of unwanted side-effects, such as tolerance. The molecular mechanisms of morphine anti-nociceptive tolerance are still unclear. The mitochondrial calcium uniporter (MCU) is involved in painful hyperalgesia, but the role of MCU in morphine tolerance has not been uncharacterised. METHODS: Rats received intrathecal injection of morphine for 7 days to induce morphine tolerance. The mechanical withdrawal threshold was measured using von Frey filaments, and thermal latency using the hotplate test. The effects of an MCU inhibitor, antisense oligodeoxynucleotide against cyclic adenosine monophosphate response element (CRE)-binding protein (CREB) or cytoplasmic polyadenylation element-binding protein 1 (CPEB1) in morphine tolerance were examined. RESULTS: Spinal morphine tolerance was associated with an increased expression of neuronal MCU, phospho-CREB (pCREB), and CPEB1 in the spinal cord dorsal horn. MCU inhibition increased the mechanical threshold and thermal latency, and reduced the accumulation of mitochondrial calcium in morphine tolerance. Intrathecal antisense oligodeoxynucleotide against CREB or CPEB1 restored the anti-nociceptive effects of morphine compared with mismatch oligodeoxynucleotide in von Frey test and hotplate test. Chromatin immunoprecipitation with quantitative PCR assay showed that CREB knockdown reduced the interaction of pCREB with the ccdc109a gene (encoding MCU expression) promoter and decreased the MCU mRNA transcription. RNA immunoprecipitation assay suggested that CPEB1 binds to the MCU mRNA 3' untranslated region. CPEB1 knockdown decreased the expression of MCU protein. CONCLUSIONS: These findings suggest that spinal MCU is regulated by pCREB and CPEB1 in morphine tolerance, and that inhibition of MCU, pCREB, or CPEB1 may be useful in preventing the development of opioid tolerance.


Assuntos
Proteína de Ligação a CREB/genética , Canais de Cálcio/metabolismo , Tolerância a Medicamentos/genética , Morfina/farmacologia , Proteínas de Ligação a RNA/genética , Corno Dorsal da Medula Espinal/metabolismo , Analgésicos Opioides/farmacologia , Animais , Masculino , Modelos Animais , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley
15.
Cell Mol Life Sci ; 76(20): 4071-4102, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31254043

RESUMO

Mammalian spermatogenesis is a highly complex multi-step process sustained by a population of mitotic germ cells with self-renewal potential known as spermatogonial stem cells (SSCs). The maintenance and regulation of SSC function are strictly dependent on a supportive niche that is composed of multiple cell types. A detailed appreciation of the molecular mechanisms underpinning SSC activity and fate is of fundamental importance for spermatogenesis and male fertility. However, different models of SSC identity and spermatogonial hierarchy have been proposed and recent studies indicate that cell populations supporting steady-state germline maintenance and regeneration following damage are distinct. Importantly, dynamic changes in niche properties may underlie the fate plasticity of spermatogonia evident during testis regeneration. While formation of spermatogenic colonies in germ-cell-depleted testis upon transplantation is a standard assay for SSCs, differentiation-primed spermatogonial fractions have transplantation potential and this assay provides readout of regenerative rather than steady-state stem cell capacity. The characterisation of spermatogonial populations with regenerative capacity is essential for the development of clinical applications aimed at restoring fertility in individuals following germline depletion by genotoxic treatments. This review will discuss regulatory mechanisms of SSCs in homeostatic and regenerative testis and the conservation of these mechanisms between rodent models and man.


Assuntos
Fertilidade/genética , Infertilidade Masculina/genética , Espermatogênese/genética , Espermatogônias/citologia , Células-Tronco/citologia , Testículo/citologia , Animais , Diferenciação Celular , Regulação da Expressão Gênica , Homeostase/genética , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Infertilidade Masculina/terapia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Modelos Genéticos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espermatogônias/metabolismo , Nicho de Células-Tronco/genética , Células-Tronco/metabolismo , Testículo/metabolismo
16.
Cell Mol Life Sci ; 76(19): 3745-3752, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31165201

RESUMO

RNA-binding proteins (RBPs) and microRNAs (miRNAs) are the most important regulators of mRNA stability and translation in eukaryotic cells; however, the complex interplay between these systems is only now coming to light. RBPs and miRNAs regulate a unique set of targets in either a positive or negative manner and their regulation is mainly opposed to each other on overlapping targets. In some cases, the levels of RBPs or miRNAs regulate the cellular levels of one another and decreased levels of either results in changes in translation of their targets. There is growing evidence that these regulatory circuits are crucial in the development and progression of cancer; however, the rules underlying synergism and antagonism between miRNAs and RNA-binding proteins remain unclear. Synthetic biology seeks to develop artificial systems to better understand their natural counterparts and to develop new, useful technologies for manipulation of gene expression at the RNA level. The recent development of artificial RNA-binding proteins promises to enable a much greater understanding of the importance of the functional interactions between RNA-binding proteins and miRNAs, as well as enabling their manipulation for therapeutic purposes.


Assuntos
MicroRNAs/metabolismo , Neoplasias/genética , Proteínas de Ligação a RNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Doenças Genéticas Inatas/terapia , Humanos , Neoplasias/metabolismo , Neoplasias/terapia , Engenharia de Proteínas , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética
17.
RNA ; 25(9): 1078-1090, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31164480

RESUMO

RNAi mediated by small-interfering RNAs (siRNAs) operates via transcriptional (TGS) and posttranscriptional gene silencing (PTGS). In Arabidopsis thaliana, TGS relies on DICER-LIKE-3 (DCL3)-dependent 24-nt siRNAs loaded into AGO4-clade ARGONAUTE effector proteins. PTGS operates via DCL4-dependent 21-nt siRNAs loaded into AGO1-clade proteins. We set up and validated a medium-throughput, semi-automatized procedure enabling chemical screening, in a 96-well in vitro format, of Arabidopsis transgenic seedlings expressing an inverted-repeat construct from the phloem companion cells. The ensuing quantitative PTGS phenotype was exploited to identify molecules, which, upon topical application, either inhibit or enhance siRNA biogenesis/activities. The vast majority of identified modifiers were enhancers, among which Sortin1, Isoxazolone, and [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) provided the most robust and consistent results, including upon their application onto soil-grown plants in which their effect was nonautonomous and long lasting. The three molecules increased the RNAi potency of the inverted-repeat construct, in large part by enhancing 21-nt siRNA accumulation and loading into AGO1, and concomitantly reducing AGO4 and DCL3 levels in planta. A similar, albeit not identical effect, was observed on 22-nt siRNAs produced from a naturally occurring inverted-repeat locus, demonstrating that the molecules also enhance endogenous PTGS. In standardized assays conducted in seedling extracts, the three enhancers selectively increased DCL4-mediated processing of in vitro-synthesized double-stranded RNAs, indicating the targeting of a hitherto unknown PTGS component probably independent of the DCL4-cofactor DOUBLE-STRANDED RNA-BINDING 4 (DRB4). This study establishes the proof-of-concept that RNAi efficacy can be modulated by chemicals in a whole organism. Their potential applications and the associated future research are discussed.


Assuntos
Arabidopsis/genética , Indenos/farmacologia , Piperidinas/farmacologia , Piridinas/farmacologia , Interferência de RNA/efeitos dos fármacos , Tionas/farmacologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas Argonauta/genética , Avaliação Pré-Clínica de Medicamentos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética
18.
Nat Commun ; 10(1): 2593, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197139

RESUMO

Prion-like domains (PLDs), defined by their low sequence complexity and intrinsic disorder, are present in hundreds of human proteins. Although gain-of-function mutations in the PLDs of neuronal RNA-binding proteins have been linked to neurodegenerative disease progression, the physiological role of PLDs and their range of molecular functions are still largely unknown. Here, we show that the PLD of Drosophila Imp, a conserved component of neuronal ribonucleoprotein (RNP) granules, is essential for the developmentally-controlled localization of Imp RNP granules to axons and regulates in vivo axonal remodeling. Furthermore, we demonstrate that Imp PLD restricts, rather than promotes, granule assembly, revealing a novel modulatory function for PLDs in RNP granule homeostasis. Swapping the position of Imp PLD compromises RNP granule dynamic assembly but not transport, suggesting that these two functions are uncoupled. Together, our study uncovers a physiological function for PLDs in the spatio-temporal control of neuronal RNP assemblies.


Assuntos
Transporte Axonal/fisiologia , Grânulos Citoplasmáticos/metabolismo , Proteínas de Drosophila/metabolismo , Domínios Proteicos/fisiologia , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Animais Geneticamente Modificados , Axônios/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Linhagem Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Microscopia de Fluorescência , Modelos Animais , Príons/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
19.
Nat Commun ; 10(1): 2682, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213602

RESUMO

RNA-protein complexes play essential regulatory roles at nearly all levels of gene expression. Using in vivo crosslinking and RNA capture, we report a comprehensive RNA-protein interactome in a metazoan at four levels of resolution: single amino acids, domains, proteins and multisubunit complexes. We devise CAPRI, a method to map RNA-binding domains (RBDs) by simultaneous identification of RNA interacting crosslinked peptides and peptides adjacent to such crosslinked sites. CAPRI identifies more than 3000 RNA proximal peptides in Drosophila and human proteins with more than 45% of them forming new interaction interfaces. The comparison of orthologous proteins enables the identification of evolutionary conserved RBDs in globular domains and intrinsically disordered regions (IDRs). By comparing the sequences of IDRs through evolution, we classify them based on the type of motif, accumulation of tandem repeats, conservation of amino acid composition and high sequence divergence.


Assuntos
Evolução Molecular , Proteômica/métodos , Motivos de Ligação ao RNA/genética , Proteínas de Ligação a RNA/genética , RNA/metabolismo , Sequência de Aminoácidos/genética , Animais , Linhagem Celular , Sequência Conservada/genética , Reagentes para Ligações Cruzadas/química , Drosophila , Humanos , Peptídeos/química , Peptídeos/genética , Ligação Proteica/genética , Proteoma/genética , RNA/química , Proteínas de Ligação a RNA/química
20.
Nat Commun ; 10(1): 2691, 2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31217428

RESUMO

The MUSASHI (MSI) family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of cancers including acute myeloid leukemia. We find that the small molecule Ro 08-2750 (Ro) binds directly and selectively to MSI2 and competes for its RNA binding in biochemical assays. Ro treatment in mouse and human myeloid leukemia cells results in an increase in differentiation and apoptosis, inhibition of known MSI-targets, and a shared global gene expression signature similar to shRNA depletion of MSI2. Ro demonstrates in vivo inhibition of c-MYC and reduces disease burden in a murine AML leukemia model. Thus, we identify a small molecule that targets MSI's oncogenic activity. Our study provides a framework for targeting RNA binding proteins in cancer.


Assuntos
Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Experimental/tratamento farmacológico , Leucemia Mieloide Aguda/tratamento farmacológico , Pteridinas/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Leucemia Experimental/sangue , Leucemia Mieloide Aguda/sangue , Masculino , Camundongos , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-myc/metabolismo , Pteridinas/uso terapêutico , RNA/metabolismo , Motivo de Reconhecimento de RNA/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transcriptoma/efeitos dos fármacos , Células Tumorais Cultivadas
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