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1.
PLoS Genet ; 15(6): e1008111, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31194729

RESUMO

Signal transduction activated by Wingless/Wnt ligands directs cell proliferation and fate specification in metazoans, and its overactivation underlies the development of the vast majority of colorectal cancers. In the conventional model, the secretion and movement of Wingless to cells distant from its source of synthesis are essential for long-range signaling in tissue patterning. However, this model was upended recently by an unanticipated finding: replacement of wild-type Drosophila Wingless with a membrane-tethered form produced viable adults with largely normal external morphology, which suggested that Wingless secretion and movement are dispensable for tissue patterning. Herein, we tested this foundational principle in the adult intestine, where Wingless signaling gradients coincide with all major boundaries between compartments. We find that the critical roles of Wingless during adult intestinal development, which include regulation of target gene activation, boundary formation, stem cell proliferation, epithelial cell fate specification, muscle differentiation, gut folding, and signaling crosstalk with the Decapentaplegic pathway, are all disrupted by Wingless tethering. These findings provide new evidence that supports the requirement for the direct, long-range action of Wingless in tissue patterning, with relevance for animal development, tissue homeostasis and Wnt-driven disease.


Assuntos
Padronização Corporal/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteína Wnt1/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Proliferação de Células/genética , Drosophila melanogaster/crescimento & desenvolvimento , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Homeostase , Intestinos/crescimento & desenvolvimento , Transdução de Sinais/genética , Células-Tronco/metabolismo
2.
PLoS Pathog ; 15(4): e1007575, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31002735

RESUMO

High-risk human papillomavirus (HPV) E6 proteins associate with the cellular ubiquitin ligase E6-Associated Protein (E6AP), and then recruit both p53 and certain cellular PDZ proteins for ubiquitination and degradation by the proteasome. Low-risk HPV E6 proteins also associate with E6AP, yet fail to recruit p53 or PDZ proteins; their E6AP-dependent targets have so far been uncharacterized. We found a cellular PDZ protein called Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) is targeted for degradation by both high and low-risk HPV E6 proteins as well as E6 proteins from diverse non-primate mammalian species. NHERF1 was degraded by E6 in a manner dependent upon E6AP ubiquitin ligase activity but independent of PDZ interactions. A novel structural domain of E6, independent of the p53 recognition domain, was necessary to associate with and degrade NHERF1, and the NHERF1 EB domain was required for E6-mediated degradation. Degradation of NHERF1 by E6 activated canonical Wnt/ß-catenin signaling, a key pathway that regulates cell growth and proliferation. Expression levels of NHERF1 increased with increasing cell confluency. This is the first study in which a cellular protein has been identified that is targeted for degradation by both high and low-risk HPV E6 as well as E6 proteins from diverse animal papillomaviruses. This suggests that NHERF1 plays a role in regulating squamous epithelial growth and further suggests that the interaction of E6 proteins with NHERF1 could be a common therapeutic target for multiple papillomavirus types.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Proteínas Oncogênicas Virais/genética , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Fosfoproteínas/genética , Filogenia , Complexo de Endopeptidases do Proteassoma , Proteólise , Proteínas Repressoras/genética , Trocadores de Sódio-Hidrogênio/genética , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologia , Proteína Wnt1/genética , beta Catenina/genética
3.
Biomed Pharmacother ; 113: 108671, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30875657

RESUMO

OBJECTIVE: Hypoxic-ischemic brain damage (HIBD) is a major cause of acute mortality and chronic neurological morbidity in infants and children. Dexmedetomidine (DEX) is an effective choice in HIBD treatment. Recent findings have revealed that microRNA-128 (miR-128) is implicated in cerebral ischemia reperfusion. Hence, this study aimed to investigate the role of miR-128 in HIBD. METHODS: HIBD models of neonatal mice were established. HIBD mice were treated with DEX, and injected with agomir (ago)-miR-128 or antagomir (anti)-miR-128 into the lateral ventricles to explore the influence of miR-128 on the neuroprotective effects of DEX on HIBD. Subsequently, the mice body weight, left/right (L/R) brain weight ratio, left-brain water content as well as learning and memory abilities were measured. Furthermore, the pathological changes of brain tissues and apoptosis rate of nerve cells were determined. The potential relationship between miR-128 and WNT1 was analyzed. RESULTS: Over-expression of miR-128 caused an increase in mouse body weight, L/R brain weight ratio, and learning and memory abilities, while led to a decline in left-brain water content, brain tissue injury and apoptosis rate of nerve cells in DEX-treated HIBD mice. WNT1 was targeted and negatively regulated by miR-128. Silencing of WNT1 exerted the same effect as miR-128 on enhancing the neuroprotective effect of DEX on HIBD mice. CONCLUSION: Collectively, miR-128 enhanced neuroprotective effect of DEX on HIBD neonatal mice by inhibiting WNT1.


Assuntos
Encéfalo/efeitos dos fármacos , Dexmedetomidina/farmacologia , Hipóxia-Isquemia Encefálica/tratamento farmacológico , MicroRNAs/metabolismo , Fármacos Neuroprotetores/farmacologia , Proteína Wnt1/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/fisiopatologia , Injeções Intraventriculares , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Tamanho do Órgão/efeitos dos fármacos , Proteína Wnt1/genética
4.
Oncogene ; 38(20): 3871-3885, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30683884

RESUMO

Metastasis begins with a subset of local tumor cells acquiring the potential to invade into surrounding tissues, and remains to be a major obstacle for cancer treatments. More than 90% of cancer patients died from tumor metastasis, instead of primary tumor growth. The canonical Wnt/ß-catenin pathway plays essential roles in promoting tumor formation, yet its function in regulating tumor metastasis and the underlying mechanisms remain controversial. Here we employed well-established Drosophila tumor models to investigate the regulating mechanism of Wingless (Wg) pathway in tumor invasion. Our results showed that Wg signaling is necessary and sufficient for cell polarity disruption-induced cell migration and molecular changes reminiscent of epithelial-mesenchymal transition (EMT). Moreover, reducing Wg signaling suppressed lgl-/-/RasV12-induced tumor invasion, and cooperation between Arm and RasV12 is sufficient to induce tumor invasion. Mechanistically, we found that cell polarity disruption activates JNK signaling, which in turn upregulate wg expression through transcription factor activator protein-1 (AP-1). We identified a consensus AP-1 binding site located in the 2nd intron of wg, and confirmed that it is essential for AP-1 induced wg transcription both in vitro and in vivo. Lastly, we confirmed that the transcriptional activation of WNT by AP-1 is conserved in human cancer cells. These evidences reveal a positive role of Wnt/ß-catenin pathway in tumor invasion, and provide a conserved mechanism that connects JNK and Wnt signaling in regulating tumor progression.


Assuntos
Proteínas de Drosophila/metabolismo , Neoplasias/patologia , Fator de Transcrição AP-1/metabolismo , Proteína Wnt1/metabolismo , Células A549 , Animais , Animais Geneticamente Modificados , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Sítios de Ligação , Movimento Celular/genética , Polaridade Celular , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/genética , Células HeLa , Humanos , IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Íntrons , Sistema de Sinalização das MAP Quinases , Células MCF-7 , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Proteína Wnt1/genética
5.
J Exp Clin Cancer Res ; 38(1): 40, 2019 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-30691485

RESUMO

BACKGROUND: Zinc-finger protein-326 (ZNF326) was initially found in the NIH3T3 cell line to regulate cell growth, however, the expression and underlying role of ZNF326 in human tumours, especially in glioma, is not fully understood. METHODS: Immunohistochemistry was applied to detect the expression of ZNF326 in glioma tissues, and statistical analysis was used to analyse the relationship between ZNF326 expression and clinicopathological factors. The effect of ZNF326 on glioma cells proliferation and invasion was conducted by functional experiments both in vivo and in vitro. Chromatin immunoprecipitation and dual-luciferase assays were performed to demonstrate that histone deacetylase enzyme-7 (HDAC7) is the target gene of ZNF326. Immunoblotting, real-time PCR, GST-pulldown and co-immunoprecipitation assays were used to clarify the underlying role of ZNF326 on Wnt pathway activation. RESULTS: High nuclear expression of ZNF326 was observed in glioma cell lines and tissues, and closely related with advanced tumour grade in the patients. Moreover, ectopic ZNF326 expression promoted the proliferation and invasiveness of glioma cells. Mechanistically, ZNF326 could activate HDAC7 transcription by binding to a specific promoter region via its transcriptional activation domain and zinc-finger structures. The interaction of the up-regulated HDAC7 with ß-catenin led to a decrease in ß-catenin acetylation level at Lys-49, followed by a decrease in ß-catenin phosphorylation level at Ser-45. These changes in ß-catenin posttranscriptional modification levels promoted its redistribution and import into the nucleus. Additionally, ZNF326 directly associated with ß-catenin in the nucleus, and enhanced the binding of ß-catenin to TCF-4, serving as a co-activator in stimulating Wnt pathway. CONCLUSIONS: Our findings elucidated ZNF326 promotes the malignant phenotype of human glioma via ZNF326-HDAC7-ß-catenin signalling. This study reveals the vital role and mechanism of ZNF326 in the malignant progression of glioma, and provides the reference for finding biomarkers and therapeutic targets for glioma.


Assuntos
Neoplasias Encefálicas/patologia , Proteínas de Transporte/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Histona Desacetilases/metabolismo , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Proteínas de Transporte/genética , Movimento Celular , Proliferação de Células , Feminino , Seguimentos , Glioma/genética , Glioma/metabolismo , Histona Desacetilases/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Fenótipo , Prognóstico , Células Tumorais Cultivadas , Proteína Wnt1/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética
6.
J Hum Genet ; 64(4): 291-296, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30692598

RESUMO

A rare form of osteogenesis imperfecta (OI) caused by Wingless-type MMTV integration site family 1 (WNT1) mutations combines central nervous system (CNS) anomalies with the characteristic increased susceptibility to fractures. We report an additional case where arachnoid cysts extend the phenotype, and that also confirms the association of intellectual disabilities with asymmetric cerebellar hypoplasia here. Interestingly, if the cerebellum is normal in this disorder, intelligence is as well, analogous to an association with similar delays in a subset of patients with sporadic unilateral cerebellar hypoplasia. Those cases typically appear to represent vascular disruptions, and we suggest that most brain anomalies in WNT1-associated OI have vascular origins related to a role for WNT1 in CNS angiogenesis. This unusual combination of benign cerebellar findings with effects on higher functions in these two situations raises the possibility that WNT1 is involved in the pathogenesis of the associated sporadic cases as well. Finally, our patient reacted poorly to pamidronate, which appears ineffective with this form of OI, so that a lack of improvement is an indication for molecular testing that includes WNT1.


Assuntos
Sistema Nervoso Central/fisiopatologia , Deficiência Intelectual/genética , Osteogênese Imperfeita/genética , Proteína Wnt1/genética , Cistos Aracnóideos/diagnóstico por imagem , Cistos Aracnóideos/fisiopatologia , Sistema Nervoso Central/anormalidades , Sistema Nervoso Central/diagnóstico por imagem , Cerebelo/anormalidades , Cerebelo/diagnóstico por imagem , Cerebelo/fisiopatologia , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/fisiopatologia , Lobo Frontal/diagnóstico por imagem , Lobo Frontal/fisiopatologia , Humanos , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/tratamento farmacológico , Deficiência Intelectual/fisiopatologia , Mutação , Malformações do Sistema Nervoso/diagnóstico por imagem , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/fisiopatologia , Osteogênese Imperfeita/diagnóstico por imagem , Osteogênese Imperfeita/tratamento farmacológico , Osteogênese Imperfeita/fisiopatologia , Pamidronato/administração & dosagem , Pamidronato/efeitos adversos
7.
PLoS One ; 14(1): e0209349, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30615641

RESUMO

This work provides theoretical tools to analyse the transcriptional effects of certain biochemical mechanisms (i.e. affinity and cooperativity) that have been proposed in previous literature to explain the proper spatial expression of Hedgehog target genes involved in Drosophila development. Specifically we have focused on the expression of decapentaplegic, wingless, stripe and patched. The transcription of these genes is believed to be controlled by enhancer modules able to interpret opposing gradients of the activator and repressor forms of the transcription factor Cubitus interruptus (Ci). This study is based on a thermodynamic approach, which provides expression rates for these genes. These expression rates are controlled by transcription factors which are competing and cooperating for common binding sites. We have made mathematical representations of the different expression rates which depend on multiple factors and variables. The expressions obtained with the model have been refined to produce simpler equivalent formulae which allow for their mathematical analysis. Thanks to this, we can evaluate the correlation between the different interactions involved in transcription and the biological features observed at tissular level. These mathematical models can be applied to other morphogenes to help understand the complex transcriptional logic of opposing activator and repressor gradients.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Proteínas Hedgehog/genética , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Expressão Gênica , Genes de Insetos , Proteínas Hedgehog/metabolismo , Modelos Genéticos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética , Proteína Wnt1/genética , Proteína Wnt1/metabolismo
8.
Oncol Rep ; 41(1): 599-607, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30365079

RESUMO

Esophageal cancer (EC) is one of the leading causes of death among malignancies. Radiotherapy for esophageal squamous cell carcinoma (ESCC) patients is limited by resistance to ionizing radiation (IR). An increasing body of evidence has demonstrated that aberrant expression of microRNA­301a (miR­301a) contributes to cancer progression and sensitivity to radiation. The aim of the present study was to investigate the exact functions and potential mechanisms of miR­301a in ESCC radioresistance. Initially, the miR­301a­transfected radioresistant ESCC cells KYSE­150R exhibited a decreased proliferation rate, and enhanced radiosensitivity and migration, whereas downregulation of miR­301a in radiosensitive KYSE­150 cells produced the opposite results. miR­301a regulates WNT1 expression at both the mRNA and protein levels. Furthermore, dual­luciferase reporter assays revealed that WNT1 was a target gene of miR­301a. In addition, the expression of miR­301a markedly affected the expression of Wnt/ß­catenin­related proteins such as ß­catenin and cyclin D1. Finally, overexpression of miR­301a inhibited epithelial­mesenchymal transition (EMT) conversion by directly targeting Snail and vimentin in radioresistant­ESCC cell lines; however, no inhibitory effects were exerted on Twist. Collectively, these results indicated that miR­301a increased the radiosensitivity and inhibited the migration of radioresistant­ESCC cells by targeting WNT1, thereby inactivating the Wnt/ß­catenin signaling pathway and EMT reversal. Thus, miR­301a may be a potential therapeutic target for the treatment of EC radioresistance.


Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Esofágicas/genética , MicroRNAs/genética , Tolerância a Radiação/genética , Proteína Wnt1/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Ciclina D1/genética , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Esofágicas/patologia , Humanos , Transdução de Sinais/genética , beta Catenina/genética
9.
Life Sci ; 210: 224-234, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30193944

RESUMO

AIM: To investigate the effect of fructose-1,6-bisphosphatase 1 (FBP1) on the malignant phenotypes of cholangiocarcinoma (CCA) cells, and to explore the underlying mechanism. MAIN METHODS: The expression of FBP1 in clinical CCA tissues was detected by real-time PCR, Western blot and immunohistochemistry staining. FBP1 was overexpressed by transfection of a forced expression plasmid. MTT, plate colony formation assay, Hoechst staining, flow cytometry, Western blot, wound healing, transwell assays and xenograft were performed to detect the growth, proliferation, cell cycle, apoptosis, migration, invasion and tumorigenesis in RBE and HCCC-9801 cells. In addition, the Wnt/ß-catenin signaling was detected. KEY FINDINGS: FBP1 was downregulated in clinical CCA specimens and cell lines, compared to paired para-carcinoma tissues or normal cholangetic epithelial cells. Gain-of-function experiments demonstrated that the forced expression of FBP1 inhibited the proliferation, colony formation, and blocked cell cycle of RBE and HCCC-9801 cells. Apoptosis of CCA cells was significantly enhanced by FBP1 overexpression, evidenced by upregulation of cleaved caspase-3, cleaved PARP and Bax levels, while downregulation of Bcl-2 level. Moreover, overexpression of FBP1 decreased the migratory and invasive ability in RBE and HCCC-9801 cells. However, FBP1-induced phenotypic changes were eliminated by overexpression of ß-catenin. Finally, the forced overexpression of FBP1 inhibited tumorigenesis in vivo. SIGNIFICANCE: Our findings demonstrate that FBP1 is downregulated in CCA tissues and cell lines, and the overexpression of FBP1 inhibits the proliferation, migration, invasion and tumorigenesis of CCA cells partly via inactivation of Wnt/ß-catenin pathway. FBP1 may be a novel early diagnosis marker and therapeutic target for CCA.


Assuntos
Neoplasias dos Ductos Biliares/prevenção & controle , Movimento Celular , Proliferação de Células , Colangiocarcinoma/prevenção & controle , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Animais , Apoptose , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/secundário , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Estudos de Casos e Controles , Transformação Celular Neoplásica , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Células Tumorais Cultivadas , Proteína Wnt1/genética , Cicatrização , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética
10.
Elife ; 72018 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-30095068

RESUMO

Wingless/Wnts are signalling molecules, traditionally considered to pattern tissues as long-range morphogens. However, more recently the spread of Wingless was shown to be dispensable in diverse developmental contexts in Drosophila and vertebrates. Here we demonstrate that release and spread of Wingless is required to pattern the proximo-distal (P-D) axis of Drosophila Malpighian tubules. Wingless signalling, emanating from the midgut, directly activates odd skipped expression several cells distant in the proximal tubule. Replacing Wingless with a membrane-tethered version that is unable to diffuse from the Wingless producing cells results in aberrant patterning of the Malpighian tubule P-D axis and development of short, deformed ureters. This work directly demonstrates a patterning role for a released Wingless signal. As well as extending our understanding about the functional modes by which Wnts shape animal development, we anticipate this mechanism to be relevant to patterning epithelial tubes in other organs, such as the vertebrate kidney.


Assuntos
Padronização Corporal , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Túbulos Renais Distais/fisiologia , Túbulos Renais Proximais/fisiologia , Proteína Wnt1/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Túbulos Renais Distais/embriologia , Túbulos Renais Proximais/embriologia , Morfogênese , Via de Sinalização Wnt , Proteína Wnt1/genética
11.
Mol Med Rep ; 18(3): 2571-2580, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30015876

RESUMO

MicroRNA (miR)­200b­3p is downregulated in multiple human cancer types. Wnt signaling serves a role in human colorectal cancer (CRC). The present study aimed to examine the effect of miR­200b­3p on human CRC and its potential association with Wnt signaling. The Cell Counting Kit­8 (CCK­8) was employed to assess cell viability. A flow cytometric assay was conducted to examine cell proliferation and apoptosis. The regulation model of miR­200b­3p and Wnt1 was assessed by a luciferase reporter assay. A commercial kit was used to evaluate the activity of caspase­3 following treatment of the cells by miR­200b­3p or Wnt1. The expression of target factors was determined by a quantitative real­time polymerase chain reaction and western blot analysis. The expression of miR­200b­3p was decreased in human CRC tissues and in cell lines. The bioinformatics analysis and the luciferase reporter assay revealed that Wnt1 may be a direct target of miR­200b­3p. Moreover, the viability and proliferation of CRC cells was suppressed by miR­200b­3p. miR­200b­3p additionally induced apoptosis in CRC cells. Furthermore, the caspase­3 activity was enhanced in the miR­200b­3p mimics group. The expression of antigen Ki­67 (additionally termed KI­67) and ß­catenin was decreased, while the expression of cleaved caspase­3 was increased by miR­200b­3p. In conclusion, miR­200b­3p inhibited proliferation and induced apoptosis in CRC cells by inactivating Wnt/ß­catenin signaling. The present study provided potential biomarkers and candidate modalities for the management of CRC.


Assuntos
Apoptose , Proliferação de Células , Neoplasias Colorretais/patologia , MicroRNAs/metabolismo , Proteína Wnt1/metabolismo , Regiões 3' não Traduzidas , Adulto , Idoso , Antagomirs/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Via de Sinalização Wnt , Proteína Wnt1/antagonistas & inibidores , Proteína Wnt1/genética , beta Catenina/genética , beta Catenina/metabolismo
12.
Oncol Rep ; 40(3): 1261-1274, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30015909

RESUMO

HES6 is a member of the hairy-enhancer of the split homolog family, which has been implicated in oncogenesis and cancer progression in a variety of human cancers, including prostate and breast cancer. However, its clinical significance and biological role in colorectal cancer (CRC) remain unclear. In the present study, the expression of HES6 was significantly upregulated in CRC cell lines and CRC tissues at both the mRNA and protein levels. The present study also reported high expression of HES6 in 138/213 (64.8%) paraffin-embedded archived CRC specimens. HES6 expression was significantly correlated with T classification (P<0.001), N classification (P=0.020), and distant metastasis (P<0.001). Patients with higher HES6 expression levels exhibited a reduced overall survival (P<0.001). In addition, a multivariate analysis revealed that the expression of HES6 may be a novel prognostic marker for the survival of patients with CRC. Furthermore, the present study demonstrated that ectopic expression of HES6 enhanced the migration and invasive abilities of CRC cells. These abilities were significantly inhibited upon knockdown of endogenous HES6 expression by specific short hairpin RNAs. Additionally, the present study reported that the effects of HES6 on metastasis may be associated with the activation of the Wnt/ß-catenin signaling pathway. Collectively, the findings of the present study revealed that overexpression of HES6 played a key role in the progression of CRC, leading to a poor prognosis and clinical outcome.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores Tumorais/metabolismo , Movimento Celular , Neoplasias Colorretais/patologia , Neoplasias Hepáticas/secundário , Proteínas Repressoras/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biomarcadores Tumorais/genética , Proliferação de Células , Neoplasias Colorretais/metabolismo , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Repressoras/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Adulto Jovem , beta Catenina/genética , beta Catenina/metabolismo
13.
Genetics ; 208(4): 1311-1336, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29618590

RESUMO

This FlyBook chapter summarizes the history and the current state of our understanding of the Wingless signaling pathway. Wingless, the fly homolog of the mammalian Wnt oncoproteins, plays a central role in pattern generation during development. Much of what we know about the pathway was learned from genetic and molecular experiments in Drosophila melanogaster, and the core pathway works the same way in vertebrates. Like most growth factor pathways, extracellular Wingless/Wnt binds to a cell surface complex to transduce signal across the plasma membrane, triggering a series of intracellular events that lead to transcriptional changes in the nucleus. Unlike most growth factor pathways, the intracellular events regulate the protein stability of a key effector molecule, in this case Armadillo/ß-catenin. A number of mysteries remain about how the "destruction complex" destabilizes ß-catenin and how this process is inactivated by the ligand-bound receptor complex, so this review of the field can only serve as a snapshot of the work in progress.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Drosophila/fisiologia , Morfogênese/genética , Via de Sinalização Wnt , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Animais , Biomarcadores , Padronização Corporal/genética , Proteínas de Drosophila/química , Evolução Molecular , Estudos de Associação Genética , Humanos , Fenótipo , Proteína Wnt1/química
14.
Development ; 145(8)2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29691225

RESUMO

Epithelial folding shapes embryos and tissues during development. Here, we investigate the coupling between epithelial folding and actomyosin-enriched compartmental boundaries. The mechanistic relationship between the two is unclear, because actomyosin-enriched boundaries are not necessarily associated with folds. Also, some cases of epithelial folding occur independently of actomyosin contractility. We investigated the shallow folds called parasegment grooves that form at boundaries between anterior and posterior compartments in the early Drosophila embryo. We demonstrate that formation of these folds requires the presence of an actomyosin enrichment along the boundary cell-cell contacts. These enrichments, which require Wingless signalling, increase interfacial tension not only at the level of the adherens junctions but also along the lateral surfaces. We find that epithelial folding is normally under inhibitory control because different genetic manipulations, including depletion of the Myosin II phosphatase Flapwing, increase the depth of folds at boundaries. Fold depth correlates with the levels of Bazooka (Baz), the Par-3 homologue, along the boundary cell-cell contacts. Moreover, Wingless and Hedgehog signalling have opposite effects on fold depth at the boundary that correlate with changes in Baz planar polarity.


Assuntos
Actomiosina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Proteína Wnt1/metabolismo , Junções Aderentes/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Bactérias/genética , Padronização Corporal , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Epitélio/embriologia , Técnicas de Silenciamento de Genes , Genes de Insetos , Proteínas de Fluorescência Verde/genética , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Luminescentes/genética , Mutação , Miosina Tipo II/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/antagonistas & inibidores , Fosfatase de Miosina-de-Cadeia-Leve/genética , Transdução de Sinais , Proteína Wnt1/genética
15.
PLoS One ; 13(4): e0196365, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29702674

RESUMO

The ability to express a gene of interest in a spatio-temporal manner using Gal4-UAS system has allowed the use of Drosophila model to study various biological phenomenon. During Drosophila eye development, a synchronous wave of differentiation called Morphogenetic furrow (MF) initiates at the posterior margin resulting in differentiation of retinal neurons. This synchronous differentiation is also observed in the differentiating retina of vertebrates. Since MF is highly dynamic, it can serve as an excellent model to study patterning and differentiation. However, there are not any Gal4 drivers available to observe the gain- of- function or loss- of- function of a gene specifically along the dynamic MF. The decapentaplegic (dpp) gene encodes a secreted protein of the transforming growth factor-beta (TGF-beta) superfamily that expresses at the posterior margin and then moves with the MF. However, unlike the MF associated pattern of dpp gene expression, the targeted dpp-Gal4 driver expression is restricted to the posterior margin of the developing eye disc. We screened GMR lines harboring regulatory regions of dpp fused with Gal4 coding region to identify MF specific enhancer of dpp using a GFP reporter gene. We employed immuno-histochemical approaches to detect gene expression. The rationale was that GFP reporter expression will correspond to the dpp expression domain in the developing eye. We identified two new dpp-Gal4 lines, viz., GMR17E04-Gal4 and GMR18D08-Gal4 that carry sequences from first intron region of dpp gene. GMR17E04-Gal4 drives expression along the MF during development and later in the entire pupal retina whereas GMR18D08-Gal4 drives expression of GFP transgene in the entire developing eye disc, which later drives expression only in the ventral half of the pupal retina. Thus, GMR18D08-Gal4 will serve as a new reagent for targeting gene expression in the ventral half of the pupal retina. We compared misexpression phenotypes of Wg, a negative regulator of eye development, using GMR17E04-Gal4, GMR18D08-Gal4 with existing dpp-Gal4 driver. The eye phenotypes generated by using our newly identified MF specific driver are not similar to the ones generated by existing dpp-Gal4 driver. It suggests that misexpression studies along MF needs revisiting using the new Gal4 drivers generated in our studies.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Retina/embriologia , Retina/crescimento & desenvolvimento , Fatores de Transcrição/genética , Proteína Wnt1/genética , Animais , Animais Geneticamente Modificados , Padronização Corporal , Diferenciação Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Neurônios Retinianos/fisiologia , Transativadores/genética , Fator de Crescimento Transformador beta/metabolismo
16.
PLoS Genet ; 14(4): e1007339, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29641560

RESUMO

Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in Drosophila embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.


Assuntos
Proteínas do Domínio Armadillo/metabolismo , Complexo de Sinalização da Axina/metabolismo , Proteínas de Drosophila/metabolismo , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt , Animais , Animais Geneticamente Modificados , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/genética , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/genética , Proteína Axina/química , Proteína Axina/genética , Proteína Axina/metabolismo , Complexo de Sinalização da Axina/química , Complexo de Sinalização da Axina/genética , Linhagem Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteólise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição Genética , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismo
17.
J Forensic Leg Med ; 56: 66-72, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29533208

RESUMO

The arrival of arthropods at a corpse exhibits specific temporal patterns, and Diptera play a key role in the initial stages of the decomposition process. Thus, the correct species assignment of the insect larvae found on a decomposing body is an important step in forensic investigations. Here, we describe a molecular procedure to define the species at larval age found on a corpse more quickly and easily than current systems. Our method involves a unique PCR amplification of a DNA segment within the evolutionarily conserved wingless gene, involved in embryo development. The amplified DNA segment contains the fourth intron of wingless, which we found to be variable in length, from about 800 to 3000 bp, among species of necrophagous Diptera. The identification of the amplified segment size in species from Lucilia, Calliphora and Sarcophaga genera, allowed us to determine the species at larval age collected in the early stages of a decomposing body, with a simple PCR amplification and subsequent electrophoresis. This procedure may help in forensic investigations to estimate the minimum Post Mortem Interval (PMI-min) of a body colonized by these larvae, avoiding the use of time-consuming and/or more expensive procedures.


Assuntos
Dípteros/genética , Íntrons , Proteína Wnt1/genética , Animais , Código de Barras de DNA Taxonômico , Complexo IV da Cadeia de Transporte de Elétrons/genética , Entomologia , Ciências Forenses , Larva , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
18.
Proc Natl Acad Sci U S A ; 115(14): E3173-E3181, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29559533

RESUMO

Wnts and R-spondins (RSPOs) support intestinal homeostasis by regulating crypt cell proliferation and differentiation. Ex vivo, Wnts secreted by Paneth cells in organoids can regulate the proliferation and differentiation of Lgr5-expressing intestinal stem cells. However, in vivo, Paneth cell and indeed all epithelial Wnt production is completely dispensable, and the cellular source of Wnts and RSPOs that maintain the intestinal stem-cell niche is not known. Here we investigated both the source and the functional role of stromal Wnts and RSPO3 in regulation of intestinal homeostasis. RSPO3 is highly expressed in pericryptal myofibroblasts in the lamina propria and is several orders of magnitude more potent than RSPO1 in stimulating both Wnt/ß-catenin signaling and organoid growth. Stromal Rspo3 ablation ex vivo resulted in markedly decreased organoid growth that was rescued by exogenous RSPO3 protein. Pdgf receptor alpha (PdgfRα) is known to be expressed in pericryptal myofibroblasts. We therefore evaluated if PdgfRα identified the key stromal niche cells. In vivo, Porcn excision in PdgfRα+ cells blocked intestinal crypt formation, demonstrating that Wnt production in the stroma is both necessary and sufficient to support the intestinal stem-cell niche. Mice with Rspo3 excision in the PdgfRα+ cells had decreased intestinal crypt Wnt/ß-catenin signaling and Paneth cell differentiation and were hypersensitive when stressed with dextran sodium sulfate. The data support a model of the intestinal stem-cell niche regulated by both Wnts and RSPO3 supplied predominantly by stromal pericryptal myofibroblasts marked by PdgfRα.


Assuntos
Células Epiteliais/citologia , Intestinos/citologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/fisiologia , Nicho de Células-Tronco/fisiologia , Células-Tronco/citologia , Células Estromais/citologia , Trombospondinas/metabolismo , Proteína Wnt1/metabolismo , Aciltransferases/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Células Epiteliais/metabolismo , Homeostase , Mucosa Intestinal/metabolismo , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organoides/citologia , Organoides/metabolismo , Células-Tronco/metabolismo , Células Estromais/metabolismo , Trombospondinas/genética , Proteína Wnt1/genética
19.
Proc Natl Acad Sci U S A ; 115(15): E3491-E3500, 2018 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-29581309

RESUMO

The jointed appendages of arthropods have facilitated the spectacular diversity and success of this phylum. Key to the regulation of appendage outgrowth is the Krüppel-like factor (KLF)/specificity protein (Sp) family of zinc finger transcription factors. In the fruit fly, Drosophila melanogaster, the Sp6-9 homolog is activated by Wnt-1/wingless (wg) and establishes ventral appendage (leg) fate. Subsequently, Sp6-9 maintains expression of the axial patterning gene Distal-less (Dll), which promotes limb outgrowth. Intriguingly, in spiders, Dll has been reported to have a derived role as a segmentation gap gene, but the evolutionary origin and regulation of this function are not understood because functional investigations of the appendage-patterning regulatory network are restricted to insects. We tested the evolutionary conservation of the ancestral appendage-patterning network of arthropods with a functional approach in the spider. RNAi-mediated knockdown of the spider Sp6-9 ortholog resulted in diminution or loss of Dll expression and truncation of appendages, as well as loss of the two body segments specified by the early Dll function. In reciprocal experiments, Dll is shown not to be required for Sp6-9 expression. Knockdown of arrow (Wnt-1 coreceptor) disrupted segmentation and appendage development but did not affect the early Sp6-9 expression domain. Ectopic appendages generated in the spider "abdomen" by knockdown of the Hox gene Antennapedia-1 (Antp-1) expressed Sp6-9 comparably to wild-type walking legs. Our results support (i) the evolutionary conservation of an appendage-patterning regulatory network that includes canonical Wnt signaling, Sp6-9, and Dll and (ii) the cooption of the Sp6-9/Dll regulatory cassette in arachnid head segmentation.


Assuntos
Padronização Corporal/genética , Aranhas/genética , Proteína Wnt1/genética , Animais , Aracnídeos/genética , Evolução Biológica , Evolução Molecular , Extremidades/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fenótipo , Interferência de RNA , Aranhas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco
20.
Mol Cells ; 41(2): 110-118, 2018 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-29385674

RESUMO

The objective of this study was to induce the production of isthmic organizer (IsO)-like cells capable of secreting fibroblast growth factor (FGF) 8 and WNT1 from human embryonic stem cells (ESCs). The precise modulation of canonical Wnt signaling was achieved in the presence of the small molecule CHIR99021 (0.6 µM) during the neural induction of human ESCs, resulting in the differentiation of these cells into IsO-like cells having a midbrain-hindbrain border (MHB) fate in a manner that recapitulated their developmental course in vivo. Resultant cells showed upregulated expression levels of FGF8 and WNT1. The addition of exogenous FGF8 further increased WNT1 expression by 2.6 fold. Gene ontology following microarray analysis confirmed that IsO-like cells enriched the expression of MHB-related genes by 40 fold compared to control cells. Lysates and conditioned media of IsO-like cells contained functional FGF8 and WNT1 proteins that could induce MHB-related genes in differentiating ESCs. The method for generating functional IsO-like cells described in this study could be used to study human central nervous system development and congenital malformations of the midbrain and hindbrain.


Assuntos
Diferenciação Celular/genética , Perfilação da Expressão Gênica/métodos , Células-Tronco Embrionárias Humanas/metabolismo , Neurônios/metabolismo , Linhagem Celular , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Proteína Wnt1/genética , Proteína Wnt1/metabolismo
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