RESUMO
INTRODUCTION: Colorectal cancer (CRC), a common digestive tract tumor that contains colon and rectal cancer, is one of the three most common cancers globally. circRNAs are involved in the occurrence and development of CRC, but the mechanism of how they participate in this process remains unclear. METHODS: We adopted PCR for expression measure, CCK-8 for cell proliferation detection, Transwell for cell migration and invasion detection, and dual-luciferase reporter assays to detect the potential downstream targets of CCDC66 in CRC. RESULTS: This study showed that circRNA CCDC66 was overexpressed in CRC tissues, and after knockdown, it inhibited the proliferation, migration, and invasion of CRC cells (RKO and HCT-116) in vitro. In addition, the dual-luciferase reporter assay showed that there was a binding site between circCCDC66 and miR-370, as well as between miR-370 and murine double minute 4 (MDM4). That is, circCCDC66 upregulated the expression of MDM4 through competitively binding to miR-370. The expression of circCCDC66 in CRC tissues was positively correlated with MDM4 and negatively correlated with miR-370. CONCLUSION: In summary, our results indicate that circCCDC66 is a key upregulation of CRC. circCCDC66 upregulates MDM4 through competitive binding to miR-370, thereby enhancing the metastatic ability of CRC cells and promoting the development of CRC.
Assuntos
Proteínas de Ciclo Celular/genética , Neoplasias Colorretais/genética , Proteínas do Olho/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas/genética , RNA Circular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Biologia Computacional , Proteínas do Olho/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Invasividade Neoplásica/genética , RNA Circular/antagonistas & inibidores , RNA Circular/metabolismo , Regulação para CimaRESUMO
Diabetic retinopathy (DR) remains high incidence and accounts for severe impact on vision in diabetics, but its mechanism is still poorly understood. Abnormal migration and proliferation of endothelial cells (ECs) drive neovascular retinopathies, which has an important role in promoting the occurrence and development of DR. In this study, we designed and synthesized a series of PEDF-derived peptides as angiogenesis inhibitors. Especially, compound G24 significantly inhibited the cell proliferation in VEGF-activated human umbilical vein endothelial cells (HUVECs) with IC50 values of 2.88 ± 0.19 µM. Further biological evaluation demonstrated that compound G24 exhibited strong inducing-effects on cell apoptosis and internalization of 67LR, and advanced inhibitory potency in cell migration and angiogenesis formed by HUVECs in vitro. In summary, the optimal compound G24 as a novel angiogenesis inhibitor showed the potentiality in the further research for the treatment for DR.
Assuntos
Inibidores da Angiogênese/farmacologia , Proteínas do Olho/antagonistas & inibidores , Neovascularização Patológica/tratamento farmacológico , Fatores de Crescimento Neural/antagonistas & inibidores , Peptídeos/farmacologia , Receptores de Laminina/antagonistas & inibidores , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Proteínas do Olho/metabolismo , Humanos , Estrutura Molecular , Fatores de Crescimento Neural/metabolismo , Peptídeos/síntese química , Peptídeos/química , Receptores de Laminina/metabolismo , Serpinas/metabolismo , Relação Estrutura-AtividadeRESUMO
Some related reports indicate that the outer retinal membrane protein 1 (ROM1) functions importantly in the regulation of the biological process of tumor. Nevertheless, studies towards the role of ROM1 in lung cancer are few. Here, our data demonstrated that ROM1 displayed a relation with lung cancer tumorigenesis and development. In the Tumor Genome Atlas (TCGA) cohort, reduced ROM1 level was observed in lung cancer tissues, instead of normal tissues. After bioinformatics analysis, the data revealed that ROM1 level was associated with the tumor stage. Additional results indicated that highly expressed ROM1 exhibited a positive correlation with the overall survival rate, and ROM1 was probably a promising prognostic biomarker of lung cancer. Additionally, our results indicated that knocking out ROM1 could promote cell proliferation, migration, and invasion. Our data conclusively demonstrated that ROM1 modulated lung cancer tumorigenesis and development, as a prognosis and treatment biomarker.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas do Olho/metabolismo , Neoplasias Pulmonares/metabolismo , Tetraspaninas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Biologia Computacional , Bases de Dados Genéticas , Progressão da Doença , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Técnicas de Inativação de Genes , Ontologia Genética , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Invasividade Neoplásica/genética , Prognóstico , Tetraspaninas/antagonistas & inibidores , Tetraspaninas/genética , Transcriptoma , Proteínas Supressoras de Tumor/genéticaRESUMO
Lung cancer is one of the leading causes of death in cancer patients. Epithelial-mesenchymal transition (EMT) plays an important role in lung cancer progression. Therefore, for lung cancer treatment, it is crucial to find substances that inhibit EMT. Ethacrynic acid (ECA) is a diuretic that inhibits cellular ion flux and exerts anticancer effects. However, the effects of ECA on EMT in lung cancer remain unclear. We examined the effects of ECA on sphingosylphosphorylcholine (SPC) or TGF-ß1-induced EMT process in A549 and H1299 cells via reverse transcription polymerase chain reaction and Western blotting. We found that ECA inhibited SPC-induced EMT and SPC-induced WNT signalling in EMT. We observed that SPC induces the expression of NDP [Norrie disease protein] and WNT-2, whereas ECA suppressed their expression. SPC-induced WNT activation, EMT, migration, and invasion were suppressed by NDP small-interfering RNA (siNDP), but NDP overexpression (pNDP) enhanced these events in A549 and H1299 cells. Accordingly, NDP expression may influence lung cancer prognosis. In summary, our results revealed that ECA inhibited SPC or TGF-ß1-induced EMT in A549 and H1299 lung cancer cells by downregulating NDP expression and inhibiting WNT activation. Therefore, ECA might be a new drug candidate for lung cancer treatment.
Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Ácido Etacrínico/farmacologia , Proteínas do Olho/farmacologia , Neoplasias Pulmonares/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Células A549 , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Relação Dose-Resposta a Droga , Transição Epitelial-Mesenquimal/fisiologia , Ácido Etacrínico/uso terapêutico , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/biossíntese , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/biossíntese , RNA Interferente Pequeno/farmacologia , Inibidores de Simportadores de Cloreto de Sódio e Potássio/uso terapêutico , Via de Sinalização Wnt/fisiologiaRESUMO
The homeodomain transcription factors (TFs) Pax6 (OMIM: 607108) and Prox1 (OMIM: 601546) critically regulate gene expression in lens development. While PAX6 mutations in humans can cause cataract, aniridia, microphthalmia, and anophthalmia, among other defects, Prox1 deletion in mice causes severe lens abnormalities, in addition to other organ defects. Furthermore, the optimal dosage/spatiotemporal expression of these key TFs is essential for development. In lens development, Pax6 expression is elevated in cells of the anterior epithelium compared to fiber cells, while Prox1 exhibits the opposite pattern. Whether post-transcriptional regulatory mechanisms control these precise TF expression patterns is unknown. Here, we report the unprecedented finding that the cataract-linked RNA-binding protein (RBP), Celf1 (OMIM: 601074), post-transcriptionally regulates Pax6 and Prox1 protein expression in lens development. Immunostaining shows that Celf1 lens-specific conditional knockout (Celf1cKO) mice exhibit abnormal elevation of Pax6 protein in fiber cells and abnormal Prox1 protein levels in epithelial cells-directly opposite to their normal expression patterns in development. Furthermore, RT-qPCR shows no change in Pax6 and Prox1 transcript levels in Celf1cKO lenses, suggesting that Celf1 regulates these TFs on the translational level. Indeed, RNA-immunoprecipitation assays using Celf1 antibody indicate that Celf1 protein binds to Pax6 and Prox1 transcripts. Furthermore, reporter assays in Celf1 knockdown and Celf1-overexpression cells demonstrate that Celf1 negatively controls Pax6 and Prox1 translation via their 3' UTRs. These data define a new mechanism of RBP-based post-transcriptional regulation that enables precise control over spatiotemporal expression of Pax6 and Prox1 in lens development, thereby uncovering a new etiological mechanism for Celf1 deficiency-based cataract.
Assuntos
Proteínas CELF1/genética , Catarata/genética , Proteínas de Homeodomínio/genética , Cristalino/metabolismo , Fator de Transcrição PAX6/genética , Proteínas Supressoras de Tumor/genética , Animais , Proteínas CELF1/antagonistas & inibidores , Proteínas CELF1/deficiência , Catarata/patologia , Diferenciação Celular/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Cristalino/crescimento & desenvolvimento , Camundongos , Camundongos Knockout , Proteínas de Ligação a RNA/genéticaRESUMO
We investigated the effects of matairesinol (MAT) in the experimental autoimmune uveitis (EAU), a classical animal model of uveitis. We found that treatment with MAT could alleviate intraocular inflammation of EAU. Notably, Th17 cells in eyes of EAU mice could be predominantly restrained by MAT. Furthermore, MAT could inhibit Th17 differentiation in vitro. In addition, MAT inhibited the signaling of MAPK and ROR-γt, a pivotal transcription factor for Th17 cell differentiation in vitro and in vivo. Taken together, these results suggested that MAT had immune-suppressive effects on autoimmune inflammation through Th17 cells.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Proteínas do Olho/antagonistas & inibidores , Furanos/uso terapêutico , Lignanas/uso terapêutico , Proteínas de Ligação ao Retinol/antagonistas & inibidores , Células Th17/efeitos dos fármacos , Uveíte/tratamento farmacológico , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Proteínas do Olho/imunologia , Proteínas do Olho/metabolismo , Feminino , Adjuvante de Freund/toxicidade , Furanos/farmacologia , Lignanas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação ao Retinol/imunologia , Proteínas de Ligação ao Retinol/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Uveíte/imunologia , Uveíte/metabolismoRESUMO
Abdominal aortic aneurysm (AAA) is fatal meanwhile unpredictable asymptomatic cardiovascular disease. Available data suggests the potential participation of circular RNAs (circRNAs) in AAA pathogenesis. But direct evidence is limited. The present study is to functionally and mechanically characterize circRNA CCDC66 (circCCDC66) in AAA. Previous work indicated the differentially expressed circCCDC66 in AAA. At molecular level, circCCDC66, miR-342-3p and CCDC66 transcript were measured through real-time quantitative polymerase chain reaction assay. Functionally, we examined the cellular behaviours of circCCDC66-depleted or CCDC66-depleted vascular smooth muscle cells (VSMCs) including proliferation and apoptosis. It elucidated that depletion of circCCDC66 induced proliferation facilitation and apoptosis reduction. Mechanically, we addressed the interplay among circCCDC66, miR-342-3p and CCDC66 transcript using RNA immunoprecipitation, RNA pull-down and luciferase reporter experiments. Through mechanical validation, we discovered the positive regulation of circCCDC66 on its host gene CCDC66. Loss of CCDC66 mimicked the effects of circCCDC66 silencing on VSMC growth. Moreover, it uncovered that circCCDC66 regulated CCDC66-dependent VSMC growth through sponging miR-342-3p. Rescue experiments aimed to address the functional role of regulatory network formed by circCCDC66, miR-342-3p and CCDC66 in VSMC growth and apoptosis. Suppressing miR-342-3p or overexpressing CCDC66 could reverse VSMC growth caused by circCCDC66 deficiency. Our study further emphasized and first unveiled the function of circCCDC66 in VSMC proliferation. CircCCDC66 upregulated its host gene through its role of miR-342-3p sponge, and hinted a novel molecular mechanism in AAA. SIGNIFICANCE OF THE STUDY: It was firstly displayed in our study that depletion of circCCDC66 induced proliferation augmentation and apoptosis reduction of vascular smooth muscle cells (VSMCs). Meanwhile, circCCDC66/miR-342-3p/CCDC66 axis was proved can play the function of modulating the cell proliferation and apoptosis of VSMCs, which provided us a novel molecular mechanism in AAA.
Assuntos
Proteínas do Olho/metabolismo , RNA Circular/metabolismo , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , Apoptose , Sequência de Bases , Linhagem Celular , Proliferação de Células , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Humanos , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Interferência de RNA , RNA Circular/antagonistas & inibidores , RNA Circular/genética , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Regulação para CimaRESUMO
Neural stem cells (NSCs) are pluripotent cells that are capable of differentiating into neurons and considered as the most promising cell source for cell replacement therapy. However, the difficulty in inducing neuronal differentiation and maturation from NSCs is a major challenge for their clinical application. Clarifying the molecular mechanisms underlying the neuronal differentiation of NSCs can provide a basis for expanding their uses. Brain 4 (Brn4) is a member of the POU domain family of transcription factors and can induce the neuronal differentiation of NSCs, but its precise function in NSCs is unclear. To address this question, in this study we isolated and expanded radial glial cells (RGCs), a type of NSC, from the cerebral cortex of 14-day embryonic rats and used lentivirus carrying the human Brn4 gene to overexpress Brn4 in these cells. This induced the differentiation of RGCs into neurons and inhibited the expression of C-terminal binding protein 2 (CtBP2), a transcriptional co-repressor. CtBP2 overexpression in RGCs suppressed their differentiation into neurons, whereas CtBP2 knockdown had the opposite effect. These results indicated that Brn4 promoted the neuronal differentiation of NSCs via inhibition of CtBP2 and is a potential tool for generating neurons in cell replacement therapy of neurodegenerative diseases and brain injury.
Assuntos
Diferenciação Celular/fisiologia , Proteínas do Olho/antagonistas & inibidores , Proteínas do Tecido Nervoso/fisiologia , Neuroglia/citologia , Neurônios/citologia , Fatores do Domínio POU/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Proteínas do Olho/metabolismo , Feminino , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Mounting evidence suggests that immunological mechanisms play a fundamental role in the pathogenesis of diabetic retinopathy (DR) and diabetic macular edema (DME). Upregulation of cytokines and other proinflammatory mediators leading to persistent low-grade inflammation is believed to actively contribute to the DR-associated damage to the retinal vasculature, inducing breakdown of the blood-retinal barrier, subsequent macular edema formation, and promotion of retinal neovascularization. This review summarizes the current knowledge of the biological processes providing an inflammatory basis for DR and DME. In addition, emerging therapeutic approaches targeting inflammation are discussed, including blockade of angiopoietin 2 and other molecular targets such as interleukin (IL)-6, IL-1ß, plasma kallikrein, and integrins.
Assuntos
Anti-Inflamatórios/uso terapêutico , Barreira Hematorretiniana , Retinopatia Diabética , Edema Macular , Vasos Retinianos , Angiopoietina-2/antagonistas & inibidores , Angiopoietina-2/metabolismo , Animais , Barreira Hematorretiniana/metabolismo , Barreira Hematorretiniana/patologia , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/metabolismo , Interleucina-6/antagonistas & inibidores , Interleucina-6/metabolismo , Edema Macular/tratamento farmacológico , Edema Macular/metabolismo , Edema Macular/patologia , Vasos Retinianos/metabolismo , Vasos Retinianos/patologiaRESUMO
Human tumorigenesis resembles embryogenesis by aberrant activation of several developmental pathways including Wnt/ß-catenin signaling. Norrin is an atypical ligand for Frizzled receptor that is preferentially expressed in the endothelium to promote retinal vascularization during development. However, its expression pattern and potential roles in human cancers remain unclear. Here we report that Norrin expression is elevated in the parenchymal cells, but not endothelial cells, in gastric cancer (GC). Moreover, Norrin is required for growth and invasion of GC cells and its expression status is associated with unfavorable outcomes. However, analysis of the TGCA database demonstrates that Norrin expression status is not correlated with key target genes of Wnt/ß-catenin signaling. Among several signaling pathways hyperactivated in cancer, Norrin-depleted GC cells also display down-regulated AKT signaling except the canonical Wnt/ß-catenin signaling. Consistently, small molecule-induced cytosolic activation of AKT partially rescues the proliferative and invasive capability of Norrin-depleted cells. Together, these findings suggest a novel role of Norrin in gastric tumorigenesis that could be exploited for adjuvant therapy against the deadly malignancy.
Assuntos
Proteínas do Olho/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Gástricas/metabolismo , Adulto , Idoso , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Via de Sinalização WntRESUMO
Several studies have shown that RNAi-mediated depletion of splicing factors (SFs) results in mitotic abnormalities. However, it is currently unclear whether these abnormalities reflect defective splicing of specific pre-mRNAs or a direct role of the SFs in mitosis. Here, we show that two highly conserved SFs, Sf3A2 and Prp31, are required for chromosome segregation in both Drosophila and human cells. Injections of anti-Sf3A2 and anti-Prp31 antibodies into Drosophila embryos disrupt mitotic division within 1 min, arguing strongly against a splicing-related mitotic function of these factors. We demonstrate that both SFs bind spindle microtubules (MTs) and the Ndc80 complex, which in Sf3A2- and Prp31-depleted cells is not tightly associated with the kinetochores; in HeLa cells the Ndc80/HEC1-SF interaction is restricted to the M phase. These results indicate that Sf3A2 and Prp31 directly regulate interactions among kinetochores, spindle microtubules and the Ndc80 complex in both Drosophila and human cells.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas do Olho/genética , Mitose , Proteínas Nucleares/genética , Fatores de Processamento de RNA/genética , Animais , Anticorpos Neutralizantes/farmacologia , Segregação de Cromossomos/efeitos dos fármacos , Sequência Conservada , Proteínas do Citoesqueleto , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Cinetocoros/efeitos dos fármacos , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Mitose/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Ligação Proteica , Fatores de Processamento de RNA/antagonistas & inibidores , Fatores de Processamento de RNA/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Fuso Acromático/ultraestruturaRESUMO
The assembly of new herpes simplex virus 1 (HSV-1) particles takes place in the nucleus. These particles then travel across the two nuclear membranes and acquire a final envelope from a cellular compartment. The contribution of the cell to the release of the virus is, however, little known. We previously demonstrated, using a synchronized infection, that the host protein kinase D and diacylglycerol, a lipid that recruits the kinase to the trans-Golgi network (TGN), promote the release of the virus from that compartment. Given the role this cellular protein plays in the herpes simplex virus 1 life cycle and the many molecules that modulate its activity, we aimed to determine to what extent this virus utilizes the protein kinase D pathway during a nonsynchronized infection. Several molecular protein kinase D (PKD) regulators were targeted by RNA interference and viral production monitored. Surprisingly, many of these modulators negatively impacted the extracellular release of the virus. Overexpression studies, the use of pharmacological reagents, and assays to monitor intracellular lipids implicated in the biology of PKD suggested that these effects were oddly independent of total intracellular diacylglycerol levels. Instead, mapping of the viral intermediates by electron microscopy suggested that some of these modulators could regulate distinct steps along the viral egress pathway, notably nuclear egress. Altogether, this suggests a more complex contribution of PKD to HSV-1 egress than originally anticipated and new research avenues to explore.IMPORTANCE Viruses are obligatory parasites that highjack numerous cellular functions. This is certainly true when it comes to transporting viral particles within the cell. Herpesviruses share the unique property of traveling through the two nuclear membranes by subsequent budding and fusion and acquiring their final envelope from a cellular organelle. Albeit disputed, the overall evidence from many laboratories points to the trans-Golgi network (TGN) as the source of that membrane. Moreover, past findings revealed that the host protein kinase D (PKD) plays an important role at that stage, which is significant given the known implication of that protein in vesicular transport. The present findings suggest that the PKD machinery not only affects the late stages of herpes simplex virus I egress but also modulates earlier steps, such as nuclear egress. This opens up new means to control these viruses.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Olho/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Proteínas de Membrana/metabolismo , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Liberação de Vírus , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transporte Vesicular/antagonistas & inibidores , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Herpes Simples/genética , Herpes Simples/metabolismo , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Células Tumorais Cultivadas , Células Vero , Rede trans-GolgiRESUMO
BACKGROUD/AIMS: Abnormal activation of the Wnt/ß-catenin signaling, which may be antagonized by the members of secreted frizzled-related proteins family (SFRPs), is implicated in tumor occurrence and development. However, the function of SFRP5 relating to Wnt/ß-catenin pathway in chondrosarcoma is not clear yet. This study was undertaken to investigate the potential role of SFRP5 promoter methylation in chondrosarcoma metastasis and invasion through activating canonical Wnt signaling pathway. METHODS AND RESULTS: The results demonstrated that SFRP5 promoter was hypermethylated and SFRP5 expression was significantly reduced in chondrosarcoma cell lines at the mRNA and protein levels. The canonical Wnt/ß-catenin signaling was observably activated with ß-catenin stabilization by dephosphorylation and translocation into the nuclear. 5-Aza-2'-deoxycytidine (5-Aza-dC), the DNA methyltransferase inhibitor, significantly inhibited the proliferation of chondrosarcoma cells by cell cycle arrest through repressing the methylation of SFRP5 and promoting its expression. Both 5-Aza-dC treatment and SFRP5 overexpression could significantly inhibited the metastasis and invasion of chondrosarcoma cells by inactivating Wnt/ß-catenin signaling pathway and promoting chondrosarcoma cells mesenchymal-epithelial transition (MET). 5-Aza-dC also inhibited the xenograft growth and lung metastasis of chondrosarcoma cells in vivo via suppressing SFRP5 promotor methylation, inactivating Wnt/ß-catenin pathway and inducing epithelial markers expression. CONCLUSION: All of our results revealed the epigenetic silencing of SFRP5 by promoter methylation plays pivotal roles in chondrosarcoma development and metastasis through SFRP5/Wnt/ß-catenin signaling axis. Modulation of their levels may serve as potential targets and diagnostic tools for novel therapeutic strategies of chondrosarcoma.
Assuntos
Condrossarcoma/genética , Condrossarcoma/patologia , Epigênese Genética/genética , Proteínas do Olho/genética , Proteínas de Membrana/genética , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Via de Sinalização Wnt/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antimetabólitos Antineoplásicos/química , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Azacitidina/química , Azacitidina/farmacologia , Células Cultivadas , Condrossarcoma/tratamento farmacológico , Condrossarcoma/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Epigênese Genética/efeitos dos fármacos , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/metabolismo , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Relação Estrutura-Atividade , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Corneal neovascularization is a sight-threatening condition caused by angiogenesis in the normally avascular cornea. Neovascularization of the cornea is often associated with an inflammatory response, thus targeting VEGF-A alone yields only a limited efficacy. The NF-κB signaling pathway plays important roles in inflammation and angiogenesis. Here, we study consequences of the inhibition of NF-κB activation through selective blockade of the IKK complex IκB kinase ß (IKK2) using the compound IMD0354, focusing on the effects of inflammation and pathological angiogenesis in the cornea. In vitro, IMD0354 treatment diminished HUVEC migration and tube formation without an increase in cell death and arrested rat aortic ring sprouting. In HUVEC, the IMD0354 treatment caused a dose-dependent reduction in VEGF-A expression, suppressed TNFα-stimulated expression of chemokines CCL2 and CXCL5, and diminished actin filament fibers and cell filopodia formation. In developing zebrafish embryos, IMD0354 treatment reduced expression of Vegf-a and disrupted retinal angiogenesis. In inflammation-induced angiogenesis in the rat cornea, systemic selective IKK2 inhibition decreased inflammatory cell invasion, suppressed CCL2, CXCL5, Cxcr2, and TNF-α expression and exhibited anti-angiogenic effects such as reduced limbal vessel dilation, reduced VEGF-A expression and reduced angiogenic sprouting, without noticeable toxic effect. In summary, targeting NF-κB by selective IKK2 inhibition dampened the inflammatory and angiogenic responses in vivo by modulating the endothelial cell expression profile and motility, thus indicating an important role of NF-κB signaling in the development of pathologic corneal neovascularization.
Assuntos
Benzamidas/farmacologia , Córnea/metabolismo , Neovascularização da Córnea/tratamento farmacológico , Proteínas do Olho/antagonistas & inibidores , Quinase I-kappa B/antagonistas & inibidores , Ceratite/tratamento farmacológico , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Córnea/patologia , Neovascularização da Córnea/genética , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Ceratite/genética , Ceratite/metabolismo , Ceratite/patologia , Masculino , NF-kappa B/genética , Ratos , Ratos Wistar , Transdução de Sinais/genética , Peixe-ZebraRESUMO
Investigating stimulation of endogenous wound healing in corneal endothelial cells (CECs) may help address the global shortage of donor corneas by decreasing the number of transplants performed for blindness because of endothelial dysfunction. We previously reported that IL-1ß stimulation leads to fibroblast growth factor (FGF2) expression, enhancing migration and proliferation of mammalian CECs. However, FGF2 also promotes the endothelial-mesenchymal transition, which can lead to retrocorneal membrane formation and blindness. This prompted us to investigate downstream FGF2 signaling targets that could be manipulated to prevent retrocorneal membrane formation. FGF2 stimulation altered cell morphology and induced expression of mesenchymal transition marker genes such as snail family transcriptional repressor 1 (SNAI1), SNAI2, zinc finger E-box-binding homeobox 1 (ZEB1), and ZEB2 This, in turn, induced expression of fibronectin, vimentin, and type I collagen, and suppressed E-cadherin in CECs in vitro and ex vivo siRNA-mediated SNAI1 knockdown revealed that SNAI1 induces ZEB1 expression, in turn inducing expression of type I collagen, the major component of retrocorneal membranes, and of cyclin-dependent kinase 2 (CDK2) and cyclin E1, promoting cell proliferation. siRNA-mediated knockdown of SNAI1 or ZEB1, but not of CDK2, inhibited FGF2-dependent expression of fibronectin, vimentin, and type I collagen and of suppression of E-cadherin expression. We conclude that SNAI1 is a key regulator of FGF2-dependent mesenchymal transition in human ex vivo corneal endothelium, with ZEB1 regulating type I collagen expression and CDK2 regulating cell proliferation. These results suggest that SNAI1 promotes fibrosis and cell proliferation in human corneal endothelium through ZEB1 and CDK2.
Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Endotélio Corneano/metabolismo , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/agonistas , Fatores de Transcrição da Família Snail/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Biomarcadores/metabolismo , Movimento Celular , Proliferação de Células , Forma Celular , Transdiferenciação Celular , Células Cultivadas , Colágeno Tipo I/agonistas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/genética , Endotélio Corneano/citologia , Endotélio Corneano/patologia , Ativação Enzimática , Proteínas do Olho/agonistas , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Humanos , Interferência de RNA , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição da Família Snail/antagonistas & inibidores , Fatores de Transcrição da Família Snail/genética , Cicatrização , Homeobox 2 de Ligação a E-box com Dedos de Zinco/agonistas , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/agonistas , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Background/ Aims: This study was performed to reveal signaling pathways exploited by pigment epithelium-derived factor (PEDF) derived from retinal (glial) Müller cells to protect retinal ganglion cells (RGCs) from cell death. METHODS: The survival of RGCs was determined in the presence of conditioned culture media (MCM) from or in co-cultures with Müller cells. The significance of PEDF-induced STAT3 activation was evaluated in viability assays and using Western blotting analyses and siRNA-transfected cells. RESULTS: Secreted mediators of Müller cells increased survival of RGCs under normoxia or hypoxia to a similar degree as of PEDF- or IL-6-exposed cells. PEDF and MCM induced an increased STAT3 activation in RGCs and R28 cells, and neutralization of PEDF in MCM attenuated STAT3 activation. Inhibition of STAT3 reduced PEDF-promoted survival of RGCs. Similar to IL-6, PEDF induced STAT3 activation, acting in a dose-dependent manner via the PEDF receptor (PEDF-R) encoded by the PNPLA2 gene. Ablation of PEDF-R attenuated MCM-induced STAT3 activation and compromised the viability of PEDF-exposed R28 cells. CONCLUSIONS: Müller cells are an important source of PEDF, which promotes RGC survival through STAT3 activation and, at least in part, via PEDF-R. Enhancing the secretory function of Müller cells may be useful to promote RGC survival in retinal neurodegenerative diseases.
Assuntos
Células Ependimogliais/metabolismo , Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/metabolismo , Fator de Transcrição STAT3/metabolismo , Serpinas/metabolismo , Animais , Hipóxia Celular , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Óxidos S-Cíclicos/farmacologia , Células Ependimogliais/citologia , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Proteínas do Olho/farmacologia , Interleucina-6/farmacologia , Lipase/antagonistas & inibidores , Lipase/genética , Lipase/metabolismo , Camundongos , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/farmacologia , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Receptores de Neuropeptídeos/metabolismo , Células Ganglionares da Retina/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Serpinas/genética , Serpinas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
The cornea is densely innervated to sustain the integrity of the ocular surface. Corneal nerve damage produced by aging, diabetes, refractive surgeries, and viral or bacterial infections impairs tear production, the blinking reflex, and epithelial wound healing, resulting in loss of transparency and vision. A combination of the known neuroprotective molecule, pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA), has been shown to stimulate corneal nerve regeneration, but the mechanisms involved are unclear. Here, we sought to define the molecular events of this effect in an in vivo mouse injury model. We first confirmed that PEDF + DHA increased nerve regeneration in the mouse cornea. Treatment with PEDF activates the phospholipase A2 activity of the PEDF-receptor (PEDF-R) leading to the release of DHA; this free DHA led to enhanced docosanoid synthesis and induction of bdnf, ngf, and the axon growth promoter semaphorin 7a (sema7a), and as a consequence, their products appeared in the mouse tears. Surprisingly, corneal injury and treatment with PEDF + DHA induced transcription of neuropeptide y (npy), small proline-rich protein 1a (sprr1a), and vasoactive intestinal peptide (vip) in the trigeminal ganglia (TG). The PEDF-R inhibitor, atglistatin, blocked all of these changes in the cornea and TG. In conclusion, we uncovered here an active cornea-TG axis, driven by PEDF-R activation, that fosters axon outgrowth in the cornea.
Assuntos
Córnea/inervação , Ácidos Docosa-Hexaenoicos/uso terapêutico , Proteínas do Olho/uso terapêutico , Modelos Neurológicos , Fatores de Crescimento Neural/uso terapêutico , Regeneração Nervosa/efeitos dos fármacos , Receptores de Neuropeptídeos/agonistas , Serpinas/uso terapêutico , Nervo Trigêmeo/efeitos dos fármacos , Administração Oftálmica , Animais , Córnea/efeitos dos fármacos , Córnea/patologia , Córnea/fisiologia , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácidos Docosa-Hexaenoicos/metabolismo , Quimioterapia Combinada , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Proteínas do Olho/administração & dosagem , Proteínas do Olho/agonistas , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas do Olho/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Injeções Intraperitoneais , Masculino , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/administração & dosagem , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Técnicas de Cultura de Órgãos , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/farmacologia , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/metabolismo , Serpinas/administração & dosagem , Serpinas/farmacologia , Gânglio Trigeminal/efeitos dos fármacos , Gânglio Trigeminal/patologia , Gânglio Trigeminal/fisiologia , Nervo Trigêmeo/patologia , Nervo Trigêmeo/fisiologia , Traumatismos do Nervo Trigêmeo/tratamento farmacológicoRESUMO
The interface between the neural retina and the retinal pigment epithelium (RPE) is critical for several processes, including visual pigment regeneration and retinal attachment to the RPE. One of its most important functions is the exchange of metabolites between the photoreceptors and RPE because photoreceptor cells have very high energy demands, largely satisfied by oxidative metabolism. The riboflavin (RF) cofactors, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), are two key cofactors involved in oxidative metabolism. We have previously shown that retbindin is a photoreceptor-specific RF-binding protein exclusively expressed in the rods and present in the interphotoreceptor matrix at the interface between the RPE and photoreceptor outer segments. Here, we show that retbindin ablation in mice causes a retinal phenotype characterized by time- and dose-dependent declines in rod and cone photoreceptor functions as early as 120 days of age. Whereas minor retinal ultrastructural defects were observed at all ages examined, a significant decline occurred in photoreceptor nuclei at 240 days of age (â¼36.8% rods and â¼19.9% cones). Interestingly, significant reductions in FAD and FMN levels were observed before the onset of degeneration (â¼46.1% FAD and â¼45% FMN). These findings suggest that the reduced levels of these flavins result in the disruption of intracellular mechanisms, leading to photoreceptor cell death. Altogether, our results suggest that retbindin is a key player in the acquisition and retention of flavins in the neural retina, warranting future investigation into retbindin's role in photoreceptor cell death in models of retinal degenerative disorders.
Assuntos
Proteínas do Olho/metabolismo , Flavinas/metabolismo , Degeneração Retiniana/etiologia , Animais , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Retina/metabolismo , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologiaRESUMO
OBJECTIVE: To seek efficient aldose reductase inhibitors (ARIs) with excellent in vitro and in vivo biological activities against rat galactosemic cataract. METHODS: The method was firstly optimized to screen strong ARIs from nonoriented synthetic compounds and natural extracts. Then, diosgenin was assessed on osmotic expansion of primarily cultured lens epithelial cells (LECs) induced by galactose (50 mM). Diosgenin was administered to galactosemic rats by oral (100 and 200 mg/kg) or direct drinking (0.1%) to evaluate its anticataract effects. RESULTS: Diosgenin was found as the strongest ARI with IC50 of 4.59 × 10-6 mol/L. Diosgenin (10 µM) evidently inhibited the formation of tiny vacuoles and upregulation of AR mRNA in LECs. In vivo, diosgenin delayed lens opacification, inhibited the increase of ratio of lens weight to body weight, and decreased AR activity, galactitol level, and AR mRNA expression, especially in the diosgenin drinking (0.1%) group. CONCLUSIONS: Diosgenin was an efficient ARI, which not only significantly decreased the LECs' osmotic expansion in vitro but also markedly delayed progression of rat galactosemic cataract in vivo. Thus, diosgenin rich food can be recommended to diabetic subjects as dietary management to postpone the occurrence of sugar cataract, and diosgenin deserves further investigation for chronic diabetic complications.
Assuntos
Aldeído Redutase/antagonistas & inibidores , Catarata/prevenção & controle , Suplementos Nutricionais , Diosgenina/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Proteínas do Olho/antagonistas & inibidores , Cristalino/metabolismo , Aldeído Redutase/genética , Aldeído Redutase/isolamento & purificação , Aldeído Redutase/metabolismo , Animais , Animais Endogâmicos , Catarata/etiologia , Catarata/metabolismo , Catarata/patologia , Tamanho Celular , Sobrevivência Celular , Células Cultivadas , Dieta da Carga de Carboidratos/efeitos adversos , Diosgenina/administração & dosagem , Diosgenina/metabolismo , Cães , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/isolamento & purificação , Proteínas do Olho/metabolismo , Galactitol/metabolismo , Galactose/efeitos adversos , Regulação Enzimológica da Expressão Gênica , Cristalino/citologia , Cristalino/patologia , Masculino , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos Sprague-Dawley , Vacúolos/patologiaRESUMO
Traditional structure and ligand based virtual screening approaches rely on the availability of structural and ligand binding information. To overcome this limitation, hybrid approaches were developed that relied on extraction of ligand binding information from proteins sharing similar folds and hence, evolutionarily relationship. However, they cannot target a chosen pocket in a protein. To address this, a pocket centric virtual ligand screening approach is required. Here, we employ a new, iterative implementation of a pocket and ligand-similarity based approach to virtual ligand screening to predict small molecule binders for the olfactomedin domain of human myocilin implicated in glaucoma. Small-molecule binders of the protein might prevent the aggregation of the protein, commonly seen during glaucoma. First round experimental assessment of the predictions using differential scanning fluorimetry with myoc-OLF yielded 7 hits with a success rate of 12.7%; the best hit had an apparent dissociation constant of 99nM. By matching to the key functional groups of the best ligand that were likely involved in binding, the affinity of the best hit was improved by almost 10,000 fold from the high nanomolar to the low picomolar range. Thus, this study provides preliminary validation of the methodology on a medically important glaucoma associated protein.