RESUMO
Colorectal cancer (CRC) is a common gastrointestinal tumour with increasing incidence worldwide. However, the underlying molecular mechanism of CRC proliferation is not completely clear. Diversinï¼as an ankyrin repeat-containing protein, is upregulated in various solid tumours and accelerates cancer progression by promoting cell proliferation and increasing S phase fraction of cells. In this study, 71 CRC samples and corresponding adjacent tissue samples were included. The expression of diversin in tissues was verified via immunohistochemical analysis. The MTS assay and flow cytometry (FCM) was used to measure cell proliferation and cell cycle. Results of immunohistochemical analysis revealed that diversin was highly expressed in human CRC tissues and was significantly associated with tumour differentiation, clinical stage and lymph node metastasis. The analysis based on the CRC data from The Cancer Genome Atlas (TCGA) database showed that a high expression of diversin correlated with the poor prognosis of CRC. Results of the MTS assay indicated that the overexpression of diversin promoted the proliferation of CRC cells, while its downregulation had an inhibitory effect on CRC cell proliferation. FCM analysises presented that diversin increased the flux of the CRC cell cycle from G1 to S and regulated cycle-related proteins, namely, P21, P27, cyclin E, CDK2, cyclin D and CDK4. The results suggest that diversin contributes to CRC proliferation that involves the distribution of the cell cycle. In CRC tissues, the expression of diversin has closely related to the prognosis. The higher the expression levels of diversin, the worse the prognosis. In vitro, diversin could increase the proliferative ability of CRC cells through the G1-S checkpoint and JNK signalling pathway, confirming that diversin contributes to CRC development.
Assuntos
Proteínas de Ciclo Celular , Neoplasias Colorretais , Proteínas do Citoesqueleto , Humanos , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
The coordinated polarization of neighboring cells within the plane of the tissue, known as planar cell polarity (PCP), is a recurring theme in biology. It is required for numerous developmental processes for the form and function of many tissues and organs across species. The genetic pathway regulating PCP was first discovered in Drosophila, and an analogous but distinct pathway is emerging in vertebrates. It consists of membrane protein complexes known as core PCP proteins that are conserved across species. Here we report that the over-expression of the murine Ankrd6 (mAnkrd6) gene that shares homology with Drosophila core PCP gene diego causes a typical PCP phenotype in Drosophila, and mAnkrd6 can rescue the loss of function of diego in Drosophila. In mice, mAnkrd6 protein is asymmetrically localized in cells of the inner ear sensory organs, characteristic of components of conserved core PCP complexes. The loss of mAnkrd6 causes PCP defects in the inner ear sensory organs. Moreover, canonical Wnt signaling is significantly increased in mouse embryonic fibroblasts from mAnkrd6 knockout mice in comparison to wild type controls. Together, these results indicated that mAnkrd6 is a functional homolog of the Drosophila diego gene for mammalian PCP regulation and act to suppress canonical Wnt signaling.
Assuntos
Padronização Corporal/fisiologia , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Orelha Interna/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Padronização Corporal/genética , Proteínas de Transporte/genética , Polaridade Celular/genética , Polaridade Celular/fisiologia , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas de Drosophila/genética , Orelha Interna/citologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Olho/citologia , Olho/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/metabolismo , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos Knockout , Microscopia Confocal , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Asas de Animais/citologia , Asas de Animais/metabolismo , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologiaRESUMO
BACKGROUND: Planar cell polarity (PCP) is a major branch of Wnt signaling that controls the process of convergent extension in gastrulation and neurulation. PCP defects were associated with neural tube defects (NTDs) that are the most common central nervous system anomalies. PCP signaling is highly dosage sensitive and exhibits an antagonistic relationship with the canonical Wnt/ß-catenin pathway. Diversin, encoded by Ankrd6, is an ankyrin repeat protein that activates the non canonical PCP signaling and simultaneously inhibits the canonical pathway. METHODS: In this study, we analyzed this dual role of ANKRD6 in NTDs. We sequenced its coding region in 473 NTD patients and 150 controls, and we validated the effect of the identified variants on Wnt signaling using reporter assays in mammalian cells. RESULTS: We identified four rare missense mutations in 0.8% of the NTD patients and two rare missense mutations in 1.3% of the controls. Notably, when all six mutations were validated, only two mutations identified in NTD patients, p.Pro548Leu, p.Arg632His, significantly altered DIVERSIN activity in Wnt signaling assays in a hypomorphic manner. CONCLUSION: Rare missense mutations in ANKRD6 could affect a balanced reciprocal antagonism between both Wnt pathways in neurulation and act as predisposing factors to NTDs in a subset of patients.
Assuntos
Polaridade Celular , Proteínas do Citoesqueleto/genética , Mutação de Sentido Incorreto/genética , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/patologia , Via de Sinalização Wnt , Estudos de Casos e Controles , Pré-Escolar , Proteínas do Citoesqueleto/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Masculino , Defeitos do Tubo Neural/metabolismo , Mapas de Interação de Proteínas , Transdução de Sinais , Técnicas do Sistema de Duplo-HíbridoRESUMO
In the present study the binding of diversin (DIV), a prenylated coumarin isolated from Ferula diversivittata, to bovine serum albumin (BSA) was investigated using surface plasmon resonance (SPR), spectrofluorimetry, and molecular docking approaches. Following the activation of carboxylic groups, via NHS/EDC, BSA was immobilized on the carboxymethyl dextran (CMD) hydrogel coated Au sensor, and was used for real-time monitoring of the interactions between DIV and BSA. KD value of DIV binding to BSA increased with increasing temperature, confirmed that the affinity between BSA and DIV decreases with rising temperature. In addition, the fluorescence and synchronous fluorescence spectroscopic data revealed that the intrinsic emission intensity of BSA was quenched via a dynamic mechanism. In addition, the micro-region around BSA tyrosine residue was changed upon interaction with DIV. The thermodynamic parameter findings suggested that the hydrophobic interactions were dominant in the binding and formation of the BSA and DIV complex. The molecular docking outputs indicated that there is only one binding site on BSA for DIV, in agreement with experimental data, and DIV bind BSA in subdomain IB.
Assuntos
Cumarínicos/química , Cumarínicos/metabolismo , Ferula/metabolismo , Fluorescência , Simulação de Acoplamento Molecular , Monoterpenos/química , Monoterpenos/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Ressonância de Plasmônio de Superfície , Animais , Sítios de Ligação , Bovinos , Interações Hidrofóbicas e Hidrofílicas , Ligação Proteica , Espectrometria de Fluorescência , TermodinâmicaRESUMO
The expression and significance of Diversin in human tumors remains unclear. We found that Diversin was overexpressed in NSCLC, and exhibited direct correlation to poor differentiation, advanced TNM stage, lymph node metastasis and survival time. Overexpression of Diversin lead to a significant increase in proliferation and invasion of NSCLC cells, possibly through activation of JNK, cyclin B and MMP9, and the effects were blocked by JNK inhibitor. These results suggest Diversin is overexpressed in NSCLC and predict poor prognosis. Diversin may promote cell proliferation and invasion through JNK pathway.