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1.
Gene ; 730: 144299, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-31881249

RESUMO

The function and mechanism of RNA editing proteins have been extensively studied, but its association with cellular processes and signaling pathways remained unaddressed. Here, we explored the function of RNA editing complementary protein- Apobec-1 Complementation Factor (A1CF) in the proliferation and colony formation of renal cell carcinoma (RCC) cells. Decreased A1CF expression inhibits the proliferation and colony formation of 786-O cells; and further signaling pathway screening demonstrated that A1CF increases ERK activation and DKK1 expression. Moreover, knockdown of DKK1 has similar phenotypes with A1CF deficiency in 786-O cells on cell proliferation and colony formation and ERK activation. Decreasing of DKK1 expression reduces the phosphorylation of ERK1/2 and MEK1/2 increased by A1CF overexpression; further, inhibiting of the phosphorylation of MEK1/2 by U0126 also decreases the ERK activation upregulated by A1CF overexpression. Deficiency of DKK1 or U0126 treatment suppresses the cell proliferation promoted by A1CF overexpression in 786-O cells; furthermore, U0126 treatment inhibits DKK1-increased cell proliferation in 786-O cells. Our results reveal that DKK1 mediates A1CF to activate ERK in promotion renal carcinoma cell proliferation and colony formation. For the important function of ERK signaling pathway in tumor metastasis and key position of DKK1 in Wnt signaling pathway, we associate RNA editing protein-A1CF with multiple cellular processes and signaling pathways through DKK1, and the key node of A1CF-DKK1-MEK/ERK axis is a potential targeting site for RCC therapy.

2.
Gene ; 725: 144159, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31629818

RESUMO

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide due to its frequent metastasis, tumor recurrence, and lack of curative treatment. However, the underlying molecular mechanisms involved in HCC progression remain unclear. Here, we analyzed the global gene expression of spontaneous liver tumor tissue from CBA/CaJ mice by RNA-Seq and identified 10,706 and 10,374 genes in the normal and liver tumor groups, respectively. Only 9793 genes were expressed in both, 913 genes were identified in only the liver tumor group, and 581 genes were found in normal liver tissues. There were 2054 differentially expressed genes (DEGs), with 975 down-regulated genes and 1079 up-regulated genes. Gene ontology (GO) term enrichment analysis showed that 43 up-regulated genes were significantly associated with cell cycle regulation and hundreds of up-regulated genes were related to cell migration, adhesion, or metabolic processes. KEGG pathway enrichment also demonstrated that some DEGs were tightly associated with the cell cycle, extracellular matrix (ECM)-receptor interactions, as well as protein digestion and absorption pathways, indicating that the activation of these oncogenic cascades was closely related to tumor liver progression in CBA/CaJ mice. Ninety-three genes with elevated expression levels preferentially localized in microtubules, kinetochores, and spindles play an important role during mitosis and meiosis and are associated with the reorganization of the cytoskeleton in cancer cells during migration and invasion. Some ECM-related genes were significantly different in the tumor group, including collagen types I, III, IV, V, and VI, non-collagenous glycoproteins, laminin, and fibronectin. We further validated the functions of upregulated genes, such as cyclin-dependent kinase 1 (CDK1) and polo-like kinase 1 (PLK1), with regards to cell cycle regulation, apoptosis, and proliferation in normal human liver or liver tumor-derived cell lines. Our results indicated that the cell cycle dysregulation, ECM-receptor interaction, and cytoskeleton-associated genes in mouse livers may promote HCC progression and deciphering the function of the genes will help investigators understand the underlying molecular mechanism of HCC.


Assuntos
Neoplasias Hepáticas Experimentais/genética , Animais , Apoptose/genética , Proteína Quinase CDC2/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos CBA , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Transcrição Genética , Transcriptoma
3.
Biomed Eng Online ; 18(1): 97, 2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578149

RESUMO

BACKGROUND: Silk fibroin hydrogel, derived from Bombyx mori cocoons, has been shown to have potential effects on wound healing due to its excellent biocompatibility and less immunogenic and biodegradable properties. Many studies suggest silk fibroin as a promising material of wound dressing and it can support the adhesion and proliferation of a variety of human cells in vitro. However, lack of translational evidence has hampered its clinical applications for skin repair. Herein, a heparin-immobilized fibroin hydrogel was fabricated to deliver FGF1 (human acidic fibroblast growth factor 1) on top of wound in rats with full-thickness skin excision by performing comprehensive preclinical studies to fully evaluate its safety and effectiveness. The wound-healing efficiency of developed fibroin hydrogels was evaluated in full-thickness wound model of rats, compared with the chitosan used clinically. RESULTS: The water absorption, swelling ratio, accumulative FGF1 releasing rate and biodegradation ratio of fabricated hydrogels were measured. The regenerated fibroin hydrogels with good water uptake properties rapidly swelled to a 17.3-fold maximum swelling behavior over 12 h and a total amount of 40.48 ± 1.28% hydrogels was lost within 15 days. Furthermore, accumulative releasing data suggested that heparinized hydrogels possessed effective release behavior of FGF1. Then full-thickness skin excision was created in rats and left untreated or covered with heparinized fibroin hydrogels-immobilized recombinant human FGF1. The histological evaluation using hematoxylin and eosin (HE) and Masson's trichrome (MT) staining was performed to observe the dermic formation and collagen deposition on the wound-healing site. To evaluate the wound-healing mechanisms induced by fibroin hydrogel treatment, wound-healing scratch and cell proliferation assay were performed. it was found that both fibroin hydrogels and FGF1 can facilitate the migration of fibroblast L929 cells proliferation and migration. CONCLUSION: This study provides systematic preclinical evidence that the silk fibroin promotes wound healing as a wound-healing dressing, thereby establishing a foundation toward its further application for new treatment options of wound repair and regeneration.

4.
Gene ; 695: 42-50, 2019 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-30763669

RESUMO

Glucocorticoid hormones have been widely used in clinical practice as potent anti-inflammatory and immunosuppressive agents. However, the underlying mechanisms of how they work remain unaddressed. Here, we used RNA-set to profile spleen gene expression in adult mice after consistent intraperitoneal injection of dexamethasone. We identified 13565 genes in control (injected with 0.9% NaCl) and 13702 genes in dexamethasone-injected group, 12920 genes were expressed in both, but 645 genes were identified only in control and 782 genes were identified only in the dexamethasone group. In the dexamethasone-injected group 101 and 67 genes were down-and up-regulated, respectively. Among these, 129 were coding genes, 19 were identified as non-coding genes or pseudogenes, and the remaining 20 were TEC (to be experimentally confirmed) genes. Gene ontology (GO) and KEGG pathway analysis revealed that the cytokine-cytokine receptor interaction pathway was highly enriched in these differentially expressed genes, and that a majority of the 129 identified coding genes were involved in immune system and cell adhesion-associated processes. Moreover, systemic lupus erythematosus, renin-angiotensin system, fat digestion and absorption, and glycerolipid metabolism pathways were significantly affected in the dexamethasone-treatment group. No obvious signaling pathway was enriched in the control group. Additionally, 20 immunoglobulin heavy or light chain variable region genes (IGH(L)Vs) were down regulated in the dexamethasone-injected group. IGH(L)Vs encode the variable region of immunoglobulin heavy chain and determine the diversity and specificity of antibodies. We were unable to determine the function of the 19 non-coding genes with differential expression following dexamethasone treatment. Our findings indicate that the expression of IGH(L)Vs and non-coding genes play an important role in the anti-inflammatory and immunosuppressive effects of dexamethasone and could be developed as potential agents in clinical practice.


Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Baço/efeitos dos fármacos , Animais , Dexametasona/administração & dosagem , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Camundongos , RNA Longo não Codificante/genética , RNA Longo não Codificante/imunologia , Baço/imunologia , Baço/metabolismo
5.
Drug Dev Res ; 80(2): 209-217, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30379338

RESUMO

Hepatocellular carcinoma (HCC) accounts for majority of cancer related deaths. Two major risk factors in induction of HCC are chemical and virus, however, the possible mechanisms of their differences remain indefinable. The current study focused on protective role of Fucoxanthin (Fx) in liver affected by diethylnitrosamine (DEN)-induced HCC. In this study, levels of liver enzymes, oxidative stressors, antioxidant status, and lipoproteins were compared both in tissue and blood. Tissues were also analyzed extensively by histological studies using H and E staining and transmission electron microscopy (TEM). Rats were clustered into four groups of six experimental animals. Group I: Control rats were administered isotonic saline intraperitoneal Group II: Animals received 0.01% DEN through drinking water to induce hepatocellular carcinoma. Group III: Animals received 0.01% DEN simultaneously oral supplementation of Fx (50 mg/kg b.w). Group IV: Rats were given Fx alone (50 mg/kg b.w) orally and the treatment is for 15 weeks. Results showed the decrease in body weight, serum albumin, antioxidant enzymes, and increased all the liver enzymes, serum bilirubin, and stress markers in DEN induced rats, where as the simultaneous supplementation of Fx reverted them to normal levels. Administration of only Fx did not show any change. Therefore, Fx may serve as a chemotherapeutic agent against liver cancer.


Assuntos
Antineoplásicos/uso terapêutico , Antioxidantes/uso terapêutico , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Xantofilas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Dietilnitrosamina , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Ratos Wistar , Xantofilas/farmacologia
6.
Free Radic Biol Med ; 129: 268-278, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30248444

RESUMO

Copper oxide nanoparticles (CuONPs) have been widely used in the industrial and pharmaceutical fields; however, their toxicity profile is deeply concerning. Currently, nanomaterials-induced toxicity in the cardiovascular system is receiving increased attention. Our previous toxicological study found that lysosomal deposition of CuONPs triggered vascular endothelial cell death, indicating that the involvement of autophagic dysfunction was crucial for CuONPs-induced toxicity in human umbilical vein endothelial cells (HUVECs). In the current study, we investigated the detailed mechanism underlying the autophagic dysfunction induced by CuONPs. We demonstrated that CuONPs exposure caused accumulation of superoxide anions, which likely resulted from mitochondrial dysfunctions. MnTBAP, a superoxide anions scavenger, alleviated CuONPs-induced HUVECs death, indicating that excessive superoxide anions were directly related to the CuONPs cytotoxicity in HUVECs. Interestingly, we found that mitophagy (a protective mechanism for clearance of damaged mitochondria and excessive superoxide anions) was initiated but failed to be cleared in CuONPs-treated cells, resulting in the accumulation of damaged mitochondria. Inhibition of mitophagy through Atg5 knockout or blocking of mitochondria fission with Mdivi-1 significantly aggravated CuONPs-induced superoxide anions accumulation and cell death, suggesting that mitophagy is a protective mechanism against CuONPs cytotoxicity in HUVECs. In summary, we demonstrate that superoxide anions (originating from damaged mitochondria) are involved in CuONPs-associated toxicity and that impaired mitophagic flux aggravates the accumulation of excessive superoxide anions, which leads to HUVECs death. Our findings indicate that there are crucial roles for superoxide anions and mitophagy in CuONPs-induced toxicity in vascular endothelial cells.


Assuntos
Morte Celular/efeitos dos fármacos , Cobre/toxicidade , Mitocôndrias/efeitos dos fármacos , Nanopartículas/toxicidade , Superóxidos/metabolismo , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Depuradores de Radicais Livres/farmacologia , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Metaloporfirinas/farmacologia , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Quinazolinonas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Superóxidos/antagonistas & inibidores
7.
Biomacromolecules ; 16(10): 3119-25, 2015 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-26302212

RESUMO

Silks are widely used biomaterials, but there are still weaknesses in their mechanical properties. Here we report a method for improving the silk fiber mechanical properties by genetic disruption of the ionic environment for silk fiber formation. An anterior silk gland (ASG) specific promoter was identified and used for overexpressing ion-transporting protein in the ASG of silkworm. After isolation of the transgenic silkworms, we found that the metal ion content, conformation and mechanical properties of transgenic silk fibers changed accordingly. Notably, overexpressing endoplasmic reticulum Ca2+-ATPase in ASG decreased the calcium content of silks. As a consequence, silk fibers had more α-helix and ß-sheet conformations, and their tenacity and extension increased significantly. These findings represent the in vivo demonstration of a correlation between metal ion content in the spinning duct and the mechanical properties of silk fibers, thus providing a novel method for modifying silk fiber properties.


Assuntos
Íons/análise , Seda/química , Animais , Animais Geneticamente Modificados , Bombyx/genética , Perfilação da Expressão Gênica , Espectroscopia de Infravermelho com Transformada de Fourier
8.
Am J Physiol Cell Physiol ; 309(6): C373-82, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26108663

RESUMO

Na-K-ATPase is a fundamental component of ion transport. Four α isoforms of the Na-K-ATPase catalytic α subunit are expressed in human cells. The ubiquitous Na-K-ATPase α1 was recently discovered to also mediate signal transduction through Src kinase. In contrast, α2 expression is limited to a few cell types including myocytes, where it is coupled to the Na(+)/Ca(2+) exchanger. To test whether rat Na-K-ATPase α2 is capable of cellular signaling like its α1 counterpart in a recipient mammalian system, we used an α1 knockdown pig renal epithelial cell (PY-17) to create an α2-expressing cell line with no detectable level of α1 expression. These cells exhibited normal ouabain-sensitive ATPase, but failed to effectively regulate Src. In contrast to α1-expressing cells, ouabain did not stimulate Src kinase or downstream effectors such as ERK and Akt in α2 cells, although their signaling apparatus was intact as evidenced by EGF-mediated signal transduction. Additionally, α2 cells were unable to rescue caveolin-1. Unlike the NaKtide sequence derived from Na-K-ATPase α1, which downregulates basal Src activity, the corresponding α2 NaKtide was unable to inhibit Src in vitro. Finally, coimmunoprecipitation of cellular Src was diminished in α2 cells. These findings indicate that Na-K-ATPase α2 does not regulate Src and, therefore, may not serve the same role in signal transduction as α1. This further implies that the signaling mechanism of Na-K-ATPase is isoform specific, thereby supporting a model where α1 and α2 isoforms play distinct roles in mediating contraction and signaling in myocytes.


Assuntos
Células Epiteliais/metabolismo , Bombas de Íon/metabolismo , Transdução de Sinais/fisiologia , ATPase Trocadora de Sódio-Potássio/deficiência , Sequência de Aminoácidos , Animais , Caveolina 1/metabolismo , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Células Epiteliais/efeitos dos fármacos , Rim/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Dados de Sequência Molecular , Ouabaína/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Suínos , Quinases da Família src/metabolismo
9.
J Insect Physiol ; 73: 53-9, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25602367

RESUMO

Calcium ions (Ca(2+)) are crucial for the conformational transition of silk fibroin in vitro, and silk fibroin conformations correlate with the mechanical properties of silk fibers. To investigate the relationship between Ca(2+) and mechanical properties of silk fibers, CaCl2 was injected into silkworms (Bombyx mori). Fourier-transform infrared spectroscopy (FTIR) analysis and mechanical testing revealed that injection of CaCl2 solution (7.5mg/g body weight) significantly increased the levels of α-helix and random coil structures of silk proteins. In addition, extension of silk fibers increased after CaCl2 injection. In mammals, sarcoplasmic reticulum Ca(2+)-ATPase in muscle and endoplasmic reticulum Ca(2+)-ATPase in other tissues (together denoted by SERCA) are responsible for calcium balance. Therefore, we analyzed the expression pattern of silkworm SERCA (BmSERCA) in silk glands and found that BmSERCA was abundant in the anterior silk gland (ASG). After injection of thapsigargin (TG) to block SERCA activity, silkworms showed a silk-spinning deficiency and their cocoons had higher calcium content compared to that of controls. Moreover, FTIR analysis revealed that the levels of α-helix and ß-sheet structures increased in silk fibers from TG-injected silkworms compared to controls. The results provide evidence that BmSERCA has a key function in calcium transportation in ASG that is related to maintaining a suitable ionic environment. This ionic environment with a proper Ca(2+) concentration is crucial for the formation of silk fibers with favorable mechanical performances.


Assuntos
Bombyx/metabolismo , Cálcio/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Seda/química , Animais , Cloreto de Cálcio/metabolismo , Fenômenos Mecânicos , Estrutura Secundária de Proteína , Seda/biossíntese , Espectroscopia de Infravermelho com Transformada de Fourier
10.
PLoS One ; 8(9): e75731, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24098719

RESUMO

Malpighian tubules (MTs) are highly specific organs of arthropods (Insecta, Myriapoda and Arachnida) for excretion and osmoregulation. In order to highlight the important genes and pathways involved in multi-functions of MTs, we performed a systematic proteomic analysis of silkworm MTs in the present work. Totally, 1,367 proteins were identified by one-dimensional gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry, and as well as by Trans Proteomic Pipeline (TPP) and Absolute protein expression (APEX) analyses. Forty-one proteins were further identified by two-dimensional gel electrophoresis. Some proteins were revealed to be significantly associated with various metabolic processes, organic solute transport, detoxification and innate immunity. Our results might lay a good foundation for future functional studies of MTs in silkworm and other lepidoptera.


Assuntos
Bombyx/genética , Túbulos de Malpighi/metabolismo , Proteoma/genética , Animais , Western Blotting , Bombyx/metabolismo , Cromatografia Líquida , Biologia Computacional , Primers do DNA/genética , Eletroforese em Gel Bidimensional , Proteoma/metabolismo , Proteômica , Espectrometria de Massas em Tandem
11.
Proteomics ; 13(17): 2657-63, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23828816

RESUMO

The Bombyx mori anterior silk gland (ASG) is a natural fiber manipulator for the material provided by the middle and posterior silk glands. In view of the significant role of the ASG in the liquid-crystal spinning process, a shotgun proteomics approach was taken to study the relationship between the function of proteins in the silkworm ASG and the spinning mechanism. A total of 1132 proteins with 7647 unique peptides were identified in the ASG dataset including some involved in the cuticle, ion transportation, energy metabolism, and apoptosis. Two putative cuticle-specific proteins were highly and specifically expressed in the ASG; therefore, the ASG dataset could provide clues for comprehensive understanding of the natural silk spinning mechanism in the silkworm. All MS data have been deposited in the ProteomeXchange with identifier PXD000090.


Assuntos
Bombyx/metabolismo , Glândulas Exócrinas/química , Proteínas de Insetos/análise , Proteoma/análise , Proteômica/métodos , Seda/biossíntese , Animais , Perfilação da Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo
12.
BMB Rep ; 45(11): 665-70, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23187007

RESUMO

The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. We used gel electrophoresis and mass spectrometry to identify the extracted proteins from the silkworm PM to obtain an in-depth understanding of the biological function of the silkworm PM components. A total of 305 proteins, with molecular weights ranging from 8.02 kDa to 788.52 kDa and the isoelectric points ranging from 3.39 to 12.91, were successfully identified. We also found several major classes of PM proteins, i.e. PM chitin-binding protein, invertebrate intestinal mucin, and chitin deacetylase. The protein profile provides a basis for further study of the physiological events in the PM of Bombyx mori.


Assuntos
Bombyx/metabolismo , Membrana Celular/metabolismo , Sistema Digestório/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Membrana/metabolismo , Proteômica , Animais , Biologia Computacional , Eletroforese em Gel Bidimensional , Espectrometria de Massas em Tandem
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