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1.
Microb Cell Fact ; 18(1): 131, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31400768

RESUMO

BACKGROUND: The overexpression and purification of membrane proteins is a bottleneck in biotechnology and structural biology. E. coli remains the host of choice for membrane protein production. To date, most of the efforts have focused on genetically tuning of expression systems and shaping membrane composition to improve membrane protein production remained largely unexplored. RESULTS: In E. coli C41(DE3) strain, we deleted two transporters involved in fatty acid metabolism (OmpF and AcrB), which are also recalcitrant contaminants crystallizing even at low concentration. Engineered expression hosts presented an enhanced fitness and improved folding of target membrane proteins, which correlated with an altered membrane fluidity. We demonstrated the scope of this approach by overproducing several membrane proteins (4 different ABC transporters, YidC and SecYEG). CONCLUSIONS: In summary, E. coli membrane engineering unprecedentedly increases the quality and yield of membrane protein preparations. This strategy opens a new field for membrane protein production, complementary to gene expression tuning.


Assuntos
Proteínas de Escherichia coli/biossíntese , Escherichia coli/metabolismo , Lipídeos/química , Proteínas de Membrana/biossíntese , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Engenharia Metabólica , Canais de Translocação SEC/química , Canais de Translocação SEC/genética
2.
Nat Neurosci ; 22(8): 1258-1268, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31308530

RESUMO

The deposition of aggregated amyloid-ß peptides derived from the pro-amyloidogenic processing of the amyloid precurson protein (APP) into characteristic amyloid plaques (APs) is distinctive to Alzheimer's disease (AD). Alternative APP processing via the metalloprotease ADAM10 prevents amyloid-ß formation. We tested whether downregulation of ADAM10 activity by its secreted endogenous inhibitor secreted-frizzled-related protein 1 (SFRP1) is a common trait of sporadic AD. We demonstrate that SFRP1 is significantly increased in the brain and cerebrospinal fluid of patients with AD, accumulates in APs and binds to amyloid-ß, hindering amyloid-ß protofibril formation. Sfrp1 overexpression in an AD-like mouse model anticipates the appearance of APs and dystrophic neurites, whereas its genetic inactivation or the infusion of α-SFRP1-neutralizing antibodies favors non-amyloidogenic APP processing. Decreased Sfrp1 function lowers AP accumulation, improves AD-related histopathological traits and prevents long-term potentiation loss and cognitive deficits. Our study unveils SFRP1 as a crucial player in AD pathogenesis and a promising AD therapeutic target.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína ADAM10/biossíntese , Proteína ADAM10/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/biossíntese , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Anticorpos Bloqueadores/uso terapêutico , Química Encefálica/genética , Regulação para Baixo , Humanos , Potenciação de Longa Duração , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Transgênicos , Neuritos/patologia , Placa Amiloide/tratamento farmacológico , Placa Amiloide/genética , Placa Amiloide/patologia
3.
Oncol Rep ; 42(4): 1459-1466, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31322270

RESUMO

The expression of CDR1­AS, a representative circular RNA, is closely linked with poor prognosis in gastrointestinal cancers, such as colon, liver, and pancreatic cancers. Although it is well known that CDR1­AS antagonizes microRNA­7 function through its sequence similarities in the brain, its biological function and link with the malignant potential of cancer cells remain unclear, partly due to the difficulties of ectopic expression of circular RNAs. In the present study, SW620, a colon cancer cell line that stably expresses CDR1­AS RNA circularized, was established using the laccase 2 gene cassette, and its biological function associated with malignant behavior was determined. In contrast to previous studies, cell growth or invasion ability was not altered by CDR1­AS expression. However, the expression levels of CMTM4 and CMTM6, which were recently recognized as critical regulators of PD­L1 protein expression at the cell surface, were significantly increased. Accordingly, the cell surface PD­L1 protein levels were increased in CDR1­AS­expressing cells. Notably, the effects were not canceled out by overexpressing microRNA­7, indicating that the increase in cell surface PD­L1 in CDR1­AS­expressing cells was not dependent on microRNA­7 function. These results indicated that expression of this circular RNA in cancer cells may lead to poor prognosis by increasing cell surface PD­L1 levels through microRNA­7­independent mechanisms.


Assuntos
Antígeno B7-H1/biossíntese , Neoplasias Colorretais/metabolismo , RNA Longo não Codificante/biossíntese , Animais , Antígeno B7-H1/genética , Células CACO-2 , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células HEK293 , Humanos , Imuno-Histoquímica , Proteínas com Domínio MARVEL/biossíntese , Proteínas com Domínio MARVEL/genética , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/metabolismo , Invasividade Neoplásica , Prognóstico , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
Mol Cell Biochem ; 459(1-2): 189-204, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31172368

RESUMO

G3BPs are members of an RNA-binding protein family and their aberrant expression is common in various cancers and there is growing evidence that G3BPs possess antiviral activities and are targeted by various viruses. G3BPs have also been implicated in both stabilization and degradation of specific mRNAs as well as translational control of mRNA targets. G3BPs have been shown to control translation of interferon-stimulated genes (ISGs), implying that G3BPs are involved in the regulation of the interferon system in response to viral infections and/or cellular stress. The interferon induced transmembrane (IFITM1, IFITM2 and IFITM3) proteins are antiviral proteins, and are also involved in cancer progression and metastasis. Therefore, these genes were selected in the studies reported here as potential transcript targets of G3BPs. Furthermore, G3BPs are involved in the regulation of the MEK pathway which also impacts on the translation of ISGs. Therefore, the role of this pathway was also analysed in regulation of IFITM1-3 proteins. Overall, this research study suggests that G3BPs are essential for the accumulation of IFITM1-3 proteins and intersect twice in the regulation of IFITM1-3 expression, first through MEK pathway and then through an interaction with the 3'-UTRs of its target transcripts. However, it is still to be determined whether the two apparent functions are part of a single control mechanism or the two functions are mutually exclusive.


Assuntos
Antígenos de Diferenciação/biossíntese , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Biossíntese de Proteínas , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/biossíntese , Antígenos de Diferenciação/genética , Proteínas de Transporte/genética , DNA Helicases/genética , Humanos , Células MCF-7 , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas de Ligação a RNA/genética
5.
PLoS One ; 14(5): e0216793, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31150395

RESUMO

Renal carcinoma is the 20th most common cancer worldwide. Clear cell renal cell carcinoma is the most frequent type of renal cancer. Even in patients diagnosed at an early stage, characteristics of disease progression remain heterogeneous. Up-to-date molecular classifications stratify the ccRCC samples into two clusters. We analyzed gene expression in 23 T1 or T3 ccRCC samples. Unsupervised clustering divided this group into three clusters, two of them contained pure T1 or T3 samples while one contained a mixed group. We defined a group of 36 genes that discriminate the mixed cluster. This gene set could be associated with tumor classification into a higher stage and it contained significant number of genes coding for molecular transporters, channel and transmembrane proteins. External data from TCGA used to test our findings confirmed that the expression levels of those 36 genes varied significantly between T1 and T3 tumors. In conclusion, we found a clustering pattern of gene expression, informative for heterogeneity among T1 and T3 tumors of clear cell renal cell carcinoma.


Assuntos
Biomarcadores Tumorais/biossíntese , Carcinoma de Células Renais/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/biossíntese , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Feminino , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética
6.
Malar J ; 18(1): 197, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31196098

RESUMO

BACKGROUND: Vivax malaria is the predominant form of malaria outside Africa, affecting about 14 million people worldwide, with about 2.5 billion people exposed. Development of a Plasmodium vivax vaccine is a priority, and merozoite surface protein 7 (MSP-7) has been proposed as a plausible candidate. The P. vivax genome contains 12 MSP-7 genes, which contribute to erythrocyte invasion during blood-stage infection. Previous analysis of MSP-7 sequence diversity suggested that not all paralogs are functionally equivalent. To explore MSP-7 functional diversity, and to identify the best vaccine candidate within the family, MSP-7 expression and antigenicity during bloodstream infections were examined directly from clinical isolates. METHODS: Merozoite surface protein 7 gene expression was profiled using RNA-seq data from blood samples isolated from ten human patients with vivax malaria. Differential expression analysis and co-expression cluster analysis were used to relate PvMSP-7 expression to genetic markers of life cycle stage. Plasma from vivax malaria patients was also assayed using a custom peptide microarray to measure antibody responses against the coding regions of 12 MSP-7 paralogs. RESULTS: Ten patients presented diverse transcriptional profiles that comprised four patient groups. Two MSP-7 paralogs, 7A and 7F, were expressed abundantly in all patients, while other MSP-7 genes were uniformly rare (e.g. 7J). MSP-7H and 7I were significantly more abundant in patient group 4 only, (two patients having experienced longer patency), and were co-expressed with a schizont-stage marker, while negatively associated with liver-stage and gametocyte-stage markers. Screening infections with a PvMSP-7 peptide array identified 13 linear B-cell epitopes in five MSP-7 paralogs that were recognized by plasma from all patients. CONCLUSIONS: These results show that MSP-7 family members vary in expression profile during blood infections; MSP-7A and 7F are expressed throughout the intraerythrocytic development cycle, while expression of other paralogs is focused on the schizont. This may reflect developmental regulation, and potentially functional differentiation, within the gene family. The frequency of B-cell epitopes among paralogs also varies, with MSP-7A and 7L consistently the most immunogenic. Thus, MSP-7 paralogs cannot be assumed to have equal potential as vaccines. This analysis of clinical infections indicates that the most abundant and immunogenic paralog is MSP-7A.


Assuntos
Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Vivax/imunologia , Malária Vivax/prevenção & controle , Proteínas de Membrana/biossíntese , Proteínas de Membrana/imunologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/imunologia , Adolescente , Adulto , África , Idoso , Idoso de 80 Anos ou mais , Alelos , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana/genética , Análise em Microsséries , Pessoa de Meia-Idade , Plasmodium vivax/imunologia , Proteínas de Protozoários/genética , Análise de Sequência de RNA , Adulto Jovem
7.
Life Sci ; 231: 116548, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31181228

RESUMO

AIMS: Multidrug resistance is a major obstacle in chemotherapy, which is mainly caused by the overexpression of ATP-binding cassette (ABC) transporters. Breast cancer resistance protein (BCRP) is one of the ABC transporters and is strongly associated with multidrug resistance. Results of studies on BCRP and multidrug resistance are always uncomparable and contradictory, which may be stem from the disadvantages of qualitative and semi-quantitative techniques. In addition, there are few literatures studying at low resistance level which is more similar to the clinical situation. Thus, it is imperative to develop a quantitative method to quantitate the expression of BCRP accurately and reveal its relationship with multidrug resistance. METHODS: SMMC-7721, MCF-7 and HepG-2 were induced by different concentrations of mitoxantrone, doxorubicin and methotrexate respectively to establish resistance cells. An advanced liquid chromatography linked to tandem mass spectrometry (LC-MS/MS) based method with surrogate peptide was developed and validated for determining BCRP at low resistant cells. The amount of BCRP was also evaluated by real-time-polymerase chain reaction (RT-PCR) and Western Blot (WB). KEY FINDINGS: The LC-MS/MS-based method we developed is more sensitive and stable than the similar methods and can monitor the slight variation of BCRP expression accurately and sensitively, while RT-PCR and WB cannot. SIGNIFICANCE: This study provides a solid foundation for understanding the development of drug resistance in cells and can be used to explain the conflicting results of published studies. Moreover, clinical multidrug resistances are mostly at low levels, which have not been discussed in current quantitative studies of BCRP.


Assuntos
Neoplasias da Mama/química , Neoplasias da Mama/metabolismo , Cromatografia Líquida/métodos , Proteínas de Membrana/análise , Proteínas de Membrana/biossíntese , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Feminino , Células Hep G2 , Humanos , Células MCF-7 , Proteínas de Neoplasias/metabolismo
8.
Respir Res ; 20(1): 118, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186017

RESUMO

OBJECTIVE: The mechanisms of lung injury in acute respiratory distress syndrome (ARDS) are not well understood.Piezo1 was recently identified as a mechanotransduction protein. The present study found the expression of Piezo1 in type II pneumocytes and investigated its role in mediating ARDS-related lung injury. METHODS: Sprague-Dawley rats were used to establish an ARDS model, the expression of Piezo1,lung injuries, apoptosis as well as calcium influx were assessed. RESULTS: Piezo1 was expressed in type II pneumocytes as shown by immunofluorescence staining and expression was increased in the ARDS model. Knockdown of Piezo1 reduced apoptosis which was related to the elevation of Bcl-2.Calcium influx played a vital role in Piezo1-induced apoptosis. CONCLUSION: Piezo1 was expressed in type II pneumocytes. Mechanical stretch of alveoli during ARDS induced activation of the Piezo1 channel,which resulted in calcium influx. The increased intracellular Ca2+ induced the apoptosis of type II pneumocytes, which may be related to the Bcl-2 pathway.


Assuntos
Células Epiteliais Alveolares/metabolismo , Apoptose/fisiologia , Proteínas de Membrana/biossíntese , Síndrome do Desconforto Respiratório do Adulto/metabolismo , Estresse Mecânico , Células A549 , Células Epiteliais Alveolares/patologia , Animais , Humanos , Ratos , Ratos Sprague-Dawley , Síndrome do Desconforto Respiratório do Adulto/patologia
9.
Virchows Arch ; 475(4): 407-414, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31201505

RESUMO

Nuclear membrane proteins reportedly play important roles in maintaining nuclear structures and coordinating cell activities. Studying profiles of nuclear membrane proteins may help us evaluate the biological and/or clinical nature of malignant tumors. Using immunohistochemistry with antibodies for emerin, lamin A/C, lamin B, and LAP2, we examined 105 lung cancer tissues from 33 small cell lung carcinomas (SCLCs) and 72 non-SCLCs (34 adenocarcinomas, 30 squamous cell carcinomas, and 8 large cell carcinomas). Emerin had negative or local/weak positivity in 79% of SCLCs and 1% of non-SCLCs, and lamin A/C had similar positivity in 91% of SCLCs and 3% of non-SCLCs. LAP2's expression was similar between SCLCs and non-SCLCs. RT-PCR analyses for these four nuclear membrane proteins over 7 cell lines showed that mRNA of emerin and lamin A/C were distinctly downregulated in the SCLC cell lines, supporting the immunohistochemical results. In conclusion, we suggest that downregulation of the nuclear membrane proteins emerin and lamin A/C is characteristic of SCLC cells, and this constitutional abnormality of the nuclear membrane may be related to the biological and/or clinical nature of SCLC. In addition, knowing the nuclear protein profile in SCLC cells may contribute to our understanding of nuclear fragility known as the crush artifact in pulmonary biopsy specimens.


Assuntos
Lamina Tipo A/biossíntese , Neoplasias Pulmonares/patologia , Proteínas de Membrana/biossíntese , Proteínas Nucleares/biossíntese , Carcinoma de Pequenas Células do Pulmão/patologia , Biomarcadores Tumorais/análise , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/biossíntese , Humanos , Lamina Tipo A/análise , Lamina Tipo B/análise , Lamina Tipo B/biossíntese , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/análise , Proteínas Nucleares/análise , Carcinoma de Pequenas Células do Pulmão/metabolismo
10.
J Neuroinflammation ; 16(1): 130, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31248427

RESUMO

BACKGROUND: Multiple sclerosis (MS) involves a misdirected immune attack against myelin in the brain and spinal cord, leading to profound neuroinflammation and neurodegeneration. While the mechanisms of disease pathogenesis have been widely studied, the suppression mechanisms that lead to the resolution of the autoimmune response are still poorly understood. Here, we investigated the role of the C-type lectin receptor macrophage galactose-type lectin (MGL), usually expressed on tolerogenic antigen-presenting cells (APCs), as a negative regulator of autoimmune-driven neuroinflammation. METHODS: We used in silico, immunohistochemical, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry analysis to explore the expression and functionality of MGL in human macrophages and microglia, as well as in MS post-mortem tissue. In vitro, we studied the capacity of MGL to mediate apoptosis of experimental autoimmune encephalomyelitis (EAE)-derived T cells and mouse CD4+ T cells. Finally, we evaluated in vivo and ex vivo the immunomodulatory potential of MGL in EAE. RESULTS: MGL plays a critical role in the resolution phase of EAE as MGL1-deficient (Clec10a-/-) mice showed a similar day of onset but experienced a higher clinical score to that of WT littermates. We demonstrate that the mouse ortholog MGL1 induces apoptosis of autoreactive T cells and diminishes the expression of pro-inflammatory cytokines and inflammatory autoantibodies. Moreover, we show that MGL1 but not MGL2 induces apoptosis of activated mouse CD4+ T cells in vitro. In human settings, we show that MGL expression is increased in active MS lesions and on alternatively activated microglia and macrophages which, in turn, induces the secretion of the immunoregulatory cytokine IL-10, underscoring the clinical relevance of this lectin. CONCLUSIONS: Our results show a new role of MGL-expressing APCs as an anti-inflammatory mechanism in autoimmune neuroinflammation by dampening pathogenic T and B cell responses, uncovering a novel clue for neuroprotective therapeutic strategies with relevance for in MS clinical applications.


Assuntos
Assialoglicoproteínas/biossíntese , Encefalomielite Autoimune Experimental/metabolismo , Lectinas Tipo C/biossíntese , Proteínas de Membrana/biossíntese , Microglia/metabolismo , Animais , Células Cultivadas , Encefalomielite Autoimune Experimental/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/imunologia , Ratos
11.
PLoS One ; 14(5): e0216405, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31071151

RESUMO

Statins exert pleiotropic and beneficial anti-inflammatory and antioxidant effects. We have previously reported that macrophages treated with statins increased the expression of heme oxygenase-1 (HO-1), an inducible anti-inflammatory and cytoprotective stress protein, responsible for the degradation of heme. In the present study, we investigated the effects of atorvastatin on inflammation in mice and analyzed its mechanism of action in vivo. Air pouches were established in 8 week-old female C57BL/6J mice. Atorvastatin (5 mg/kg, i.p.) and/or tin protoporphyrin IX (SnPPIX), a heme oxygenase inhibitor (12 mg/kg, i.p.), were administered for 10 days. Zymosan, a cell wall component of Saccharomyces cerevisiae, was injected in the air pouch to trigger inflammation. Cell number and levels of inflammatory markers were determined in exudates collected from the pouch 24 hours post zymosan injection by flow cytometry, ELISA and quantitative PCR. Analysis of the mice treated with atorvastatin alone displayed increased expression of HO-1, arginase-1, C-type lectin domain containing 7A, and mannose receptor C-type 1 in the cells of the exudate of the air pouch. Flow cytometry analysis revealed an increase in monocyte/macrophage cells expressing HO-1 and in leukocytes expressing MRC-1 in response to atorvastatin. Mice treated with atorvastatin showed a significant reduction in cell influx in response to zymosan, and in the expression of proinflammatory cytokines and chemokines such as interleukin-1α, monocyte chemoattractant protein-1 and prostaglandin E2. Co-treatment of mice with atorvastatin and tin protoporphyrin IX (SnPPIX), an inhibitor of heme oxygenase, reversed the inhibitory effect of statin on cell influx and proinflammatory markers, suggesting a protective role of HO-1. Flow cytometry analysis of air pouch cell contents revealed prevalence of neutrophils and to a lesser extent of monocytes/macrophages with no significant effect of atorvastatin treatment on the modification of their relative proportion. These findings identify HO-1 as a target for the therapeutic actions of atorvastatin and highlight its potential role as an in vivo anti-inflammatory agent.


Assuntos
Anti-Inflamatórios/farmacologia , Atorvastatina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/biossíntese , Proteínas de Membrana/biossíntese , Zimosan/toxicidade , Animais , Movimento Celular/efeitos dos fármacos , Feminino , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/epidemiologia , Inflamação/patologia , Macrófagos/enzimologia , Macrófagos/patologia , Metaloporfirinas/farmacologia , Camundongos , Monócitos/enzimologia , Monócitos/patologia , Neutrófilos/enzimologia , Neutrófilos/patologia , Protoporfirinas/farmacologia
12.
Med Sci Monit ; 25: 3238-3246, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31044775

RESUMO

BACKGROUND The TMEFF2 gene encodes the transmembrane protein with EGF like and two follistatin-like domains 2 and has been reported to be a tumor suppressor gene, but its role remains unknown in pancreatic cancer. This study aimed to investigate the expression of TMEFF2 in human pancreatic cancer tissue and the effects of knockdown of TMEFF2 on cell, proliferation, and apoptosis in human pancreatic cell lines. MATERIAL AND METHODS Thirty-five samples of human pancreatic tissue and adjacent normal pancreatic tissue, and five human pancreatic cancer cell lines, CAPAN1, ASPC1, BXPC3, SW1990, and CFPAC were studied. RNA expression, protein expression, cell proliferation, and apoptosis were studied using real-time polymerase chain reaction (RT-PCR), Western blot, the cell counting kit-8 (CCK-8) assay, and flow cytometry, respectively. A co-immunoprecipitation assay evaluated protein interactions. RESULTS TMEFF2 expression was down-regulated in pancreatic cancer tissue compared with normal pancreas. In human pancreatic cancer cell lines, overexpression of TMEFF2 suppressed cell proliferation and enhanced apoptosis, suppressed the expression of p-STAT3, MCL1, VEGF and increased the expression of the tyrosine-specific protein phosphatase, SHP-1. The co-immunoprecipitation assay showed that TMEFF2 interacted with SHP-1. Knockdown of expression of TMEFF2 resulted in the increased expression of p-STAT3, MCL1, and VEGF, increased cell proliferation and decreased cell apoptosis, which were reversed by overexpression of SHP-1. CONCLUSIONS In pancreatic cancer, TMEFF2 exerted as a tumor suppressor effect by regulating p-STAT3, MCL1, and VEGF via SHP-1.


Assuntos
Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias Pancreáticas/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Apoptose/fisiologia , Pontos de Checagem do Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Regulação para Baixo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
Med Sci Monit ; 25: 3374-3389, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31063460

RESUMO

BACKGROUND The protein 4.1 family is a family of cytoskeletal proteins that play an important role in maintaining normal cell morphology and cell adhesion, migration, division, and intercellular signaling. The main aim of this study was to explore the prognostic significance of the protein 4.1 family in breast cancer (BC) patients and to provide new biomarkers and therapeutic targets for the diagnosis and treatment of BC. MATERIAL AND METHODS The expression of 4.1 family members in various tumor types was compared to normal controls using the ONCOMINE and GOBO databases. The prognostic significance of the 4.1 family in BC patients was determined by Kaplan-Meier Plotter. RESULTS EPB41L2 (4.1G) was expressed at higher levels in normal tissues compared with BC patients for all 4.1 family members. In survival analysis, 4.1G and EPB41 (4.1R) mRNA high expressions were associated with better survival in BC patients. Moreover, 4.1G high expression was significantly associated with longer overall survival (OS) in luminal A and protracted relapse-free survival (RFS) in luminal B subtype BC patients who received Tamoxifen treatment. In addition, high expression of each 4.1 family member also showed better prognostic value in different molecular subtypes of BC. CONCLUSIONS These results indicate that the protein 4.1 family can be regarded as novel biomarkers and potential therapeutic targets for BC. Further research is needed to explore the detailed biological functions.


Assuntos
Neoplasias da Mama/patologia , Proteínas do Citoesqueleto/biossíntese , Proteínas de Membrana/biossíntese , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas do Citoesqueleto/genética , Bases de Dados Genéticas , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Proteínas de Membrana/genética , Prognóstico , Análise de Sobrevida
14.
Pathol Res Pract ; 215(6): 152369, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30987833

RESUMO

Ovarian carcinoma is one of the most lethal malignancies, but only very few prognostic biomarkers are known. The degradome, comprising proteases, protease non-proteolytic homologues and inhibitors, have been involved in the prognosis of many cancer types, including ovarian carcinoma. The prognostic significance of the whole degradome family has not been specifically studied in high-grade serous ovarian cancer. A targeted DNA microarray known as the CLIP-CHIP microarray was used to identify potential prognostic factors in ten high-grade serous ovarian cancer women who had early recurrence (<1.6 years) or late/no recurrence after first line surgery and chemotherapy. In women with early recurrence, we identified seven upregulated genes (TMPRSS4, MASP1/3, SPC18, PSMB1, IGFBP2, CFI - encoding Complement Factor I - and MMP9) and one down-regulated gene (ADAM-10). Using immunohistochemistry, we evaluated the prognostic effect of these 8 candidate genes in an independent cohort of 112 high-grade serous ovarian cancer women. Outcomes were progression, defined according to CA-125 criteria, and death. Multivariate Cox proportional hazard regression models were done to estimate the associations between each protein and each outcome. High ADAM-10 expression (intensity of 2-3) was associated with a lower risk of progression (adjusted hazard ratio (HR): 0.51; 95% confidence interval (CI): 0.29-0.87). High complement factor I expression (intensity 2-3) was associated with a higher risk of progression (adjusted HR: 2.30, 95% CI: 1.17-4.53) and death (adjusted HR: 3.42; 95% CI: 1.72-6.79). Overall, we identified the prognostic value of two proteases, ADAM-10 and complement factor I, for high-grade serous ovarian cancer which could have clinical significance.


Assuntos
Proteína ADAM10/biossíntese , Secretases da Proteína Precursora do Amiloide/biossíntese , Fator I do Complemento/biossíntese , Cistadenocarcinoma Seroso/patologia , Proteínas de Membrana/biossíntese , Neoplasias Ovarianas/patologia , Idoso , Biomarcadores Tumorais/análise , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/mortalidade , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/mortalidade , Prognóstico , Intervalo Livre de Progressão
15.
J Biochem Mol Toxicol ; 33(7): e22333, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30980515

RESUMO

BACKGROUND: Streptococcus pneumoniae causes many human diseases including bacterial meningitis. Previous study proposed that pneumolysin (PLY), a cytotoxin from pneumococcus, is related to the infection across blood-brain barrier (BBB). However, the mechanism of how PLY break through BBB remains elusive. The present study showed that PLY can increase the permeability of BBB both in vitro and in vivo in our experiments. RESULTS: Further we found out that PLY leads to the high expression of CERB-binding protein (CBP) which can lead to releasing of tumor necrosis factor α then enhance apoptosis of cells which is a significant factor leading to permeabilization of BBB. CONCLUSION: Our findings demonstrate that CBP plays an important role in the pneumococcus infection in the brain and could be a potential therapeutic target against pneumococcal meningitis.


Assuntos
Barreira Hematoencefálica/metabolismo , Proteínas de Membrana/biossíntese , Meningite Pneumocócica/metabolismo , Fosfoproteínas/biossíntese , Streptococcus pneumoniae/metabolismo , Estreptolisinas/metabolismo , Regulação para Cima , Animais , Proteínas de Bactérias/metabolismo , Barreira Hematoencefálica/microbiologia , Barreira Hematoencefálica/patologia , Linhagem Celular , Feminino , Humanos , Meningite Pneumocócica/microbiologia , Meningite Pneumocócica/patologia , Camundongos , Permeabilidade , Streptococcus pneumoniae/patogenicidade , Fator de Necrose Tumoral alfa/metabolismo
16.
Blood ; 133(23): 2518-2528, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-30971389

RESUMO

The microRNA (miRNA) locus miR-144/451 is abundantly expressed in erythrocyte precursors, facilitating their terminal maturation and protecting against oxidant stress. However, the full repertoire of erythroid miR-144/451 target messenger RNAs (mRNAs) and associated cellular pathways is unknown. In general, the numbers of mRNAs predicted to be targeted by an miRNA vary greatly from hundreds to thousands, and are dependent on experimental approaches. To comprehensively and accurately identify erythroid miR-144/451 target mRNAs, we compared gene knockout and wild-type fetal liver erythroblasts by RNA sequencing, quantitative proteomics, and RNA immunoprecipitation of Argonaute (Ago), a component of the RNA-induced silencing complex that binds miRNAs complexed to their target mRNAs. Argonaute bound ∼1400 erythroblast mRNAs in a miR-144/451-dependent manner, accounting for one-third of all Ago-bound mRNAs. However, only ∼100 mRNAs were stabilized after miR-144/451 loss. Thus, miR-144 and miR-451 deregulate <10% of mRNAs that they bind, a characteristic that likely applies generally to other miRNAs. Using stringent selection criteria, we identified 53 novel miR-144/451 target mRNAs. One of these, Cox10, facilitates the assembly of mitochondrial electron transport complex IV. Loss of miR-144/451 caused increased Cox10 mRNA and protein, accumulation of complex IV, and increased mitochondrial membrane potential with no change in mitochondrial mass. Thus, miR-144/451 represses mitochondrial respiration during erythropoiesis by inhibiting the production of Cox10.


Assuntos
Alquil e Aril Transferases/biossíntese , Eritropoese/genética , Regulação da Expressão Gênica/genética , Proteínas de Membrana/biossíntese , MicroRNAs/genética , Alquil e Aril Transferases/genética , Animais , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout
17.
Gene ; 701: 23-31, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30898713

RESUMO

As a tumor metastasis suppressor gene, LASS2 has been found to be negatively associated with the stage of bladder cancer and overall survival of patients. However, the mechanisms regulating LASS2 in bladder cancer remain poorly understood. Here, we aim to identify a miRNA that targets LASS2 from bladder cancer-associated miRNAs and to reveal its potential functions in bladder cancer cells. Through miRNA microarray and bioinformatics analyses, we identified miR-3622a as a negative regulator of LASS2. The expression levels of miR-3622a in bladder cancer tissues were negatively correlated with the overall survival of patients. Overexpression of miR-3622a significantly increased the proliferation and invasion abilities of bladder cancer cells. In conclusion, our results indicate that miR-3622a promotes the proliferation and invasion of bladder cancer cells by downregulating LASS2.


Assuntos
Proliferação de Células , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/biossíntese , MicroRNAs/biossíntese , RNA Neoplásico/biossíntese , Esfingosina N-Aciltransferase/biossíntese , Proteínas Supressoras de Tumor/biossíntese , Neoplasias da Bexiga Urinária/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/genética , MicroRNAs/genética , Invasividade Neoplásica , RNA Neoplásico/genética , Esfingosina N-Aciltransferase/genética , Proteínas Supressoras de Tumor/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia
18.
J Cancer Res Clin Oncol ; 145(4): 895-907, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30820716

RESUMO

PURPOSE: Radiofrequency ablation (RFA) therapy has proven to be effective and feasible for early-stage hepatocellular carcinoma (HCC); however, rapid progression of residual tumor cells after RFA has been confirmed, but the molecular mechanisms of this phenomenon are poorly understood. This study evaluated the effect of the lipid raft proteins known as flotillins on the invasive and metastatic potential of residual HCC. METHODS: The human HCC cell line HCCLM3 was used to establish insufficient RFA models in vivo and in vitro. Changes in cellular morphology, soft agar colony formation, motility, metastasis, and epithelial-mesenchymal transition (EMT) markers after insufficient RFA intervention in vitro and in vivo were detected by real-time PCR, western blotting, immunohistochemistry and transwell assays. RESULTS: The results showed that flotillin-1 and flotillin-2 expression were upregulated in HCCLM3 cells following 45 °C heat treatment and in residual HCCLM3 xenografts cells after insufficient RFA. Knocking down flotillin-1 or flotillin-2 in HCCLM3 cells by shRNA significantly lowered insufficient RFA-induced tumor growth, EMT changes, and metastasis in vitro and in vivo. Furthermore, mechanism studies indicated that flotillins altered the EMT status and metastatic potential of heat-treated HCCLM3 cells by activating the Akt/Wnt/ß-catenin signaling pathway. CONCLUSIONS: Our findings present new evidence that flotillins play a key role in the aggressive behaviors of residual cancer cells after insufficient RFA and provide new insights into the regulatory mechanism of Wnt/ß-catenin signaling.


Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Proteínas de Membrana/biossíntese , Ablação por Radiofrequência/efeitos adversos , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Transição Epitelial-Mesenquimal , Células Hep G2 , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Inoculação de Neoplasia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ablação por Radiofrequência/métodos , Regulação para Cima , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismo
19.
PLoS One ; 14(2): e0211903, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30753204

RESUMO

The link between blood pressure (BP) and cerebral function is well established. However, it is not clear whether a common mechanism could underlie the relationship between elevated BP and cognitive deficits. The expression of calcyon, a gene abundant in catecholaminergic and hypothalamic nuclei along with other forebrain regions, is increased in the brain of the spontaneously hypertensive rat (SHR) which is a widely accepted animal model of essential hypertension and attention deficit hyperactivity disorder (ADHD). Previous studies demonstrated that mice with up-regulation of calcyon in forebrain (CalOE) exhibit deficits in working memory. To date, there is no evidence directly connecting calcyon to BP regulation. Here, we investigated whether forebrain up-regulation of calcyon alters BP using radiotelemetry. We found that CalOE mice exhibited higher mean arterial pressure (MAP) compared to tTA controls. Plasma norepinephrine levels were significantly higher in CalOE mice compared to tTA controls. Silencing the transgene with doxycycline normalized BP in CalOE mice, whereas challenging the mice with 4% high salt diet for 12 days exacerbated the MAP differences between CalOE and tTA mice. High salt diet challenge also increased proteinuria and urinary thiobarbituric acid reactive substances (TBARs) in tTA and CalOE; and the increases were more prominent in CalOE mice. Taken together, our data suggest that upregulation of calcyon in forebrain could increase BP via alterations in noradrenergic transmission and increased oxidative stress during high salt challenge. Overall, this study reveals that calcyon could be a novel neural regulator of BP raising the possibility that it could play a role in the development of vascular abnormalities.


Assuntos
Pressão Sanguínea , Hipertensão Essencial/metabolismo , Proteínas de Membrana/biossíntese , Estresse Oxidativo , Prosencéfalo/metabolismo , Animais , Transtorno do Deficit de Atenção com Hiperatividade , Modelos Animais de Doenças , Hipertensão Essencial/induzido quimicamente , Hipertensão Essencial/genética , Hipertensão Essencial/fisiopatologia , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Prosencéfalo/fisiopatologia , Cloreto de Sódio na Dieta/efeitos adversos , Cloreto de Sódio na Dieta/farmacologia
20.
Medicine (Baltimore) ; 98(5): e14076, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30702562

RESUMO

To explore the role of hScrib in the pathogenesis of endometriosis.This was a retrospective study of 240 women in our hospital between January 2014 and January 2017. The expression of hScrib in endometrium (EM), endometriosis (EMs), and endometrial adenocarcinoma (EC) was investigated, and compared the differences among them. Serum levels, protein expressions, localizations, and correlations of hScrib and E-cadherin were determined.The levels of serum soluble hScrib and E-cadherin were significantly highest in EC, followed by EMs, and healthy women (P < .05). hScrib protein content was opposite result in 3 tissues (P < .05), and was negatively correlated with r-AFS stage in EMs. The location changed from membrane to cytoplasm. Co-localization of hScrib with E-cadherin was found at extensive cell-cell boundaries in EMs.hScrib and E-cadherin may be as new diagnostic markers of endometriosis. Low expression of hScrib leads to the loss of cell polarity and stability. Also, hScrib may induce EMT through regulating E-cadherin, might play an important role in pathogenesis of endometriosis.


Assuntos
Adenocarcinoma/patologia , Neoplasias do Endométrio/patologia , Endometriose/patologia , Endométrio/patologia , Proteínas de Membrana/biossíntese , Proteínas Supressoras de Tumor/biossíntese , Adulto , Caderinas/biossíntese , Caderinas/sangue , Neoplasias do Endométrio/imunologia , Endometriose/imunologia , Endométrio/imunologia , Feminino , Humanos , Proteínas de Membrana/sangue , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Estudos Retrospectivos , Proteínas Supressoras de Tumor/sangue
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