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1.
Oncogene ; 38(33): 6123-6141, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285549

RESUMO

Most N6-methyladenosine (m6A) associated regulatory proteins (i.e., m6A writer, eraser, and reader proteins) are involved in the pathogenesis of various cancers, mostly in m6A-dependent manners. As a component in the m6A 'writers', KIAA1429 is reported to be an RNA-binding protein and involved in the m6A modification, mRNA splicing and processing. Till now, the functions of KIAA1429 in tumorigenesis and related mechanism have not been reported. In the present study, we found KIAA1429 was highly expressed in breast cancer tissues, but frequently down-regulated in non-cancerous breast tissues. The overall survival of breast cancer patients with high-expression KIAA1429 was significantly shorter than those with low-expression KIAA1429. Then, we demonstrated that KIAA1429 was associated with breast cancer proliferation and metastasis in vivo and in vitro. The potential targeting genes of KIAA1429 in breast cancer were identified by RNA immunoprecipitation sequencing. One of these genes is cyclin-dependent kinase 1 (CDK1), which plays an oncogenic role in cancers. Furthermore, we confirmed that KIAA1429 played its oncogenic role in breast cancer by regulating CDK1 by an m6A-independent manner. 5'-fluorouracil was found to be very effective in reducing the expression of KIAA1429 and CDK1 in breast cancer. These findings indicated that KIAA1429 could promote breast cancer progression and was correlated with pathogenesis. It may represent a promising therapeutic strategy on breast cancer, especially in combination with CDK1 treatment.


Assuntos
Adenosina/análogos & derivados , Neoplasias da Mama/genética , Proteína Quinase CDC2/genética , Oncogenes/fisiologia , Proteínas de Ligação a RNA/fisiologia , Adenosina/metabolismo , Adenosina/fisiologia , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína Quinase CDC2/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Proteínas de Ligação a RNA/genética , Células Tumorais Cultivadas
2.
Int J Mol Med ; 43(3): 1531-1541, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30664181

RESUMO

Dogs are a major source of indoor allergens. However, the prevalence of dog allergies in China remains unclear, especially in children. In the present study, Can f 7, a canine allergen belonging to the Niemann pick type C2 protein family, was selected to study its sensitization rate in Chinese children with dog allergies. The Can f 7 gene was subcloned into a pET­28a vector and expressed in Escherichia coli BL21 (DE3) cells. Recombinant Can f 7 was purified by nickel affinity chromatography, identified by SDS­PAGE electrophoresis, and had its allergenicity assessed by western blot, ELISA and basophil activation tests. Through a series of bioinformatical approaches, B­cell epitopes, secondary structures, and 3 dimensional (3D) homology modeling of Can f 7 were predicted. The activity of the B cell epitopes was verified by ELISA. The recombinant Can f 7 showed a distinct band with a molecular weight of 14 kDa. Six of 20 sera from dog­allergic children reacted positively to the Can f 7. Can f 7 induced an ~4.0­fold increase in cluster of differentiation 63 and C­C motif chemokine receptor R3 expression in basophils sensitized with the serum of dog­allergic children compared with those of non­allergic controls. The secondary structure analysis showed that Can f 7 contains 6 ß­sheets. Five B cell epitopes of Can f 7 were predicted, and two of these were confirmed by ELISA. These results indicate that Can f 7 is an important canine allergen in Chinese children and provide novel data for further research concerning the use of Can f 7 in the diagnosis and treatment of Chinese children with canine allergy symptoms.


Assuntos
Alérgenos/genética , Alérgenos/imunologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Expressão Gênica , Lipocalinas/genética , Lipocalinas/imunologia , Adolescente , Alérgenos/química , Alérgenos/isolamento & purificação , Sequência de Aminoácidos , Animais , Criança , Pré-Escolar , Códon , Cães , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/química , Feminino , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Lactente , Lipocalinas/química , Lipocalinas/isolamento & purificação , Masculino , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes
3.
Mol Med Rep ; 17(1): 394-399, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29115430

RESUMO

Platanus acerifolia is one of the major sources of outdoor allergens to humans, and can induce allergic asthma, rhinitis, dermatitis and other allergic diseases. Pla a 2 is a polygalacturonase and represents the major allergen identified in P. acerifolia pollen. The aim of the present study was to express and purify Pla a 2, and to predict B and T cell epitopes of Pla a 2. The gene encoding Pla a 2 was cloned into the pET28a vector and subsequently transfected into ArcticExpress™ (DE3) Escherichia coli cells; purified Pla a 2 was analyzed by western blot analysis. The results of the present study revealed that the Pla a 2 allergen has the ability to bind immunoglobulin E within the sera of patients allergic to P. acerifolia pollen. In addition, the B cell epitopes of Pla a 2 were predicted using the DNAStar Protean system, Bioinformatics Predicted Antigenic Peptides and BepiPred 1.0 software; T cell epitopes were predicted using NetMHCIIpan ­3.0 and ­2.2. In total, eight B cell epitopes (15­24, 60­66, 78­86, 109­124, 232­240, 260­269, 298­306 and 315­322) and five T cell epitopes (62­67, 86­91, 125­132, 217­222 and 343­350) were predicted in the present study. These findings may be used to improve allergen immunotherapies and reduce the frequency of pollen­associated allergic reactions.


Assuntos
Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Mapeamento de Epitopos , Epitopos/genética , Epitopos/imunologia , Expressão Gênica , Pólen/genética , Pólen/imunologia , Adulto , Antígenos de Plantas/química , Antígenos de Plantas/isolamento & purificação , Epitopos/química , Epitopos de Linfócito B , Epitopos de Linfócito T , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Humanos , Imunoglobulina E/imunologia , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Ligação Proteica/imunologia , Conformação Proteica
4.
Oncotarget ; 8(53): 90796-90807, 2017 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-29207604

RESUMO

Dog allergy is common worldwide. However, the allergenicity of dog allergy is still unclear in China as well as in special group, such as children. In this study, we chose Can f 6, a major dog allergen which belongs to the lipocalin to study its allergenicity in Chinese dog allergic children. Can f 6 gene was subcloned into pET-28a vector and transformed into E. coli BL21 (DE3) cells for expression. The recombinant Can f 6 was purified by nickel affinity chromatography, identified by SDS-PAGE, and tested for its allergenicity by Western blot with sera and basophil activation test. Secondary structures, B cell epitopes and homology modeling of Can f 6 were predicted by using a series of bioinformatical approaches. And the verification of B cell epitopes was detected by ELISA. The recombinant allergen showed an explicit band with the molecular weight of 20 kDa by SDS-PAGE. Sera from 56.3 % (18/32) of dog-allergic children patients reacted with Can f 6. The induction of the expression of CD63 and CCR3 of dog allergic children in passively sensitized basophils was up to approximately 5.0 times higher than healthy subjects. The secondary structure of Can f 6 contains 3 α-helices, 9 ß-sheets and random coils. Five B cell epitopes of Can f 6 were predicted and were confirmed successfully by ELISA. The results showed Can f 6 is a major allergen in Chinese children, which provides a basis for further study of Can f 6 in diagnosis and treatment of symptoms in children in China. The structural information of Can f 6 will help to form a foundation for the future design of vaccines and therapies for Can f 6 related allergies.

5.
Mol Med Rep ; 16(3): 2887-2892, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28677761

RESUMO

Platanus acerifolia pollen is considered an important source of airborne allergens in numerous cities. Pla a 1 is a major allergen from P. acerifolia pollen. The present study aimed to express and purify Pla a 1, and to prepare its monoclonal antibody. In the present study, the Pla a 1 gene was subcloned into a pET­28a vector and transformed into the ArcticExpress™ (DE3) RP Escherichia coli host strain. The purified Pla a 1 was then used to immunize BALB/c mice. When serum detection was positive, spleen cells were isolated from the mice and fused with SP2/0 myeloma cells at a ratio of 10:1. Hybridoma cells were screened by indirect ELISA and limiting dilution. Positive cells were used to induce the formation of antibody­containing ascites fluid, and the antibodies were purified using protein A­agarose. The results of the present study demonstrated that recombinant Pla a 1 was successfully expressed and purified, and exhibited positive immunoglobulin E­binding to serum from patients allergic to P. acerifolia. A total of 11 hybridomas that steadily secreted anti­Pla a 1 antibody were obtained and an immunoblotting analysis indicated that all of these monoclonal antibodies specifically recognized the Pla a 1 protein. These results suggested that specific anti­Pla a 1 antibodies may be obtained, which can be used for the rapid detection of Pla a 1 allergens and in the preparation of vaccines against P. acerifolia pollen.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Clonagem Molecular/métodos , Adolescente , Adulto , Animais , Antígenos de Plantas/isolamento & purificação , Linhagem Celular , Escherichia coli/genética , Feminino , Vetores Genéticos/genética , Humanos , Imunoglobulina E/imunologia , Magnoliopsida/genética , Magnoliopsida/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pólen , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Rinite Alérgica/imunologia , Rinite Alérgica Sazonal/imunologia , Adulto Jovem
6.
Mol Med Rep ; 16(3): 2851-2855, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28656246

RESUMO

Platanus acerifolia (P. acerifolia) is an important cause of pollinosis in cities. The use of allergen extracts on patients with allergic diseases is the most commonly applied method to attempt to treat pollinosis. Pla a 3, a non­specific lipid transfer protein, is a major allergen present in P. acerifolia pollen extracts. In the present study, the Pla a 3 gene was sub­cloned into a pSUMO­Mut vector using Stu I and Xho I sites and transformed into the Arctic Express™ (DE3) RP E. coli host strain. The purified Pla a 3 allergen was analyzed by western blotting and the results revealed that the Pla a 3 allergen has the ability to bind IgE in the P. acerifolia pollen of allergic patients' sera. Moreover, the authors predicted the potential B cell epitopes of the Pla a 3 allergen using the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides system and the BepiPred 1.0 server. In addition, the T cell epitopes were predicted by the SYFPEITHI database and the NetMHCII­2.2 server. As a result, two B cell epitopes (35­45 and 81­86) and four potential T cell epitopes including 2­15, 45­50, 55­61 and 67­73 were predicted in the present study. The current results can be used to contribute to allergen immunotherapies and useful in peptide­based vaccine designs of pollen allergy.


Assuntos
Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Imunoglobulina E/imunologia , Magnoliopsida/imunologia , Rinite Alérgica Sazonal/imunologia , Adolescente , Adulto , Antígenos de Plantas/química , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Clonagem Molecular , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/isolamento & purificação , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/isolamento & purificação , Escherichia coli/genética , Feminino , Humanos , Imunoglobulina E/sangue , Magnoliopsida/química , Magnoliopsida/genética , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Pólen/química , Pólen/genética , Pólen/imunologia , Conformação Proteica , Rinite Alérgica Sazonal/sangue , Adulto Jovem
7.
Expert Opin Ther Pat ; 27(8): 919-928, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28425830

RESUMO

INTRODUCTION: Tryptase is one of the main serine-proteinases located in the secretory granules of mast cells, and is released through degranulation, which is involved in the pathogenesis of allergic inflammatory disease, cardiovascular diseases, lung fibrosis and tumor. Therefore, inhibitors targeting tryptase may represent a new direction for the treatment of allergic inflammatory disease and other diseases. Areas covered: In this article, we discussed the history and development of tryptase inhibitors and described a variety of tryptase inhibitors via their structures and biological importance in clinical studies and drug development for tryptase-related diseases. Expert opinion: Initial tryptase inhibitors based on indole structure as the hydrophobic substituent on a benzylamine-piperidine template have low specificity and poor bioavailability. Therefore, designing new and specific inhibitors targeting tryptase should be involved in future clinical studies. Modifications toward indoles with varying N-substitution, introducing an amide bond, and growing the chain length contribute to an increase in the specific selectivity and potency of tryptase inhibitors. Tryptase has become the research hotspot to explore many related diseases. Therefore, there has been growing appreciation for the potential importance of the tryptase inhibitors as a target for treating these diseases.


Assuntos
Desenho de Drogas , Inibidores de Serino Proteinase/farmacologia , Triptases/antagonistas & inibidores , Animais , Humanos , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/enzimologia , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Mastócitos/enzimologia , Mastócitos/metabolismo , Patentes como Assunto , Inibidores de Serino Proteinase/administração & dosagem , Triptases/metabolismo
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