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1.
Ren Fail ; 41(1): 842-849, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31488014

RESUMO

Purpose: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive development of kidney cysts and enlargement and dysfunction of the kidneys. The Consortium of Radiologic Imaging Studies of the Polycystic Kidney Disease (CRISP) cohort revealed that 89.1% had either a PKD1 or PKD2 mutation. Of the CRISP patients with a genetic cause detected, mutations in PKD1 accounted for 85%, while mutations in the PKD2 accounted for the remaining 15%. Here, we report exome sequencing of 16 Saudi patients diagnosed with ADPKD and 16 ethnically matched controls. Methods: Exome sequencing was performed using combinatorial probe-anchor synthesis and improved DNA Nanoballs technology on BGISEQ-500 sequencers (BGI, China) using the BGI Exome V4 (59 Mb) Kit. Identified variants were validated with Sanger sequencing. Results: With the exception of GC-rich exon 1, we obtained excellent coverage of PKD1 (mean read depth = 88) including both duplicated and non-duplicated regions. Of nine patients with typical ADPKD presentations (bilateral symmetrical kidney involvement, positive family history, concordant imaging, and kidney function), four had protein truncating PKD1 mutations, one had a PKD1 missense mutation, and one had a PKD2 mutation. These variants have not been previously observed in the Saudi population. In seven clinically diagnosed ADPKD cases but with atypical features, no PKD1 or PKD2 mutations were identified, but rare predicted pathogenic heterozygous variants were found in cystogenic candidate genes including PKHD1, PKD1L3, EGF, CFTR, and TSC2. Conclusions: Mutations in PKD1 and PKD2 are the most common cause of ADPKD in Saudi patients with typical ADPKD. Abbreviations: ADPKD: Autosomal dominant polycystic kidney disease; CFTR: Cystic fibrosis transmembrane conductance regulator; EGF: Epidermal growth factor; MCIC: Mayo Clinic Imaging Classification; PKD: Polycystic kidney disease; TSC2: Tuberous sclerosis complex 2.


Assuntos
Rim Policístico Autossômico Dominante/genética , Adulto , Idoso , Árabes/genética , Canais de Cálcio/genética , Estudos de Casos e Controles , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Análise Mutacional de DNA , Fator de Crescimento Epidérmico/genética , Éxons/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Rim Policístico Autossômico Dominante/diagnóstico por imagem , Receptores de Superfície Celular/genética , Arábia Saudita , Canais de Cátion TRPP/genética , Tomografia Computadorizada por Raios X , Proteína 2 do Complexo Esclerose Tuberosa/genética , Sequenciamento Completo do Exoma
2.
Biomed Pharmacother ; 118: 109214, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31382129

RESUMO

OBJECTIVE: To investigate the effects of desmoglein 3 (DSG3) gene mediating epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) signaling pathway on inflammatory response and immune function of anaphylactic rhinitis (AR). METHODS: Ten of the seventy male BALB/c mice were randomly selected as the normal control group, and the remaining 60 were used to construct the AR mice model. AR model mice were divided into 6 groups: model group (instilled with 5 µL saline), empty vector group (instilled with 5 µL of liposome and empty vector mixture), siRNA-DSG3 group (instilled with 5 µL of liposome and siRNA-DSG3 carrier mixture), AG1478 group (instilled with 5 µL of EGF/EGFR inhibitor AG1478), siRNA-DSG3+AG1478 group (instilled with 5 µL of liposome and siRNA-DSG3 carrier and EGF/EGFR inhibitor AG1478 mixture) and oe-DSG3 group, 10 in each group. After taking serum, each group of mice was sacrificed to get nasal mucosa tissues. HE staining was used to observe the pathological changes of nasal mucosa tissues in each group. The expression levels of DSG3, EGF and EGFR in nasal mucosa tissues of mice in each group were detected by qRT-PCR and western blot methods respectively. TUNEL staining was used to observe the apoptosis of nasal mucosa cells in mice. The expression of IgE, INF-γ, TNF-α, IL-2, IL-4 and IL-6 in serum of mice was determined by ELISA method. The immune adhesion function of red blood cells was detected by complement sensitization yeast hemagglutination method. RESULTS: All the mice with AR showed different degrees of nasal mucosa injury and inflammatory cell infiltration, and silencing DSG3 or inhibiting the activity of EGF signaling pathway could alleviate the nasal mucosa injury. Compared with control group, the INF-γ and IL-2 levels of serum in AR model mice were significantly decreased; IgE, TNF-α, IL-4 and IL-6 levels were significantly increased (all P < 0.05); the mRNA expression levels and protein levels of DSG3, EGF and EGFR were significantly increased (all P < 0.05); C3b receptor rosette rate and Ic rosette rate were significantly decreased (all P < 0.05). Detected by ELISA method, the expression levels of IgE, TNF-α, IL-4 and IL-6 were increased, while the expression levels of INF-γ and IL-2 were decreased after DSG3 silencing or using AG1478. Detected by qRT-PCR and western blot methods, the expression of DSG3, EGF and EGFR did decrease after DSG3 silencing. There was no significant difference in the EGF and EGFR expression between DSG3 silencing and using AG1478, and the expression decreased even more under the double effect. The mRNA and protein expression levels of DSG3, EGF and EGFR in the nasal mucosa tissues of mice with overexpression of DSG3 plasmid were significantly higher than those of normal mice (all P < 0.05). CONCLUSION: Silencing DSG3 gene can inhibit the activation of EGF signaling pathway, alleviate the inflammation of AR nasal mucosa, and enhance red blood cells immune adherence function.


Assuntos
Anafilaxia/imunologia , Desmogleína 3/genética , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Rinite Alérgica/imunologia , Anafilaxia/genética , Anafilaxia/metabolismo , Animais , Modelos Animais de Doenças , Fator de Crescimento Epidérmico/genética , Receptores ErbB/genética , Regulação da Expressão Gênica , Inflamação , Camundongos Endogâmicos BALB C , Mucosa Nasal/imunologia , Mucosa Nasal/metabolismo , Rinite Alérgica/genética , Rinite Alérgica/metabolismo , Transdução de Sinais
3.
Mar Biotechnol (NY) ; 21(5): 589-595, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31346855

RESUMO

The liver is an important central organ, which controls carbohydrate metabolism through maintaining glucose homeostasis by a tightly regulated system of genes or enzymes. The microRNAs are small non-coding RNAs playing an important role in the regulation of genes associated with developmental biology, physiology, metabolism, etc. Thus, in this study, we have intended to detect liver-specific microRNAs in farmed carp, Labeo bata, upon being fed a diet with different levels of carbohydrates. Here, we have conducted the experiment for 45 days using fingerlings of farmed carp fed with 20% (control), 40%, and 60% gelatinized starch levels. The liver tissues were collected from each treatment and processed for RNA isolation, small RNA library preparation, and high-throughput sequencing using Illumina NexSeq500. Through sequencing, 15,779,417 reads in 20% CHO, 13,959,039 in 40% CHO, and 13,661,950 in 60% CHO reads were generated for control and treated fishes using three small RNA libraries. We have investigated 445 novel and 231 conserved microRNAs in 20%, 40%, and 60% carbohydrate (CHO), respectively, through computational analysis. The differential expression analysis of miRNAs was carried out between different treatments compared with control and this study depicted 117 known and 114 novel miRNA genes involved in carbohydrate metabolic pathways. Further, target prediction and gene ontology analysis revealed that miRNAs were involved in several pathways such as signaling pathway, G protein pathway, complement receptor-mediated pathway, dopamine receptor signaling pathway, epidermal growth factor pathway, and notch signaling pathway. The predicted miRNA sites in targeted genes were associated with cellular activities, developmental biology, DNA binding, Golgi apparatus, extracellular region, catalytic activity, MAPK cascade, etc. Overall, we have generated a vital resource of liver-specific miRNAs involved in metabolic gene regulation. These studies further will help develop miRNA inhibitors to study their role during carbohydrate metabolism in farmed carp.


Assuntos
Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Fígado/efeitos dos fármacos , MicroRNAs/genética , Amido/administração & dosagem , Ração Animal , Animais , Aquicultura , Carpas , Dieta/métodos , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Proteínas de Peixes/classificação , Proteínas de Peixes/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Fígado/metabolismo , MicroRNAs/classificação , MicroRNAs/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Anotação de Sequência Molecular , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Amido/metabolismo
4.
J Enzyme Inhib Med Chem ; 34(1): 1233-1246, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31286784

RESUMO

Neratinib is an oral pan HER inhibitor, that irreversibly inhibits EGFR and HER2 and was proven to be effective against multiple EGFR mutations. In previous study, we reported spiro [indoline-3, 4'-piperidine]-2-ones as anticancer agents. In this study, we designed aminopyridine-containing spiro [indoline-3,4'-piperidine] derivatives A1-A4 using Neratinib and spiro [indoline-3, 4'-piperidine]-2-one compound patented as lead structure, then replaced piperidine with cyclopropane to obtain B1-B7 and replaced indoline with benzmorpholine to get C1-C4 and D1-D2. We synthesized these compounds and evaluated their residual activities under 0.5 M drug concentration on EGFR and ERBB2. Most of compounds showed stronger inhibition on EGFR-wt and ERBB2, in which A1-A4 showed excellent inhibitory activity with inhibition percentage on EGFR-wt kinase of 7%, 6%, 19%, 27%, respectively and 9%, 5%, 12%, 34% on ERBB2 kinase compared with 2% and 6% of Neratinib.


Assuntos
Aminopiridinas/química , Descoberta de Drogas , Fator de Crescimento Epidérmico/antagonistas & inibidores , Mutação , Compostos de Espiro/farmacologia , Fator de Crescimento Epidérmico/genética , Simulação de Acoplamento Molecular , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , Compostos de Espiro/química
5.
Nutrients ; 11(6)2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31167465

RESUMO

Chili peppers are one of the most widely consumed spices worldwide. However, research on the health benefits of chili peppers and some of its constituents has raised controversy as to whether chili pepper compounds possess cancer-promoting or cancer-preventive effects. While ample studies have been carried out to examine the effect of capsaicin in carcinogenesis, the chemopreventive effect of other major components in chili pepper, including dihydrocapsaicin, capsiate, and capsanthin, is relatively unclear. Herein, we investigated the inhibitory effect of chili pepper components on malignant cell transformation. Among the tested chili pepper compounds, dihydrocapsaicin displayed the strongest inhibitory activity against epidermal growth factor (EGF)-induced neoplastic transformation. Dihydrocapsaicin specifically suppressed EGF-induced phosphorylations of the p70S6K1-S6 pathway and the expression of c-Fos. A reduction in c-Fos levels by dihydrocapsaicin led to a concomitant downregulation of AP-1 activation. Further analysis of the molecular mechanism responsible for the dihydrocapsaicin-mediated decrease in phospho-p70S6K1, revealed that dihydrocapsaicin can block amino acid-dependent mechanistic targets of rapamycin complex 1 (mTORC1)-p70S6K1-S6 signal activation. Additionally, dihydrocapsaicin was able to selectively augment amino acid deprivation-induced cell death in mTORC1-hyperactive cells. Collectively, dihydrocapsaicin exerted chemopreventive effects through inhibiting amino acid signaling and c-Fos pathways and, thus, might be a promising cancer preventive natural agent.


Assuntos
Aminoácidos/metabolismo , Capsaicina/análogos & derivados , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Genes fos/fisiologia , Animais , Capsaicina/química , Capsaicina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes fos/genética , Humanos , Camundongos , Estrutura Molecular , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Acetato de Tetradecanoilforbol/metabolismo
6.
BMC Cancer ; 19(1): 532, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159758

RESUMO

BACKGROUND: Homeobox A5 (HOXA5), a member of the HOX family, plays an important role in tumor development and morphogenesis, although opposite effects on tumorigenesis have been observed, depending on the tissue type. In this study, we aimed to investigate the role of a novel transcript from the HOXA6-HOXA5 locus in colon cancer tumorigenesis. METHODS: Human colon cancer cell lines were analyzed using next generation sequencing-based targeted mRNA capture. The effects of overexpression and silencing of HOXA5 transcripts were evaluated in vitro and using a xenograft nude mouse model. RESULTS: We identified three novel transcripts (HOXA5 short, long 1, and long 2) transcribed from the HOXA6-HOXA5 locus in HCT116 colon cancer cells using next generation sequencing-based targeted mRNA capture. Knockdown of HOXA5 long 1 and long 2 transcripts did not affect cell growth, while selective silencing of HOXA5 short RNA inhibited cell growth independent of HOXA5 expression. Stable overexpression of HOXA5 short RNA promoted proliferation and migration of colon cancer cell lines HCT116, DLD1, and HT-29 and accelerated tumor growth in the xenograft mouse model. In vitro translation assays suggested HOXA5 short RNA was a functional long non-coding RNA (lncRNA). Consistent with these observations, expression of HOXA5 short RNA was upregulated in advanced colon cancer tissues. Ingenuity Pathway Analysis of differentially expressed genes between HOXA5 short RNA overexpressed and silenced HCT116 cells revealed that HOXA5 short RNA preferentially modified expression of epidermal growth factor (EGF) signal-related genes. Western blot analysis demonstrated that stable overexpression of HOXA5 short RNA increased EGF receptor levels and facilitated its phosphorylation in both HCT116 cells and xenograft tumors. CONCLUSIONS: Our results suggested that HOXA5 short RNA, a novel lncRNA, may play a crucial role in colon tumor growth through activation of EGF signaling.


Assuntos
Neoplasias do Colo/genética , Proteínas de Homeodomínio/genética , RNA Longo não Codificante/metabolismo , Animais , Carcinogênese/genética , Movimento Celular , Proliferação de Células , Neoplasias do Colo/patologia , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Genes Homeobox/fisiologia , Células HCT116 , Células HT29 , Humanos , Camundongos , Camundongos Nus , Fosfoproteínas , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Cancer Biomark ; 25(2): 177-184, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31104010

RESUMO

BACKGROUND: Reseptor tyrosine kinases (cMET and EGFR) are important in lung cancer targeted therapy. We believe if we can use them as markers for clinicians to help decide the diagnosis of lung cancer. This parameter will be important in serum samples of patients with lung cancer diagnosis and treatment. The aim of this study is aimed to evaluate the clinical utility of serum protein and circulating mRNA of cMET and HGF in lung cancer patients. We also analyzed the correlation of mRNA expression with clinicopathologic parameters. METHODS: We performed enzyme-linked immunosorbent assay (ELISA) to measure and compare serum protein and circulating mRNA of cMET and HGF levels in peripheral blood from 60 lung cancer patients and 40 healthy control group. RESULTS: We found that both protein and gene expression levels of serum c-MET, HGF and EGFR were significantly higher in patients with lung cancer than control group. There was no association between HGF, cMET, EGF, EGFR (both protein and gene) expression levels with age, gender, smoking habit, COPD, pathological types or tumor size, stage, metastatic-non metastatic adenocarcinoma-squamous carcinoma, SCLC-NSCLC. As a result of ROC analysis, serum cMET (AUC: 0.892) and HGF protein (AUC: 0.784) were diagnosed in lung cancer patients (Fig. 1). The AUC values of serum EGF and EGFR proteins were calculated to be 0.631 and 0.692, respectively. CONCLUSION: To our knowledge this is the first study comparing the levels of protein and mRNA in the serum material of HGF, c-MET, EGF and EGFR parameters in lung cancer patients' blood samples. Further prospective studies with more participants for better understanding of mechanism and effect for HGF and c-MET inhibitors in lung cancer will help us to identify of these biomarkers role for guiding us to sellect individualized itargeted therapies.


Assuntos
DNA Tumoral Circulante , Fator de Crescimento Epidérmico/genética , Fator de Crescimento de Hepatócito/genética , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas c-met/genética , Adulto , Idoso , Biomarcadores Tumorais , Estudos de Casos e Controles , Fator de Crescimento Epidérmico/sangue , Receptores ErbB/sangue , Receptores ErbB/genética , Feminino , Fator de Crescimento de Hepatócito/sangue , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Medicina de Precisão , Proteínas Proto-Oncogênicas c-met/sangue , Curva ROC
8.
PLoS Genet ; 15(5): e1008056, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31086367

RESUMO

The six C. elegans vulval precursor cells (VPCs) are induced to form the 3°-3°-2°-1°-2°-3° pattern of cell fates with high fidelity. In response to EGF signal, the LET-60/Ras-LIN-45/Raf-MEK-2/MEK-MPK-1/ERK canonical MAP kinase cascade is necessary to induce 1° fate and synthesis of DSL ligands for the lateral Notch signal. In turn, LIN-12/Notch receptor is necessary to induce neighboring cells to become 2°. We previously showed that, in response to graded EGF signal, the modulatory LET-60/Ras-RGL-1/RalGEF-RAL-1/Ral signal promotes 2° fate in support of LIN-12. In this study, we identify two key differences between RGL-1 and RAL-1. First, deletion of RGL-1 confers no overt developmental defects, while previous studies showed RAL-1 to be essential for viability and fertility. From this observation, we hypothesize that the essential functions of RAL-1 are independent of upstream activation. Second, RGL-1 plays opposing and genetically separable roles in VPC fate patterning. RGL-1 promotes 2° fate via canonical GEF-dependent activation of RAL-1. Conversely, RGL-1 promotes 1° fate via a non-canonical GEF-independent activity. Our genetic epistasis experiments are consistent with RGL-1 functioning in the modulatory 1°-promoting AGE-1/PI3-Kinase-PDK-1-AKT-1 cascade. Additionally, animals lacking RGL-1 experience 15-fold higher rates of VPC patterning errors compared to the wild type. Yet VPC patterning in RGL-1 deletion mutants is not more sensitive to environmental perturbations. We propose that RGL-1 functions to orchestrate opposing 1°- and 2°-promoting modulatory cascades to decrease developmental stochasticity. We speculate that such switches are broadly conserved but mostly masked by paralog redundancy or essential functions.


Assuntos
Caenorhabditis elegans/genética , Fator de Crescimento Epidérmico/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/genética , Vulva/metabolismo , Animais , Padronização Corporal/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Epistasia Genética , Feminino , Fertilidade/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo , Vulva/citologia , Vulva/crescimento & desenvolvimento , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
9.
J Insect Physiol ; 116: 90-99, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31063731

RESUMO

Using the mass spectrometry analysis of cuticle casts of brown planthopper (BPH, Nilaparvata lugens) and transcriptome analysis of BPH tissues, we identified a gigantic gene (50,922 bp, 16,973 aa) tentatively called Nlegf-like. Multiple transcripts were found. Nlegf-like encodes an integral membrane protein of 16,973 amino acid residues with 260 EGF-like repeats and 16 Ca2+-binding EGF repeats type (cbEGFs) in the extracellular portion. Nlegf-like was highly expressed in the integument and tended to peak at the middle stage or late stage of each nymph instar. Phylogenetic analysis showed this gene is conserved in many other insects. Different double-stranded RNA-mediated RNA interference targeting eight different regions of the Nlegf-like gene resulted in abnormal cuticle formation or molting and lethal phenotypes. Transmission electron microscopy revealed that the newly formed endocuticle was significantly thinner for RNAi-treated BPHs with phenotype of contracted abdomen, or the old cuticle could not be digested sufficiently for those with phenotype of slender body shape or died with molting difficulty when compared with the control group. We suggest that the Nlegf-like is crucial for metabolism of the cuticle in BPH molting.


Assuntos
Fator de Crescimento Epidérmico/genética , Hemípteros/genética , Proteínas de Insetos/genética , Muda/genética , Sequência de Aminoácidos , Animais , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/metabolismo , Feminino , Hemípteros/crescimento & desenvolvimento , Hemípteros/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Masculino , Ninfa/genética , Ninfa/crescimento & desenvolvimento , Ninfa/metabolismo , Filogenia , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Alinhamento de Sequência
10.
Int J Mol Sci ; 20(8)2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31027239

RESUMO

BACKGROUND: Human epidermal growth factor (hEGF) has drawn intense research attention due to its potential ability to promote healing of serious injuries, such as cuts, burns, and diabetic ulcers. Although hEGF displays prospective clinical value, the growth factor is restricted to the treatment of chronic diabetic ulcers because of its high production cost. METHODS: Leguminous plant peanut (Arachis hypogaea L.) hairy roots contain relatively few toxic and harmful substances, and tested as an excellent production system for hEGF in our study. To explore the possibility of hEGF expression in peanut, hEGF overexpression hairy roots were obtained by infecting leaves with Agrobacterium rhizogenes R1601. RESULTS: The maximum transgenic hairy roots inducing rate was 82%. Protein purification and mass spectrometry assays showed that the protein expressed in peanut hairy roots was identified as hEGF. Furthermore, Methylthiazolyldiphenyl-tetrazolium bromide assay showed that hEGF promoted HL-7702 liver cells proliferation, which indicate that hEGF has biological activity and non-toxic on human cells. CONCLUSION: Our results demonstrate the capacity of peanut hairy root cultures as a controlled, sustainable, and scalable production system that can be induced to produce valued human proteins, such as hEGF.


Assuntos
Arachis/genética , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/isolamento & purificação , Expressão Gênica , Sequência de Aminoácidos , Fator de Crescimento Epidérmico/química , Humanos , Peptídeos/química , Peptídeos/isolamento & purificação , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas
11.
Molecules ; 24(6)2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30897725

RESUMO

Targeted cancer therapy has become a high potential cancer treatment. Epidermal growth factor receptor (EGFR), which plays an important role in cell signaling, enhanced cell survival and proliferation, has been suggested as molecular target for the development of novel cancer therapeutics. In this study, a series of chalcone derivatives was screened by in vitro cytotoxicity against the wild type (A431 and A549) and mutant EGFR (H1975 and H1650) cancer cell lines, and, subsequently, tested for EGFR-tyrosine kinase (TK) inhibition. From the experimental screening, all chalcones seemed to be more active against the A431 than the A549 cell line, with chalcones 1c, 2a, 3e, 4e, and 4t showing a more than 50% inhibitory activity against the EGFR-TK activity and a high cytotoxicity with IC50 values of < 10 µM against A431 cells. Moreover, these five chalcones showed more potent on H1975 (T790M/L858R mutation) than H1650 (exon 19 deletion E746-A750) cell lines. Only three chalcones (1c, 2a and 3e) had an inhibitory activity against EGFR-TK with a relative inhibition percentage that was close to the approved drug, erlotinib. Molecular dynamics studies on their complexes with EGFR-TK domain in aqueous solution affirmed that they were well-occupied within the ATP binding site and strongly interacted with seven hydrophobic residues, including the important hinge region residue M793. From the above information, as well as ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties, all three chalcones could serve as lead compounds for the development of EGFR-TK inhibitors.


Assuntos
Chalcona/análogos & derivados , Chalcona/farmacologia , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/genética , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Células A549 , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Mutação/genética
12.
Mol Biol Rep ; 46(2): 2417-2425, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30783937

RESUMO

Epidermal growth factor (EGF) and its receptor (EGFR) play an important role in lung carcinogenesis. A functional single nucleotide polymorphism (SNP) in EGF promoter region (EGF+61 A>G-rs4444903) has been associated with cancer susceptibility. Yet, in lung cancer, the EGF+61 A>G role is unclear. The aim of this study was to evaluate the risk of lung cancer associated with EGF+61 A>G SNP in the Brazilian population. For that, 669 lung cancer patients and 1104 controls were analyzed. EGF+61 A>G genotype was assessed by PCR-RFLP and TaqMan genotyping assay. Both patients and controls were in Hardy-Weinberg equilibrium. As expected, uni- and multivariate analyses showed that tobacco consumption and age were significant risk factors for lung cancer. The genotype frequencies in lung cancer patients were 27.3% of AA, 47.4% of AG and 25.3% of GG, and for controls were 25.3% of AA, 51.6% of AG and 23.1% of GG. The allele frequencies were 51.1% of A and 48.9% of G for both cases and controls. No significant differences for the three genotypes (AA, AG and GG-codominant model) were observed between cases and controls. We then grouped AG and GG (recessive model) genotypes, as well as AA and AG (dominant model), and again, no significant differences were also found. This is the largest study to explore EGF+61 A>G polymorphism association with lung cancer risk and suggests that this SNP is not a risk factor for lung cancer in the Brazilian population.


Assuntos
Fator de Crescimento Epidérmico/genética , Neoplasias Pulmonares/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Brasil , Estudos de Casos e Controles , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Genética Populacional/métodos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Fatores de Risco
13.
Genetics ; 211(4): 1315-1330, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30700527

RESUMO

Genetic screens in the nematode Caenorhabditis elegans identified the EGF/Ras and Notch pathways as central for vulval precursor cell fate patterning. Schematically, the anchor cell secretes EGF, inducing the P6.p cell to a primary (1°) vulval fate; P6.p in turn induces its neighbors to a secondary (2°) fate through Delta-Notch signaling and represses Ras signaling. In the nematode Oscheius tipulae, the anchor cell successively induces 2° then 1° vulval fates. Here, we report on the molecular identification of mutations affecting vulval induction in O. tipulae A single Induction Vulvaless mutation was found, which we identify as a cis-regulatory deletion in a tissue-specific enhancer of the O . tipulae lin-3 homolog, confirmed by clustered regularly interspaced short palindromic repeats/Cas9 mutation. In contrast to this predictable Vulvaless mutation, mutations resulting in an excess of 2° fates unexpectedly correspond to the plexin/semaphorin pathway. Hyperinduction of P4.p and P8.p in these mutants likely results from mispositioning of these cells due to a lack of contact inhibition. The third signaling pathway found by forward genetics in O. tipulae is the Wnt pathway; a decrease in Wnt pathway activity results in loss of vulval precursor competence and induction, and 1° fate miscentering on P5.p. Our results suggest that the EGF and Wnt pathways have qualitatively similar activities in vulval induction in C. elegans and O. tipulae, albeit with quantitative differences in the effects of mutation. Thus, the derived induction process in C. elegans with an early induction of the 1° fate appeared during evolution, after the recruitment of the EGF pathway for vulval induction.


Assuntos
Fator de Crescimento Epidérmico/metabolismo , Proteínas de Helminto/genética , Nematoides/genética , Semaforinas/genética , Vulva/crescimento & desenvolvimento , Via de Sinalização Wnt , Animais , Fator de Crescimento Epidérmico/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Helminto/metabolismo , Mutação , Nematoides/crescimento & desenvolvimento , Nematoides/metabolismo , Semaforinas/metabolismo , Vulva/metabolismo
14.
Lasers Med Sci ; 34(6): 1217-1227, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30796543

RESUMO

This research aims to observe and compare the wound healing process of urethral bladder after transurethral holmium laser resection of bladder tumor (HoLRBT) and transurethral resection of bladder tumor (TURBT) and explore the possible mechanism of wound healing and bladder re-epithelialization after HoLRBT. An animal model of canine achieving HoLRBT and TURBT was established. Cystoscopy was performed at different time points (3 days and 1, 2, 3, and 4 weeks) after operation to observe the wound healing and re-epithelialization of bladder epithelium. Bladder mucosa specimens were obtained and histopathological changes of the bladder epithelium were observed under light microscope after HE staining. Immunochemistry was used to determine the cell expression ofCK5, CK14, EGF, EGFR; microRNA expressions of CK5, CK14, EGF, and EGFR were measured by qRT-PCR. The changes of urinary EGF concentration were detected by ELISA. The bladder epithelial wound was repaired and re-epithelialized at 1 week after HoLRBT. At the 4th week, the bladder wound was basically completed and re-epithelialized; repair of bladder epithelial wounds recapitulates the wounds with the proliferation and migration of residual epithelial cells under the wound and the bladder epithelium that proliferates alongside the wound surface to complete re-epithelialization. The process begins at 1 week after surgery and basically completes at 4 weeks after surgery. CK5 and CK14 positive cells were detected in the basal cells of the bladder epithelium after HoLRBT, and the expression of CK5 and CK14 mRNA in the basal cells of the bladder epithelium under hyperplasia was significantly higher than that of the normal bladder epithelial basal cells. Bladder epithelial wound repair of TURBT group was performed by the proliferative differentiation of the peri-bladder epithelium adjacent to the wound edge and crawled to the wound surface to complete the re-epithelialization process. The wound repair and re-epithelialization were significantly slower than HoLRBT group. The CK5 and CK14 positive cells can also be detected in the basal cells of marginal hyperplasia of basal margin, and the expression of CK5 and CK14 mRNA in the basal cells of the peri-bladder hyperplasia is obviously higher than that of the normal bladder epithelial basal cells. The expression of EGF in bladder regenerating epithelium gradually increased with time after HoLRBT. Bladder basal cells and bladder regenerating epithelium express high levels of EGFR after HoLRBT. The concentration of EGF in urine after HoLRBT and TURBT increased significantly after surgery, and peaked at 3 days after operation. The urinary EGF concentration in HoLRBT group was higher than that in TURBT group at 3 and 4 weeks after operation. The re-epithelialization process can be seen 1 week after the cystectomy with holmium laser cystectomy, and the epithelialization rate is faster than the traditional transection surgery. This is because the residual bladder epithelial stem cells and wound marginal epithelial cells are involved in the process of wound repair and re-epithelialization following HoLRBT. But only the marginal epithelial tissue participates in the re-epithelialization process after TURBT, so the repair rate of TURBT is slower. The repair of bladder epithelium after HoLRBT is related to the stimulation of tissue factor EGF. The regenerated bladder epithelium also participates in the wound repair process by means of autocrine of EGF.


Assuntos
Lasers de Estado Sólido , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/radioterapia , Cicatrização/efeitos da radiação , Animais , Diferenciação Celular , Cistectomia , Cães , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/urina , Células Epiteliais/patologia , Células Epiteliais/efeitos da radiação , Receptores ErbB/genética , Receptores ErbB/metabolismo , Queratina-14/genética , Queratina-14/metabolismo , Queratina-5/genética , Queratina-5/metabolismo , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reepitelização/efeitos da radiação , Uretra/efeitos da radiação , Neoplasias da Bexiga Urinária/cirurgia
15.
Biochemistry ; 58(8): 1043-1047, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30735360

RESUMO

The epidermal growth factor receptor (EGFR) is a transmembrane protein involved in cell signaling processes, and dysregulation of its activity often drives tumor growth. EGFR is a clinically validated tumor marker and target for antibodies and tyrosine kinase inhibitors. We demonstrate that a fusion protein of the natural ligand epidermal growth factor (EGF) with the fluorescent reporter mCherry can be expressed in the cytosol of E. coli in high yields and with a high biological activity. Biophysical characterization by mass spectrometry analysis confirmed three disulfide bonds that are crucial for protein structure. Biolayer interferometry data of the protein-protein interaction of EGF-mCherry with the soluble EGFR are comparable to that of unmodified EGF. Cell culture experiments demonstrated that this fusion replicates all important features of the natural ligand. Finally, fluorescent assays based on EGF-mCherry provided a simple and convenient method to compare EGFR levels on cells and to determine competition of EGFR-binding molecules. These assays will help to rank competitive properties of EGFR inhibitors.


Assuntos
Fator de Crescimento Epidérmico/metabolismo , Escherichia coli/metabolismo , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fator de Crescimento Epidérmico/genética , Receptores ErbB/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Células HeLa , Humanos , Ligantes , Proteínas Luminescentes/genética , Fosforilação , Ligação Proteica , Proteínas Recombinantes de Fusão/genética
16.
Biomater Sci ; 7(3): 995-1010, 2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30603758

RESUMO

The objective of this study was to develop a novel delivery system for recombinant human epidermal growth factor (rhEGF) for chronic wound treatment. Such a delivery system should be of good cargo stability and system mechanical properties in order to guarantee a satisfactory wound-healing effect. rhEGF-containing lyotropic liquid crystalline precursors (rhEGF-LLCPs) with in situ gelation capability were considered as a promising candidate to achieve this aim. Various properties of the optimal formulations (rhEGF-LLCP1 and rhEGF-LLCP2) were characterized, including apparent viscosity, gelation time, in vitro release and phase behavior. The stability of rhEGF and system mechanical properties (i.e. mechanical rigidity and bioadhesive force) were verified. Interestingly, rhEGF-LLCP2 with a larger internal water channel diameter exhibited faster release rate in vitro and then better bioactivity in Balb/c 3T3 and HaCaT cell models. Moreover, rhEGF-LLCP2 showed distinct promotion effects on wound closure, inflammatory recovery and re-epithelization process in Sprague-Dawley rat models. In conclusion, rhEGF-LLCP emerged as a prospective candidate to preserve the stability and enhance the wound-healing effect of rhEGF, which might serve as a new delivery system for chronic wound therapies.


Assuntos
Fator de Crescimento Epidérmico/metabolismo , Cristais Líquidos/química , Cicatrização , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Fator de Crescimento Epidérmico/genética , Géis/química , Humanos , Masculino , Camundongos , Estabilidade Proteica , Ratos , Ratos Sprague-Dawley , Reepitelização/efeitos dos fármacos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Reologia , Pele/efeitos dos fármacos , Pele/patologia , Viscosidade , Cicatrização/efeitos dos fármacos
17.
Medicine (Baltimore) ; 98(2): e14007, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30633190

RESUMO

BACKGROUND: Colorectal cancer was a complex disease with multiple causative factors including genetic and environmental factors, as well as the interaction of the 2 factors. Relationship between epidermal growth factor (EGF) A61G polymorphism and colorectal cancer risk has been widely investigated previously, whereas results derived from these studies were inconclusive and controversial. The aim of this study was to investigate the association between the EGF A61G polymorphism and colorectal cancer using a meta-analysis of existing literature. METHODS: Literature search was conducted from PubMed, EMBASE, China National Knowledge Infrastructure, Wanfang, and Cochrane library databases before July 2017. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) were used to evaluate the strength of the association between EGF A61G and colorectal cancer. RESULTS: A total of 9 studies that involved 1448 cases and 1928 healthy controls and found allelic (OR = 1.18, P = .04) and recessive models (OR = 1.36, P = .03) of EGF A61G were significantly associated with the risk of colorectal cancer. Stratification analyses by ethnicity indicated that the EGF 61G significantly increased the risk of colorectal cancer in the Caucasian subgroup (OR = 1.24, P = .02), but not in Asian subgroup (OR = 1.12, P = .08). And the frequency of GG genotype of EGF A61G significantly increased in cases than that in healthy controls in both Caucasian (OR = 1.40, P = .04) and Asian subgroups (OR = 1.27, P = .01). Furthermore, the sample sources and genotyping methods seem to have no influence on the correction of EGF A61G and colorectal cancer susceptibility (P > .05). CONCLUSION: The results indicate that EGF A61G might increase the risk of colorectal cancers.


Assuntos
Neoplasias Colorretais/genética , Fator de Crescimento Epidérmico/genética , Predisposição Genética para Doença , Humanos
18.
J Cell Sci ; 132(3)2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30659112

RESUMO

The interaction between tumor cells and macrophages is crucial in promoting tumor invasion and metastasis. In this study, we examined a novel mechanism of intercellular communication, namely membranous actin-based tunneling nanotubes (TNTs), that occurs between macrophages and tumor cells in the promotion of macrophage-dependent tumor cell invasion. The presence of heterotypic TNTs between macrophages and tumor cells induced invasive tumor cell morphology, which was dependent on EGF-EGFR signaling. Furthermore, reduction of a protein involved in TNT formation, M-Sec (TNFAIP2), in macrophages inhibited tumor cell elongation, blocked the ability of tumor cells to invade in 3D and reduced macrophage-dependent long-distance tumor cell streaming in vitro Using an in vivo zebrafish model that recreates macrophage-mediated tumor cell invasion, we observed TNT-mediated macrophage-dependent tumor cell invasion, distant metastatic foci and areas of metastatic spread. Overall, our studies support a role for TNTs as a novel means of interaction between tumor cells and macrophages that leads to tumor progression and metastasis.


Assuntos
Neoplasias da Mama/genética , Comunicação Celular/genética , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Macrófagos/metabolismo , Neoplasias Mamárias Animais/genética , Animais , Transporte Biológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Embrião não Mamífero , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Xenoenxertos , Humanos , Macrófagos/ultraestrutura , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Cultura Primária de Células , Células RAW 264.7 , Ratos , Transdução de Sinais , Fatores de Necrose Tumoral/genética , Fatores de Necrose Tumoral/metabolismo , Peixe-Zebra
19.
Oncogene ; 38(5): 747-764, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30177836

RESUMO

Aberrant activation of EGFR represents a common event in non-small cell lung carcinoma (NSCLC) and activates various downstream signaling pathways. While EGFR activation of ß-catenin signaling was previously reported, the mediating mechanism remains unclear. Our current study found that EGFR activation in NSCLC cells releases SHC-binging protein 1 (SHCBP1) from SHC adaptor protein 1 (SHC1), which subsequently translocates into the nucleus and directly promotes the transactivating activity of ß-catenin, consequently resulting in development of NSCLC cell stemness and malignant progression. Furthermore, SHCBP1 promotes ß-catenin activity through enhancing the CBP/ß-catenin interaction, and most interestingly, a candidate drug that blocks the CBP/ß-catenin binding effectively abrogates the aforementioned biological effects of SHCBP1. Clinically, SHCBP1 level in NSCLC tumors was found to inversely correlate with patient survival. Together, our study establishes a novel convergence between EGFR and ß-catenin pathways and highlights a potential significance of SHCBP1 as a prognostic biomarker and a therapeutic target.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Núcleo Celular/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , beta Catenina/metabolismo , Transporte Ativo do Núcleo Celular/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/patologia , Fator de Crescimento Epidérmico/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/genética , Proteínas Adaptadoras da Sinalização Shc/genética , beta Catenina/genética
20.
Respir Investig ; 57(1): 3-8, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30361052

RESUMO

Bronchoscopy is one of the main techniques used for sampling lung tumor biopsies. In recent years, a large number of tumor specimens have been required to determine the best chemotherapy regimen for each patient; this personalized approach is known as precision medicine. In this review, radial endobronchial ultrasound; bronchoscopic navigation systems, including virtual bronchoscopic navigation and electromagnetic navigation; ultrathin bronchoscope,; and endobronchial ultrasound-guided transbronchial needle aspiration are highlighted as techniques used to increase the diagnostic yield. Personalized therapy includes tests for analysis of epidermal growth factor mutations, anaplastic lymphoma kinase or ROS proto-oncogene 1 fusion gene, and programmed death ligand 1 expression. In cryobiopsy, a relatively large amount of tissue is collected from endobronchial lung cancer and peripheral pulmonary lesions, and it is a promising technique for analyzing these tissues using molecular tests.


Assuntos
Broncoscopia , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biópsia por Agulha Fina , Broncoscopia/métodos , Fator de Crescimento Epidérmico/genética , Expressão Gênica , Humanos , Biópsia Guiada por Imagem , Mutação , Medicina de Precisão , Ultrassonografia
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