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1.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(9): 918-921, 2019 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-31515790

RESUMO

OBJECTIVE: To explore phenotypic and mutational characteristics of a pedigree affected with autosomal dominant Charcot-Marie-Tooth disease (CMT) and nephropathy. METHODS: Clinical data of the proband and his family members was collected. Electrophysiology, renal biopsy and next-generation sequencing were carried out for the proband. RESULTS: The proband presented with distal lower limb weakness and proteinuria in childhood. His mother and brother had similar symptoms. Electrophysiological test of the proband revealed demyelination and axonal changes in both motor and sensory nerves. Renal biopsy suggested focal segmental glomerulosclerosis. Genetic testing revealed a heterozygous c.341G>A (p.G114D) mutation in exon 2 of the INF2 gene. CONCLUSION: The phenotypic feature of the pedigree is autosomal dominant intermediate CMT and focal segmental glomerulosclerosis, which may be attributed to the c.341G>A mutation of the INF2 gene.


Assuntos
Doença de Charcot-Marie-Tooth/genética , Glomerulosclerose Segmentar e Focal/genética , Proteínas dos Microfilamentos/genética , Doença de Charcot-Marie-Tooth/complicações , Criança , Feminino , Glomerulosclerose Segmentar e Focal/complicações , Heterozigoto , Humanos , Masculino , Mutação , Linhagem
2.
Parasitol Res ; 118(9): 2509-2521, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31377908

RESUMO

In this study, we characterize the HIP-55 protein in the mosquito Aedes aegypti for the first time. HIP-55 is a 55-kDa HPK1-interacting protein that is also called SH3P7. HIP-55 constitutively binds HPK1 'via' an HPK1 proline-rich motif 2(PR2) through its C-terminal SH3 domain. HIP-55 critically interacts with ZAP-70, and this interaction was induced by TCR signalling. ZAP-70 phosphorylated HIP-55 at Tyr-334 and Tyr-344 in vitro and in vivo. In our previous findings, AaZAP gene expression strongly proved that AaZAP-70 was involved in immunity-like functions in mosquito. Northern blot analysis of HIP-55 mRNA expression confirmed that it is only expressed in the abdomen and haemocyte tissues; this prediction correlates 100% and a polyclonal antibody also confirmed its localization in haemocytes and the abdomen. We prepared extracts to show the cytoplasmic expression (CE) of this protein. Previous results had proven that this protein is secreted from the cytoplasm; thus, we confirmed here that the protein is a cytoplasmic adaptor protein in mosquitoes and mammalian systems. Furthermore, our polyclonal antibody against HIP-55 also demonstrated that this protein is found in haemocytes and abdomen tissues, which assumes that the protein may be involved in phagocytic-like functions. RNAi (siRNA) silencing studies were used to degrade mosquito HIP-55; however, silencing only slightly affected the HIP-55 sequence and the gene transcriptional level. To characterize this protein, we cloned 609 bp from the 1.6-kb full-length cDNA using a pET28 vector for polyclonal antibody production. Graphical abstract.


Assuntos
Aedes/imunologia , Proteínas dos Microfilamentos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Domínios de Homologia de src/fisiologia , Animais , Hemócitos/metabolismo , Proteínas dos Microfilamentos/genética , Fosforilação , Ligação Proteica , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Proteína-Tirosina Quinase ZAP-70/metabolismo , Domínios de Homologia de src/genética
3.
Gene ; 710: 273-278, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31200082

RESUMO

BACKGROUND: Carotid plaque is a hallmark of advanced carotid atherosclerosis and there is evidence of phosphatase and actin regulator 1 (PHACTR1) involvement in the processes that lead to atherosclerosis. PHACTR1 intronic variants have been associated with coronary artery disease and carotid dissection. Up to date the PHACTR1 haplotypes were not investigated in association with carotid plaque presence (CPP). So, the aims of this study were to investigate possible association of PHACTR1 haplotypes inferred from the intronic variants rs9349379, rs2026458 and rs2876300 with CPP in patients with advanced carotid atherosclerosis and to analyze their possible effect on PHACTR1 relative mRNA expression in carotid plaque tissue specimens. METHODS: The study group consisted of 501 patients with evidence of carotid plaque presence admitted for carotid endarterectomy and 310 healthy controls. PHACTR1 rs9349379, rs2026458, rs2876300 and relative mRNA expression were detected by TaqMan® technology. RESULTS: We have found significant and independent association of haplotype ACA with the CPP, compared to the referent haplotype GTA (adjusted OR = 1.54 95% CI = 1.07-2.21, p = 0.02). The OR was adjusted for gender, age, BMI, hypertension and total cholesterol. The relative expression of PHACTR1 mRNA in carotid plaque tissue proved to be significantly higher in carriers of the ACG haplotype compared to the referent haplotype GTA (p = 0.03). CONCLUSION: Our results suggest that PHACTR1 haplotypes inferred from the variants rs9349379, rs2026458 and rs2876300 affect PHACTR1 mRNA and bear the risk for CPP in patients with advanced carotid atherosclerosis. Further replication and validation studies are inevitable.


Assuntos
Doenças das Artérias Carótidas/genética , Proteínas dos Microfilamentos/genética , Placa Aterosclerótica/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Doenças das Artérias Carótidas/cirurgia , Estudos de Casos e Controles , Endarterectomia das Carótidas , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Haplótipos , Humanos , Íntrons , Masculino , Pessoa de Meia-Idade , Placa Aterosclerótica/cirurgia , Regulação para Cima
4.
J Ovarian Res ; 12(1): 41, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31077237

RESUMO

BACKGROUND AND OBJECTIVE: Ovarian cancer is a common gynaecological cancer with a poor prognosis that poses a serious threat to human life and health. It is essential to explore the possible prognostic biomarkers of ovarian cancer. As an important tumour suppressor gene, BCL7A actively participates in the growth of tumours. We aimed to study the prognostic role of BCL7A in ovarian cancer. RESULTS: Through data mining of RNAseq data from the Cancer Genome Atlas database (TCGA), we explored the clinical relevance of BCL7A mRNA expression. As a result, we found that BCL7A is expressed at low levels in ovarian cancer tissues and is correlated with survival status. Survival analysis showed that, compared with those who had higher levels of BCL7A expression, patients with ovarian cancer and low levels of BCL7A generally had shorter overall/relapse-free survival times. Cox regression models showed that low BCL7A expression could be used as an independent prognostication factor for ovarian cancer patients. CONCLUSIONS: Low BCL7A expression is an independent risk factor for poor prognosis in ovarian cancer patients.


Assuntos
Proteínas dos Microfilamentos/biossíntese , Proteínas Oncogênicas/biossíntese , Neoplasias Ovarianas/genética , Feminino , Humanos , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteínas Oncogênicas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Prognóstico , RNA Mensageiro/metabolismo , Fatores de Risco , Análise de Sobrevida
5.
Cancer Sci ; 110(7): 2133-2144, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31066149

RESUMO

Lymphoid-specific helicase (LSH) is overexpressed in tumor tissues and its overexpression is associated with poor prognosis in several cancers. However, the role and molecular mechanism of LSH in hepatocellular carcinoma (HCC) remains largely unknown. Herein, we report that LSH was overexpressed in tumor tissues of HCC, and overexpression of LSH was associated with poor prognosis from a public HCC database, and validated by clinical samples from our department. Ectopic LSH expression promoted the growth of HCC cells in vivo and in vitro. Mechanistically, LSH overexpression promoted tumor growth by activating transcription of centromere protein F (CENPF). Clinically, overexpression of LSH and/or CENPF correlated with shorter overall survival and higher cumulative recurrence rates of HCC. In conclusion, LSH promotes tumor growth of HCC through transcriptional regulation of CENPF expression. Therefore, LSH may be a novel predictor for prognosis and a potential therapeutic target for HCC.


Assuntos
Carcinoma Hepatocelular/patologia , Proteínas Cromossômicas não Histona/genética , DNA Helicases/metabolismo , Neoplasias Hepáticas/patologia , Proteínas dos Microfilamentos/genética , Regulação para Cima , Idoso , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Transplante de Neoplasias , Prognóstico , Análise de Sequência de RNA , Análise de Sobrevida , Análise Serial de Tecidos , Ativação Transcricional
6.
Nat Commun ; 10(1): 2193, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097705

RESUMO

Filamentous actin (F-actin) networks facilitate key processes like cell shape control, division, polarization and motility. The dynamic coordination of F-actin networks and its impact on cellular activities are poorly understood. We report an antagonistic relationship between endosomal F-actin assembly and cortical actin bundle integrity during Drosophila airway maturation. Double mutants lacking receptor tyrosine phosphatases (PTP) Ptp10D and Ptp4E, clear luminal proteins and disassemble apical actin bundles prematurely. These defects are counterbalanced by reduction of endosomal trafficking and by mutations affecting the tyrosine kinase Btk29A, and the actin nucleation factor WASH. Btk29A forms protein complexes with Ptp10D and WASH, and Btk29A phosphorylates WASH. This phosphorylation activates endosomal WASH function in flies and mice. In contrast, a phospho-mimetic WASH variant induces endosomal actin accumulation, premature luminal endocytosis and cortical F-actin disassembly. We conclude that PTPs and Btk29A regulate WASH activity to balance the endosomal and cortical F-actin networks during epithelial tube maturation.


Assuntos
Proteínas de Drosophila/metabolismo , Endossomos/metabolismo , Morfogênese/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Embrião não Mamífero/diagnóstico por imagem , Epitélio/diagnóstico por imagem , Epitélio/crescimento & desenvolvimento , Fibroblastos , Microscopia Intravital , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Fosforilação/fisiologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Sistema Respiratório/diagnóstico por imagem , Sistema Respiratório/crescimento & desenvolvimento , Proteínas de Transporte Vesicular/genética
7.
Medicine (Baltimore) ; 98(22): e15800, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31145309

RESUMO

RATIONALE: Hereditary elliptocytosis is an inherited disorder characterized by the elliptical red blood cells (RBCs) on the peripheral blood smear and related hemolysis, mainly results from a heterozygous mutation in the genes that encode protein 4.1, α-spectrin, ß-spectrin. Mutations of SPTA1 are the most common. PATIENT CONCERNS: A 21-year-old female presented with left epigastric pain and jaundice with numerous elliptical RBCs on blood film. The family history review discovered jaundice in her sibling. DIAGNOSIS: A novel heterozygous mutation of SPTA1 was detected in the proband, her brother and father, c.7220_7221del:p.Tyr2407* in exon 52. Bioinformatics analysis indicated that this mutation was likely pathogenic and results in early termination of transcription and production of defective protein. INTERVENTIONS: The proband underwent splenectomy and cholecystectomy due to symptomatic splenomegaly and gallstone. OUTCOMES: After surgery, the bilirubin levels decreased to normal (i.e., total bilirubin 16.4 µmol/L; indirect bilirubin 12.3 µmol/L), and the pain and uncomfortableness in the upper abdomen relieved completely. LESSONS: We suggest that simultaneous whole exome sequencing of causative genes of all family members is a useful strategy to identify pathogenetic mutations for hereditary RBC membrane disorders, mainly in cases with an ambiguous phenotype.


Assuntos
Proteínas de Transporte/genética , Eliptocitose Hereditária/genética , Hiperbilirrubinemia/genética , Proteínas dos Microfilamentos/genética , Grupo com Ancestrais do Continente Asiático/genética , Éxons , Feminino , Humanos , Mutação , Linhagem , Espectrina , Sequenciamento Completo do Exoma , Adulto Jovem
8.
Orphanet J Rare Dis ; 14(1): 114, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31122244

RESUMO

BACKGROUND: Current diagnostic tests for hereditary spherocytosis (HS) focus on the detection of hemolysis or indirectly assessing defects of membrane protein, whereas direct methods to detect protein defects are complicated and difficult to implement. In the present study, we investigated the patterns of genetic variation associated with HS among patients clinically diagnosed with HS. METHODS: Multi-gene targeted sequencing of 43 genes (17 RBC membrane protein-encoding genes, 20 RBC enzyme-encoding genes, and six additional genes for the differential diagnosis) was performed using the Illumina HiSeq platform. RESULTS: Among 59 patients with HS, 50 (84.7%) had one or more significant variants in a RBC membrane protein-encoding genes. A total of 54 significant variants including 46 novel mutations were detected in six RBC membrane protein-encoding genes, with the highest number of variants found in SPTB (n = 28), and followed by ANK1 (n = 19), SLC4A1 (n = 3), SPTA1 (n = 2), EPB41 (n = 1), and EPB42 (n = 1). Concurrent mutations of genes encoding RBC enzymes (ALDOB, GAPDH, and GSR) were detected in three patients. UGT1A1 mutations were present in 24 patients (40.7%). Positive rate of osmotic fragility test was 86.8% among patients harboring HS-related gene mutations. CONCLUSIONS: This constitutes the first large-scaled genetic study of Korean patients with HS. We demonstrated that multi-gene target sequencing is sensitive and feasible that can be used as a powerful tool for diagnosing HS. Considering the discrepancies of clinical and molecular diagnoses of HS, our findings suggest that molecular genetic analysis is required for accurate diagnosis of HS.


Assuntos
Fragilidade Osmótica/fisiologia , Esferócitos/metabolismo , Esferocitose Hereditária/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Anquirinas/genética , Anquirinas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Criança , Pré-Escolar , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Feminino , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Lactente , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Mutação/genética , Fragilidade Osmótica/genética , Patologia Molecular , República da Coreia , Espectrina/genética , Espectrina/metabolismo , Esferocitose Hereditária/genética , Adulto Jovem
9.
Biochim Biophys Acta Gene Regul Mech ; 1862(8): 858-869, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31075540

RESUMO

Alternative splicing (AS) events occur in the majority of human genes. AS in a single gene can give rise to different functions among multiple isoforms. Human ortholog of mammalian enabled (Mena) is a conserved regulator of actin dynamics that plays an important role in metastasis. Mena has been shown to have multiple splice variants in human tumor cells due to AS. However, the mechanism mediated Mena AS has not been elucidated. Here we showed that polypyrimidine tract-binding protein 1 (PTBP1) could modulate Mena AS. First, PTBP1 levels were elevated in metastatic lung cancer cells as well as during epithelial-mesenchymal transition (EMT) process. Then, knockdown of PTBP1 using shRNA inhibited migration and invasion of lung carcinoma cells and decreased the Mena exon11a skipping, whereas overexpression of PTBP1 had the opposite effects. The results of RNA pull-down assays and mutation analyses demonstrated that PTBP1 functionally targeted and physically interacted with polypyrimidine sequences on both upstream intron11 (TTTTCCCCTT) and downstream intron11a (TTTTTTTTTCTTT). In addition, the results of migration and invasion assays as well as detection of filopodia revealed that the effect of PTBP1 was reversed by knockdown of Mena but not Mena11a+. Overexpressed MenaΔ11a also rescued the PTBP1-induced migration and invasion. Taken together, our study provides a novel mechanism that PTBP1 modulates Mena exon11a skipping, and indicates that PTBP1 depends on the level of Mena11a- to promote lung cancer cells migration and invasion. The regulation of Mena AS may be a potential prognostic marker and a promising target for treatment of lung carcinoma.


Assuntos
Processamento Alternativo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Neoplasias Pulmonares/genética , Proteínas dos Microfilamentos/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Células A549 , Movimento Celular , Transição Epitelial-Mesenquimal , Éxons , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Invasividade Neoplásica , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Regulação para Cima
10.
J Biotechnol ; 299: 1-7, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31002855

RESUMO

Breast cancer (BC) development is caused by the interaction of environmental and genetic factors. At least 90 susceptible genetic variants with different population penetration and incidence have been associated with BC. This paper therefore analysed the individual discrimination power of 8 low penetrant common genetic variants and calculated the predictive accuracy of the genetic risk model. The study enrolled 171 women with developed breast cancer (57.06 ± 11.60 years) and 146 control subjects (50.24 ± 10.69 years). The genotyping was performed by high resolution melting method (HRM) and confirmed by Sanger sequencing, and the Random Forest algorithm provided the ROC curve with AUC values. Significant association with BC was confirmed in 2 SNPs: rs2981582 FGFR2 and rs889312 MAP3K1, and the odds ratios of homozygotes with two risk alleles in both SNP's were higher than in heterozygotes with one mutant allele, as follows: FGFR2 TT: 1.953 (95%CI 1.014-3.834, p = 0.049), CT 1.771 (95%CI 1.088-2.899, p = 0.026) and MAP3K1 CC 2.894 (95%CI 1.028-9.566, p = 0.048), AC 1.760 (95%CI 1.108-2.813, p = 0.019). FGFR2 had the best discrimination ability, followed by MAP3K1 and CASP8. Discriminative accuracy of the genetic risk model distinguishing the breast cancer patients and controls explained by AUC was 0.728, with 70.6% sensitivity and 65.1% specificity. Our study results therefore confirmed polygenic breast cancer inheritance with important involvement of FGFR2, MAP3K1, LSP1 and CASP8 gene variants.


Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Adulto , Idoso , Algoritmos , Estudos de Casos e Controles , Caspase 8/genética , Feminino , Frequência do Gene , Humanos , MAP Quinase Quinase Quinase 1/genética , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Penetrância , Estudos Prospectivos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética
11.
Mol Med Rep ; 19(6): 4919-4926, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30942422

RESUMO

Cervical cancer is a malignancy that threatens female health. The present study aimed to investigate the role of transgelin 2 (TAGLN2) in cervical cancer. Reverse transcription­quantitative polymerase chain reaction and western blotting were conducted to detect the mRNA and protein expression levels of particular factors in HeLa cells. Cell Counting kit­8, wound healing and Transwell assays were conducted to determine cell viability, and migratory and invasive abilities, respectively. The results demonstrated that the expression levels of TAGLN2 were decreased in cervical cancer tissues and were associated with the survival time of patients with cervical cancer. In addition, the expression of TAGLN2 was significantly reduced in three cervical cancer cell lines (HeLa, SiHa and C­33A) compared with in a normal cervical cell line. The present study also demonstrated that TAGLN2 overexpression in HeLa cells could inhibit cell viability, migration and invasion, and it was suggested that this may occur via upregulation of the expression levels of E­cadherin and inhibitor of nuclear factor κ­light­chain­enhancer of activated B cells (NF­κB) (IκB), and downregulation of C­X­C chemokine receptor type 4, matrix metalloproteinase (MMP)­2, MMP­9, p50 and transcription factor p65. In conclusion, TAGLN2 was revealed to inhibit cell viability, and the migratory and invasive abilities of HeLa cervical cancer cells via regulating the expression of metastasis­associated factors and the NF­κB signaling pathway. The present study proposed a novel target gene for the diagnosis, treatment and prognosis of cervical cancer.


Assuntos
Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Neoplasias do Colo do Útero/patologia , Adulto , Idoso , Caderinas/metabolismo , Linhagem Celular , Movimento Celular , Sobrevivência Celular , Feminino , Células HeLa , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Proteínas Musculares/genética , NF-kappa B/metabolismo , Estadiamento de Neoplasias , Receptores CXCR4/metabolismo , Transdução de Sinais , Regulação para Cima , Neoplasias do Colo do Útero/metabolismo
12.
Gene Expr Patterns ; 32: 53-66, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30940554

RESUMO

We have cloned and characterized an intronic fragment of zebrafish lymphocyte cytosolic protein 1 (lcp1, also called L-plastin) that drives expression to the zebrafish enveloping layer (EVL). L-plastin is a calcium-dependent actin-bundling protein belonging to the plastin/fimbrin family of proteins, and is necessary for the proper migration and attachment of several adult cell types, including leukocytes and osteoclasts. However, in zebrafish lcp1 is abundantly expressed much earlier, during differentiation of the EVL. The cells of this epithelial layer migrate collectively, spreading vegetally over the yolk. L-plastin expression persists into the larval periderm, a transient epithelial tissue that forms the first larval skin. This finding establishes that L-plastin is activated in two different embryonic waves, with a distinct regulatory switch between the early EVL and the later leukocyte. To better study L-plastin expressing cells we attempted CRISPR/Cas9 homology-driven recombination (HDR) to insert a self-cleaving peptide (Cre-P2A-EGFP-CAAX) downstream of the native lcp1 promoter. This produced a stable zebrafish line expressing Cre recombinase in EVL nuclei and green fluorescence in EVL cell membranes. In vivo tracking of these labeled cells provided enhanced views of EVL migration behavior, membrane extensions, and mitotic events. Finally, we experimentally dissected key elements of the targeted lcp1 locus, discovering a ∼300 bp intronic sequence sufficient to drive EVL expression. The lcp1: Cre-P2A-EGFP-CAAX zebrafish should be useful for studying enveloping layer specification, gastrulation movements and periderm development in this widely used vertebrate model. In addition, the conserved regulatory sequences we have isolated predict that L-plastin orthologs may have a similar early expression pattern in other vertebrate embryos.


Assuntos
Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Embrião não Mamífero/metabolismo , Epitélio/crescimento & desenvolvimento , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Sequências Reguladoras de Ácido Nucleico/genética , Transcriptoma/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
13.
Reprod Domest Anim ; 54(6): 882-891, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974481

RESUMO

Dairy cow mastitis is a detrimental factor in milk quality and food safety. Mastitis generally refers to inflammation caused by infection by pathogenic microorganisms. Our studies in recent years have revealed the role of miRNA regulation in Staphylococcus aureus-induced mastitis. In the present study, we overexpressed and suppressed miR-145 to investigate the function of miR-145 in Mac-T cells. Flow cytometry, ELISA and EdU staining were used to detect changes in the secretion of several Mac-T cytokines and in cell proliferation. We found that overexpression of miR-145 in Mac-T cells significantly reduced the secretion of IL-12 and TNF-α, but increased the secretion of IFN-γ; the proliferation of bovine mammary epithelial cells was also inhibited. Using quantitative real-time PCR (qRT-PCR), Western blotting and luciferase multiplex verification techniques, we found that miR-145 targeted and regulated FSCN1. Knock-down of FSCN1 significantly increased the secretion of IL-12, while the secretion of TNF-α was significantly downregulated in Mac-T cells. Upon S. aureus infection of mammary gland tissue, the body initiated inflammatory responses; Bta-miR-145 expression was downregulated, which reduced the inhibitory effect on the FSCN1 gene; and upregulation of FSCN1 expression promoted mammary epithelial cell proliferation to allow the recovery of damaged tissue. The results of the present study will aid in understanding the immune mechanism opposing S. aureus infection in dairy cows and will provide a laboratory research basis for the prevention and treatment of mastitis.


Assuntos
Proteínas de Transporte/metabolismo , Mastite Bovina/imunologia , MicroRNAs/metabolismo , Proteínas dos Microfilamentos/metabolismo , Infecções Estafilocócicas/veterinária , Animais , Proteínas de Transporte/genética , Bovinos , Linhagem Celular , Proliferação de Células , Citocinas/metabolismo , Células Epiteliais/fisiologia , Feminino , Proteínas dos Microfilamentos/genética , Infecções Estafilocócicas/imunologia , Staphylococcus aureus
14.
Nat Cell Biol ; 21(5): 592-602, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962575

RESUMO

Inverted formin 2 (INF2) is a member of the formin family of actin assembly factors. Dominant missense mutations in INF2 are linked to two diseases: focal segmental glomerulosclerosis, a kidney disease, and Charcot-Marie-Tooth disease, a neuropathy. All of the disease mutations map to the autoinhibitory diaphanous inhibitory domain. Interestingly, purified INF2 is not autoinhibited, suggesting the existence of other cellular inhibitors. Here, we purified an INF2 inhibitor from mouse brain tissue, and identified it as a complex of lysine-acetylated actin (KAc-actin) and cyclase-associated protein (CAP). Inhibition of INF2 by CAP-KAc-actin is dependent on the INF2 diaphanous inhibitory domain (DID). Treatment of CAP-KAc-actin-inhibited INF2 with histone deacetylase 6 releases INF2 inhibition, whereas inhibitors of histone deacetylase 6 block the activation of cellular INF2. Disease-associated INF2 mutants are poorly inhibited by CAP-KAc-actin, suggesting that focal segmental glomerulosclerosis and Charcot-Marie-Tooth disease result from reduced CAP-KAc-actin binding. These findings reveal a role for KAc-actin in the regulation of an actin assembly factor by a mechanism that we call facilitated autoinhibition.


Assuntos
Actinas/genética , Proteínas de Transporte/genética , Doença de Charcot-Marie-Tooth/genética , Glomerulosclerose Segmentar e Focal/genética , Proteínas dos Microfilamentos/genética , Actinas/química , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Transporte/química , Doença de Charcot-Marie-Tooth/patologia , Glomerulosclerose Segmentar e Focal/patologia , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/genética , Humanos , Lisina/química , Camundongos , Proteínas dos Microfilamentos/química , Mutação , Ligação Proteica , Domínios Proteicos/genética
15.
Zhonghua Zhong Liu Za Zhi ; 41(2): 91-96, 2019 Feb 23.
Artigo em Chinês | MEDLINE | ID: mdl-30862136

RESUMO

Objective: To investigate the expression of microRNA-133b (miR-133b) in esophageal squamous cell carcinoma (ESCC), and explore its effect and the underlying molecular mechanisms on cell proliferation and invasion. Methods: Real-time quantitative PCR (qPCR) was used to examine miR-133b expression in 63 ESCC tissues and paired adjacent non-cancerous tissues, several ESCC cells (Eca109, EC9706, EC1, TE1, KYSE70) and normal esophageal epithelial cell Het-1A. MiR-133b mimic, inhibitor and negative control (NC) were transfected into TE1 cells. The effect of miR-133b on cell proliferation and invasion were determined by CCK-8 and Transwell assays, respectively. Subsequently, the target gene of miR-133b was predicted by online tools TargetScan and miRDB, which was verified by dual luciferase reporter assays. Finally, Western blot was utilized to detect the effects of miR-133b overexpression on expression of target gene TAGLN2 as well as EMT-related proteins E-cadherin, N-cadherin, Snail, Slug and Vimentin. Results: Relative levels of miR-133b in ESCC tissues (0.295±0.040) were significantly lower than those in adjacent non-cancerous tissues (1.002±0.011, P<0.001). The expression of miR-133b was tightly associated with clinical staging, lymph node metastasis and prognosis. Moreover, relative levels of miR-133b in ESCC cells Eca109, EC9706, EC1, TE1 and KYSE70 (0.679±0.031, 0.391±0.008, 0.236±0.016, 0.031±0.005 and 0.099±0.020) were evidently lower than that in normal esophageal epithelial cell Het-1A (1.005±0.016, all P<0.001). In TE1 cells, miR-133b mimic significantly increased the level of miR-133b to 6.199±0.627, and suppressed cell proliferation and invasion, whereas miR-133b inhibitor obviously decreased its expression to 0.182±0.023, and promoted cell proliferation and invasion. Most notably, the relative luciferase activities of miR-133b-mimic group (0.320±0.018) in TE1 cells transfected with TAGLN-3'UTR-WT were markedly lower than that in NC group (1.010±0.036, P<0.001), whereas those in TAGLN-3'UTR-MUT transfection cells were 1.019±0.056 and 1.008±0.021, respectively, showing no significantly statistical difference (P>0.05). Furthermore, miR-133b overexpression markedly downregulated TAGLN2, N-cadherin, Snail, Slug and Vimentin levels, and increased E-cadherin expression. Conclusion: MiR-133b plays an important role in the proliferation and invasion of ESCC cells by regulating TAGLN2 expression, and it may be a potential therapeutic target for ESCC patients.


Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , MicroRNAs/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Invasividade Neoplásica , Sincalida/metabolismo
16.
Microvasc Res ; 124: 43-50, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30853343

RESUMO

Pulmonary arterial hypertension (PAH) is a devastating and fatal vascular disease for which currently there is no satisfying therapy available. Excessive cell proliferation of pulmonary arterial smooth muscle cells (PASMCs) contributes significantly to PAH pathogenesis. In this study, we found that miR-205-5p was lowly expressed in hypoxia-induced PAH mouse model and hypoxia-treated PASMCs. Restoration of miR-205-5p suppressed PASMCs proliferation. In contrast, molecule interacting with CasL 2 (MICAL2) was highly expressed in hypoxia-induced PAH mouse model and hypoxia-treated PASMCs. Overexpression of MICAL2 promoted cell proliferation. Furthermore, miR-205-5p inhibited MICAL2 expression levels by targeting the MICAL2 3' untranslated region. In addition, MICAL2 activated ERK1/2 signaling in PASMCs and ERK1/2 inhibitor blocked MICAL2-mediated-promotion effect on PASMCs proliferation. These results demonstrated that miR-205-5p suppressed PASMCs proliferation by targeting MICAL2, which activated ERK1/2 signaling. Therefore, miR-205-5p/MICAL2/Erk1/2 may serve as an ideal therapeutic target to PAH treatment.


Assuntos
Proliferação de Células , Proteínas do Citoesqueleto/metabolismo , Hipertensão Pulmonar/enzimologia , MicroRNAs/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Oxirredutases/metabolismo , Animais , Células Cultivadas , Proteínas do Citoesqueleto/genética , Modelos Animais de Doenças , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipóxia/complicações , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Proteínas dos Microfilamentos/genética , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Oxirredutases/genética , Artéria Pulmonar/enzimologia , Artéria Pulmonar/patologia , Transdução de Sinais , Remodelação Vascular
17.
J Exp Clin Cancer Res ; 38(1): 111, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823924

RESUMO

BACKGROUND: The effect of competing endogenous RNA (ceRNA) can regulate gene expression by competitively binding microRNAs. Fascin-1 (FSCN1) plays an important role in the regulation of cellular migration and invasion during tumor progression, but how its regulatory mechanism works through the ceRNA effect is still unclear in bladder cancer (BLCA). METHODS: The role of fascin-1, miR-200b, and ZEB1-AS1 in BLCA was investigated in vitro and in vivo. The interaction between fascin-1, miR-200b, and ZEB1-AS1 was identified using bioinformatics analysis, luciferase activity assays, RNA-binding protein immunoprecipitation (RIP), quantitative PCR, and western blotting. Loss (or gain)-of-function experiments were performed to investigate the biological roles of miR-200b and ZEB1-AS1 on migration, invasion, proliferation, cell apoptosis, and cell cycle. RESULTS: ZEB1-AS1 functions as a competing endogenous RNA in BLCA to regulate the expression of fascin-1 through miR-200b. Moreover, the oncogenic long non-coding RNA ZEB1-AS1 was highly expressed in BLCA and positively correlated with high tumor grade, high TNM stage, and reduced survival of patients with BLCA. Moreover, ZEB1-AS1 downregulated the expression of miR-200b, promoted migration, invasion, and proliferation, and inhibited apoptosis in BLCA. Furthermore, we found TGF-ß1 induced migration and invasion in BLCA by regulating the ZEB1-AS1/miR-200b/FSCN1 axis. CONCLUSION: The observations in this study identify an important regulatory mechanism of fascin-1 in BLCA, and the TGF-ß1/ZEB1-AS1/miR-200b/FSCN1 axis may serve as a potential target for cancer therapeutic purposes.


Assuntos
Proteínas de Transporte/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Proteínas dos Microfilamentos/genética , RNA Longo não Codificante/genética , Neoplasias da Bexiga Urinária/patologia , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Transdução de Sinais/genética , Fator de Crescimento Transformador beta1/metabolismo , Neoplasias da Bexiga Urinária/genética
18.
Biomed Res Int ; 2019: 4379639, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30834264

RESUMO

Objective: To study the protective effect of Echinacoside for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced dopaminergic (DA) neurons injury in the subacute mouse model of Parkinson's disease (PD) and to explore its mechanism of action. Methods: We chose 10 weeks of healthy wild type C57BL/6 male mice, hypodermic MPTP 30 mg/kg/day, five days, to prepare PD subacute mouse model. Behavior indexes of open field test and pole test were applied to examine the function of ECH to PD subacute mice model of PD sample action. The effects of ECH on dopaminergic neurons and astrocyte were examined using Immunohistochemistry including tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP) expression. The total numbers of TH-positive neurons and GFAP-positive cells in the substantia nigra pars compacts (SNpc) and ventral tegmental area (VTA) were obtained stereologically using the optical fractionator method. Enzyme-linked immunosorbent assay (ELISA) method was used to detect the inflammatory cytokines in the serum, including TNF-α (Ttumor necrosis factor alpha) and IFN-γ (interferon gamma). Protein expressions of ionized calcium binding adaptor molecule 1 (IBA-1), TNF-α, Cleaved caspase-3, glial derived neurotrophic factor (GDNF), and phosphorylated and total extracellular signal-regulated kinase (p-ERK and ERK) in the anatomical region of substantia nigra (SN) were tested by protein immunoblot method (i.e., Western blotting). Results: ECH reversed the reduction of total distance in open field test in MPTP-induced PD model mice (P < 0.01), shortened the return time and total time of PD subacute model mice in pole test (P < 0.01, P < 0.05), significantly reversed the reduction of TH positive neurons induced by MPTP (P < 0.05), and reduced the activation of astrocytes (P < 0.05). Meanwhile, ECH significantly inhibited the expression of IBA-1, Cleaved caspase-3, and TNF-α in midbrain of MPTP model mice (P < 0.05, P < 0.05, and P < 0.05) and upregulated the expression of GDNF (P < 0.05). And ECH lowered the level of TNF-α and IFN-γ in serum (P < 0.05, P < 0.05). Conclusion: ECH has protective effects on the MPTP subacute model mice, its mechanism may be through inhibiting activation of microglia and astrocytes, reducing inflammatory reaction and promoting the secretion of neurotrophic factors, and eventually resulting in the reduction of the DA neurons apoptosis.


Assuntos
Glicosídeos/administração & dosagem , Intoxicação por MPTP/tratamento farmacológico , Fármacos Neuroprotetores/administração & dosagem , Doença de Parkinson/tratamento farmacológico , Animais , Proteínas de Ligação ao Cálcio/genética , Caspase 3/genética , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/genética , Humanos , Imuno-Histoquímica , Intoxicação por MPTP/genética , Intoxicação por MPTP/patologia , Camundongos , Proteínas dos Microfilamentos/genética , Microglia/efeitos dos fármacos , Microglia/patologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Substância Negra/efeitos dos fármacos , Substância Negra/patologia , Fator de Necrose Tumoral alfa/genética , Tirosina 3-Mono-Oxigenase/genética
19.
Mol Med Rep ; 19(4): 2801-2807, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816434

RESUMO

Hereditary spherocytosis (HS) is a common heterogeneous type of inherited hemolytic anemia characterized by jaundice and splenomegaly. Diagnosis of HS in neonates is considered unreliable, and is generally based on positive family history, spherocytes in peripheral smears, increased osmotic fragility, and jaundice. In the present study, routine laboratory tests, next­generation sequencing, and Sanger sequencing were applied to diagnose a neonatal patient with Coombs­negative hemolytic jaundice. The neonate had no family history of HS; however, spherocytes were observed in peripheral smears, and the patient exhibited Coombs­negative and severe hemolytic jaundice, normal mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular volume (MCV), normal glucose­6­phosphate dehydrogenase activity, negative thalassemia genetic mutation screening results, and negative autoimmune antibody tests. Novel compound heterozygous mutations in the spectrin­α, erythrocytic 1 (SPTA1) gene (c.3897­1G>C and c.5029G>A) were identified. The SPTA1 c.3897­1G>C mutation in intron 27­1, which disrupted the consensus splice site, was inherited from his asymptomatic mother, and the SPTA1 c.5029G>A (p.Gly1677Arg) mutation in trans with the SPTA1 c.3897­1G>C mutation was inherited from his asymptomatic father. Sanger sequencing of mRNA reverse transcribed into cDNA identified a deletion of the first 10 nucleotides of exon 28, confirming the splicing mutation. In conclusion, the present study reports a rare case of autosomal­recessive HS with a severe clinical phenotype, but normal MCHC and MCV.


Assuntos
Proteínas de Transporte/genética , Heterozigoto , Icterícia/diagnóstico , Proteínas dos Microfilamentos/genética , Mutação , Esferocitose Hereditária/diagnóstico , Esferocitose Hereditária/genética , Adulto , Alelos , Proteínas de Transporte/química , Biologia Computacional/métodos , Análise Mutacional de DNA , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recém-Nascido , Masculino , Proteínas dos Microfilamentos/química , Modelos Moleculares , Linhagem , Fenótipo , Relação Estrutura-Atividade
20.
Gastroenterology ; 157(1): 163-178, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30885780

RESUMO

BACKGROUND & AIMS: The peroxisome proliferator-activated receptor delta (PPARD) regulates cell metabolism, proliferation, and inflammation and has been associated with gastric and other cancers. Villin-positive epithelial cells are a small population of quiescent gastric progenitor cells. We expressed PPARD from a villin promoter to investigate the role of these cells and PPARD in development of gastric cancer. METHODS: We analyzed gastric tissues from mice that express the Ppard (PPARD1 and PPARD2 mice) from a villin promoter, and mice that did not carry this transgene (controls), by histology and immunohistochemistry. We performed cell lineage-tracing experiments and analyzed the microbiomes, chemokine and cytokine production, and immune cells and transcriptomes of stomachs of these mice. We also performed immunohistochemical analysis of PPARD levels in 2 sets of human gastric tissue microarrays. RESULTS: Thirty-eight percent of PPARD mice developed spontaneous, invasive gastric adenocarcinomas, with severe chronic inflammation. Levels of PPARD were increased in human gastric cancer tissues, compared with nontumor tissues, and associated with gastric cancer stage and grade. We found an inverse correlation between level of PPARD in tumor tissue and patient survival time. Gastric microbiomes from PPARD and control mice did not differ significantly. Lineage-tracing experiments identified villin-expressing gastric progenitor cells (VGPCs) as the origin of gastric tumors in PPARD mice. In these mice, PPARD up-regulated CCL20 and CXCL1, which increased infiltration of the gastric mucosa by immune cells. Immune cell production of inflammatory cytokines promoted chronic gastric inflammation and expansion and transformation of VGPCs, leading to tumorigenesis. We identified a positive-feedback loop between PPARD and interferon gamma signaling that sustained gastric inflammation to induce VGPC transformation and gastric carcinogenesis. CONCLUSIONS: We found PPARD overexpression in VPGCs to result in inflammation, dysplasia, and tumor formation. PPARD and VGPCs might be therapeutic targets for stomach cancer.


Assuntos
Carcinogênese/genética , Transformação Celular Neoplásica/genética , Citocinas/imunologia , Mucosa Gástrica/metabolismo , Interferon gama/imunologia , Receptores Citoplasmáticos e Nucleares/genética , Células-Tronco/metabolismo , Estômago/imunologia , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Animais , Carcinogênese/imunologia , Linhagem da Célula , Transformação Celular Neoplásica/imunologia , Quimiocina CCL20/metabolismo , Quimiocina CXCL1/metabolismo , Quimiocinas , Retroalimentação Fisiológica , Perfilação da Expressão Gênica , Inflamação , Camundongos , Microbiota/imunologia , Proteínas dos Microfilamentos/genética , Células-Tronco/imunologia , Estômago/microbiologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/imunologia
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