Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 11.283
Filtrar
1.
Curr Protoc ; 1(9): e246, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34529358

RESUMO

Natural killer (NK) cells are potent innate immune cells that provide the surveillance and elimination of infected, stressed, and malignant cells. The unique immune recognition mechanisms and functions of NK cells make them an attractive cell type for immunology research and adoptive immunotherapy. However, primary NK cells are challenging to culture ex vivo and lack efficient genetic tools, hindering the research of NK cells and the development of NK cell therapeutics. Here we describe methods for the freeze-thaw process, feeder-free ex vivo expansion, CRISPR-Cas9 genome editing, and functional characterizations of primary human NK cells. Our protocol enables ∼30-fold and ∼2000-fold average expansion rates from 1 × 107 cryopreserved NK cells in 14 and 28 days, respectively. We also detail methods for CRISPR gene knockout and knockin by nucleofection of Cas9 ribonucleoproteins (RNP) and DNA repair templates. Gene knockout by Cas9 RNP nucleofection can be multiplexed to simultaneously target three genes. The CRISPR-edited cells can be cryopreserved and rethawed with high viability for future studies. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Thawing of natural killer cells Basic Protocol 2: Ex vivo expansion of natural killer cells Basic Protocol 3: Cryopreservation of expanded natural killer cells Basic Protocol 4: Characterization of natural killer cells: Flow cytometry and surface marker analysis Basic Protocol 5: Cytotoxicity and degranulation assays Basic Protocol 6: Preparation of homology-directed repair templates Basic Protocol 7: Nucleofection of CRISPR-Cas9 ribonucleoproteins Basic Protocol 8: Genotyping of gene-edited natural killer cells Basic Protocol 9: Phenotyping of gene-edited natural killer cells.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Técnicas de Inativação de Genes , Humanos , Imunoterapia Adotiva , Células Matadoras Naturais
2.
Anticancer Res ; 41(9): 4287-4294, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34475048

RESUMO

BACKGROUND/AIM: Sunitinib continues to be administered as a first-line therapeutic agent in metastatic renal cell carcinoma (mRCC). This study examined the potential role of p53 in sunitinib resistance and as a predictive marker in mRCC. MATERIALS AND METHODS: We analysed the effects of p53 knockout on sunitinib resistance. p53 expression in 53 mRCC patients receiving first-line sunitinib was determined immunohistochemically. We performed in silico analysis to examine the predictive value of p53 in mRCC. RESULTS: WST-1 assays showed that p53 knockout decreased sensitivity to sunitinib. Sunitinib and nutlin-3 together suppressed cell growth. Immunohistochemistry revealed 11 p53-positive cases among 53 patients with mRCC. Kaplan-Meier analysis showed that p53-positive cases tended to be associated with poor progression-free survival (PFS) after first-line sunitinib treatment. In the JAVELIN 101 study, TP53 mutation was significantly associated with poor PFS after sunitinib treatment. CONCLUSION: p53 may be involved in sunitinib resistance and represent a valuable marker for sunitinib treatment in mRCC.


Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Imidazóis/farmacologia , Neoplasias Renais/tratamento farmacológico , Piperazinas/farmacologia , Sunitinibe/farmacologia , Proteína Supressora de Tumor p53/genética , Regulação para Cima , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Proliferação de Células/efeitos dos fármacos , Simulação por Computador , Sinergismo Farmacológico , Feminino , Técnicas de Inativação de Genes , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Masculino , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Estudos Retrospectivos , Resultado do Tratamento , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos
3.
Nat Commun ; 12(1): 5240, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475390

RESUMO

ß-actin is a crucial component of several chromatin remodeling complexes that control chromatin structure and accessibility. The mammalian Brahma-associated factor (BAF) is one such complex that plays essential roles in development and differentiation by regulating the chromatin state of critical genes and opposing the repressive activity of polycomb repressive complexes (PRCs). While previous work has shown that ß-actin loss can lead to extensive changes in gene expression and heterochromatin organization, it is not known if changes in ß-actin levels can directly influence chromatin remodeling activities of BAF and polycomb proteins. Here we conduct a comprehensive genomic analysis of ß-actin knockout mouse embryonic fibroblasts (MEFs) using ATAC-Seq, HiC-seq, RNA-Seq and ChIP-Seq of various epigenetic marks. We demonstrate that ß-actin levels can induce changes in chromatin structure by affecting the complex interplay between chromatin remodelers such as BAF/BRG1 and EZH2. Our results show that changes in ß-actin levels and associated chromatin remodeling activities can not only impact local chromatin accessibility but also induce reversible changes in 3D genome architecture. Our findings reveal that ß-actin-dependent chromatin remodeling plays a role in shaping the chromatin landscape and influences the regulation of genes involved in development and differentiation.


Assuntos
Actinas/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Actinas/genética , Animais , Cromatina/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Fibroblastos , Dosagem de Genes , Técnicas de Inativação de Genes , Histonas/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Ligação Proteica , Fatores de Transcrição/metabolismo
4.
Int J Mol Sci ; 22(16)2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34445101

RESUMO

Vps35 (vacuolar protein sorting 35) is a key component of retromer that consists of Vps35, Vps26, and Vps29 trimers, and sortin nexin dimers. Dysfunctional Vps35/retromer is believed to be a risk factor for development of various neurodegenerative diseases. Vps35Neurod6 mice, which selectively knock out Vps35 in Neurod6-Cre+ pyramidal neurons, exhibit age-dependent impairments in terminal differentiation of dendrites and axons of cortical and hippocampal neurons, neuro-degenerative pathology (i.e., increases in P62 and Tdp43 (TAR DNA-binding protein 43) proteins, cell death, and reactive gliosis), and neonatal death. The relationships among these phenotypes and the underlying mechanisms remain largely unclear. Here, we provide evidence that expression of low level of VPS35-mCherry fusion protein in Vps35Neurod6 mice could diminish the phenotypes in an age-dependent manner. Specifically, we have generated a conditional transgenic mouse line, LSL-Vps35-mCherry, which expresses VPS35-mCherry fusion protein in a Cre-dependent manner. Crossing LSL-Vps35-mCherry with Vps35Neurod6 to obtain TgVPS35-mCherry, Vps35Neurod6 mice prevent the neonatal death and diminish the dendritic morphogenesis deficit and gliosis at the neonatal, but not the adult age. Further studies revealed that the Vps35-mCherry transgene expression was low, and the level of Vps35 mRNA comprised only ~5-7% of the Vps35 mRNA of control mice. Such low level of VPS35-mCherry could restore the amount of other retromer components (Vps26a and Vps29) at the neonatal age (P14). Importantly, the neurodegenerative pathology presented in the survived adult TgVps35-mCherry; Vps35Neurod6 mice. These results demonstrate the sufficiency of low level of VPS35-mCherry fusion protein to diminish the phenotypes in Vps35Neurod6 mice at the neonatal age, verifying a key role of neuronal Vps35 in stabilizing retromer complex proteins, and supporting the view for Vps35 as a potential therapeutic target for neurodegenerative diseases.


Assuntos
Doenças Neurodegenerativas/genética , Neurogênese , Neurônios/patologia , Proteínas de Transporte Vesicular/genética , Animais , Animais Recém-Nascidos , Feminino , Técnicas de Inativação de Genes , Humanos , Recém-Nascido , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doenças Neurodegenerativas/patologia , Neurônios/citologia , Neurônios/metabolismo , Morte Perinatal , Proteínas Recombinantes de Fusão/genética
5.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445433

RESUMO

The cornea is an anterior eye structure specialized for vision. The corneal endothelium and stroma are derived from the periocular mesenchyme (POM), which originates from neural crest cells (NCCs), while the stratified corneal epithelium develops from the surface ectoderm. Activating protein-2ß (AP-2ß) is highly expressed in the POM and important for anterior segment development. Using a mouse model in which AP-2ß is conditionally deleted in the NCCs (AP-2ß NCC KO), we investigated resulting corneal epithelial abnormalities. Through PAS and IHC staining, we observed structural and phenotypic changes to the epithelium associated with AP-2ß deletion. In addition to failure of the mutant epithelium to stratify, we also observed that Keratin-12, a marker of the differentiated epithelium, was absent, and Keratin-15, a limbal and conjunctival marker, was expanded across the central epithelium. Transcription factors PAX6 and P63 were not observed to be differentially expressed between WT and mutant. However, growth factor BMP4 was suppressed in the mutant epithelium. Given the non-NCC origin of the epithelium, we hypothesize that the abnormalities in the AP-2ß NCC KO mouse result from changes to regulatory signaling from the POM-derived stroma. Our findings suggest that stromal pathways such as Wnt/ß-Catenin signaling may regulate BMP4 expression, which influences cell fate and stratification.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Regulação para Baixo , Epitélio Corneano/anormalidades , Deleção de Genes , Fator de Transcrição AP-2/genética , Animais , Proteína Morfogenética Óssea 4/genética , Diferenciação Celular , Epitélio Corneano/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Queratina-12/metabolismo , Queratina-15/metabolismo , Masculino , Camundongos , Crista Neural/metabolismo , Fenótipo , Fator de Transcrição AP-2/metabolismo , Via de Sinalização Wnt
6.
Nat Commun ; 12(1): 4789, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373451

RESUMO

CRISPR-based cancer dependency maps are accelerating advances in cancer precision medicine, but adequate functional maps are limited to the most common oncogenes. To identify opportunities for therapeutic intervention in other rarer subsets of cancer, we investigate the oncogene-specific dependencies conferred by the lung cancer oncogene, RIT1. Here, genome-wide CRISPR screening in KRAS, EGFR, and RIT1-mutant isogenic lung cancer cells identifies shared and unique vulnerabilities of each oncogene. Combining this genetic data with small-molecule sensitivity profiling, we identify a unique vulnerability of RIT1-mutant cells to loss of spindle assembly checkpoint regulators. Oncogenic RIT1M90I weakens the spindle assembly checkpoint and perturbs mitotic timing, resulting in sensitivity to Aurora A inhibition. In addition, we observe synergy between mutant RIT1 and activation of YAP1 in multiple models and frequent nuclear overexpression of YAP1 in human primary RIT1-mutant lung tumors. These results provide a genome-wide atlas of oncogenic RIT1 functional interactions and identify components of the RAS pathway, spindle assembly checkpoint, and Hippo/YAP1 network as candidate therapeutic targets in RIT1-mutant lung cancer.


Assuntos
Neoplasias Pulmonares/genética , Oncogenes/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Receptores ErbB/genética , Feminino , Técnicas de Inativação de Genes , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Terapia de Alvo Molecular , Mutação , Células NIH 3T3 , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras
7.
Nat Commun ; 12(1): 4988, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404781

RESUMO

Glycans are fundamental cellular building blocks, involved in many organismal functions. Advances in glycomics are elucidating the essential roles of glycans. Still, it remains challenging to properly analyze large glycomics datasets, since the abundance of each glycan is dependent on many other glycans that share many intermediate biosynthetic steps. Furthermore, the overlap of measured glycans can be low across samples. We address these challenges with GlyCompare, a glycomic data analysis approach that accounts for shared biosynthetic steps for all measured glycans to correct for sparsity and non-independence in glycomics, which enables direct comparison of different glycoprofiles and increases statistical power. Using GlyCompare, we study diverse N-glycan profiles from glycoengineered erythropoietin. We obtain biologically meaningful clustering of mutant cell glycoprofiles and identify knockout-specific effects of fucosyltransferase mutants on tetra-antennary structures. We further analyze human milk oligosaccharide profiles and find mother's fucosyltransferase-dependent secretor-status indirectly impact the sialylation. Finally, we apply our method on mucin-type O-glycans, gangliosides, and site-specific compositional glycosylation data to reveal tissues and disease-specific glycan presentations. Our substructure-oriented approach will enable researchers to take full advantage of the growing power and size of glycomics data.


Assuntos
Vias Biossintéticas , Glicômica , Polissacarídeos/biossíntese , Transporte Biológico , Vias Biossintéticas/genética , Análise por Conglomerados , Análise de Dados , Eritropoetina/metabolismo , Fucosiltransferases/genética , Gangliosídeos , Técnicas de Inativação de Genes , Glicosilação , Humanos , Mucinas
8.
Nat Commun ; 12(1): 4980, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404792

RESUMO

Proximity labeling (PL) with genetically-targeted promiscuous enzymes has emerged as a powerful tool for unbiased proteome discovery. By combining the spatiotemporal specificity of PL with methods for functional protein enrichment, we show that it is possible to map specific protein subclasses within distinct compartments of living cells. In particular, we develop a method to enrich subcompartment-specific RNA binding proteins (RBPs) by combining peroxidase-catalyzed PL with organic-aqueous phase separation of crosslinked protein-RNA complexes ("APEX-PS"). We use APEX-PS to generate datasets of nuclear, nucleolar, and outer mitochondrial membrane (OMM) RBPs, which can be mined for novel functions. For example, we find that the OMM RBP SYNJ2BP retains specific nuclear-encoded mitochondrial mRNAs at the OMM during translation stress, facilitating their local translation and import of protein products into the mitochondrion during stress recovery. Functional PL in general, and APEX-PS in particular, represent versatile approaches for the discovery of proteins with novel function in specific subcellular compartments.


Assuntos
RNA Mensageiro/metabolismo , RNA Mitocondrial/metabolismo , Proteínas de Ligação a RNA/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Fosforilação , Proteoma/metabolismo , Proteômica , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética
9.
Nat Commun ; 12(1): 4990, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404808

RESUMO

Cells can expand their plasma membrane laterally by unfolding membrane undulations and by exocytosis. Here, we describe a third mechanism involving invaginations held shut by the membrane adapter, dynamin. Compartments open when Ca activates the lipid scramblase, TMEM16F, anionic phospholipids escape from the cytoplasmic monolayer in exchange for neutral lipids, and dynamins relax. Deletion of TMEM16F or dynamins blocks expansion, with loss of dynamin expression generating a maximally expanded basal plasma membrane state. Re-expression of dynamin2 or its GTPase-inactivated mutant, but not a lipid binding mutant, regenerates reserve compartments and rescues expansion. Dynamin2-GFP fusion proteins form punctae that rapidly dissipate from these compartments during TMEM16F activation. Newly exposed compartments extend deeply into the cytoplasm, lack numerous organellar markers, and remain closure-competent for many seconds. Without Ca, compartments open slowly when dynamins are sequestered by cytoplasmic dynamin antibodies or when scrambling is mimicked by neutralizing anionic phospholipids and supplementing neutral lipids. Activation of Ca-permeable mechanosensitive channels via cell swelling or channel agonists opens the compartments in parallel with phospholipid scrambling. Thus, dynamins and TMEM16F control large plasma membrane reserves that open in response to lateral membrane stress and Ca influx.


Assuntos
Anoctaminas/metabolismo , Membrana Celular/metabolismo , Dinaminas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Anoctaminas/genética , Cálcio/metabolismo , Citoplasma , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Membranas/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Fosfolipídeos/metabolismo
10.
Sheng Li Xue Bao ; 73(4): 577-583, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34405214

RESUMO

The objective of this study was to explore the roles of arachidonic acid cytochrome P450ω hydroxylase CYP4A14 in skeletal muscle regeneration after injury. Wild-type (WT) control mice and Cyp4a14 knockout (A14-/-) mice were used to establish the muscle injury and regeneration model by intramuscular injection with cardiotoxin (CTX) on the tibial anterior (TA) muscle. The TA muscles were harvested at the time points of 0, 3, 5 and 15 days after injury. The changes in skeletal muscle regeneration and fibrosis were assessed by wheat germ agglutinin (WGA) staining and Sirius Red staining. Immunohistochemical staining was used to observe the expression of proliferation-related protein Ki-67 and macrophage marker protein Mac-2. The mRNA levels of regeneration and inflammation associated genes were analyzed by real-time PCR. The results showed that the cross-section area (CSA) of regenerated myofibers in A14-/- mice was significantly smaller (P < 0.05), while the percentage of fibrosis area was significantly higher than those in WT mice at 15 days after injury (P < 0.05). In A14-/- muscles, both the ratio of Ki-67 positive proliferating cells and the mRNA levels of differentiation associated genes Myod1 and Myog were significantly lower than those in WT muscles (P < 0.05). At 3 days after injury, the mRNA expression of inflammatory cells marker genes CD45 and CD11b and Mac-2 positive macrophages in A14-/- muscles were significantly lower than those in WT skeletal muscle (P < 0.05). Macrophages derived pro-regeneration cytokines IL-1ß, IGF-1 and SDF-1 were also significantly decreased in A14-/- muscles (P < 0.05). These results suggest that arachidonic acid cytochrome P450ω hydroxylase CYP4A14 plays a critical role in skeletal muscle regeneration after injury.


Assuntos
Oxigenases de Função Mista , Regeneração , Animais , Ácido Araquidônico , Citocromos , Técnicas de Inativação de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético
11.
Nucleic Acids Res ; 49(13): 7476-7491, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34197614

RESUMO

Poly (ADP-ribose) polymerase inhibitor (PARPi)-based therapies initially reduce tumor burden but eventually lead to acquired resistance in cancer patients with BRCA1 or BRCA2 mutation. To understand the potential PARPi resistance mechanisms, we performed whole-genome CRISPR screens to discover genetic alterations that change the gene essentiality in cells with inducible depletion of BRCA2. We identified that several RNA Polymerase II transcription Mediator complex components, especially Cyclin C (CCNC) as synthetic survival targets upon BRCA2 loss. Total mRNA sequencing demonstrated that loss of CCNC could activate the transforming growth factor (TGF)-beta signaling pathway and extracellular matrix (ECM)-receptor interaction pathway, however the inhibition of these pathways could not reverse cell survival in BRCA2 depleted CCNC-knockout cells, indicating that the activation of these pathways is not required for the resistance. Moreover, we showed that the improved survival is not due to restoration of homologous recombination repair although decreased DNA damage signaling was observed. Interestingly, loss of CCNC could restore replication fork stability in BRCA2 deficient cells, which may contribute to PARPi resistance. Taken together, our data reveal CCNC as a critical genetic determinant upon BRCA2 loss of function, which may help the development of novel therapeutic strategies that overcome PARPi resistance.


Assuntos
Proteína BRCA2/genética , Ciclina C/genética , Proteína BRCA2/metabolismo , Sistemas CRISPR-Cas , Sobrevivência Celular , Dano ao DNA , Replicação do DNA , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Complexo Mediador/genética , Complexo Mediador/fisiologia , Reparo de DNA por Recombinação , Estresse Fisiológico/genética
12.
Nucleic Acids Res ; 49(13): 7406-7423, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34214177

RESUMO

Heterochromatin binding protein HP1ß plays an important role in chromatin organization and cell differentiation, however the underlying mechanisms remain unclear. Here, we generated HP1ß-/- embryonic stem cells and observed reduced heterochromatin clustering and impaired differentiation. We found that during stem cell differentiation, HP1ß is phosphorylated at serine 89 by CK2, which creates a binding site for the pluripotency regulator KAP1. This phosphorylation dependent sequestration of KAP1 in heterochromatin compartments causes a downregulation of pluripotency factors and triggers pluripotency exit. Accordingly, HP1ß-/- and phospho-mutant cells exhibited impaired differentiation, while ubiquitination-deficient KAP1-/- cells had the opposite phenotype with enhanced differentiation. These results suggest that KAP1 regulates pluripotency via its ubiquitination activity. We propose that the formation of subnuclear membraneless heterochromatin compartments may serve as a dynamic reservoir to trap or release cellular factors. The sequestration of essential regulators defines a novel and active role of heterochromatin in gene regulation and represents a dynamic mode of remote control to regulate cellular processes like cell fate decisions.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Embrionárias/metabolismo , Heterocromatina/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Animais , Caseína Quinase II/metabolismo , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/fisiologia , Cricetinae , Células-Tronco Embrionárias/citologia , Técnicas de Inativação de Genes , Humanos , Camundongos , Fosforilação , Serina/metabolismo , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/fisiologia
13.
Sheng Li Xue Bao ; 73(3): 482-490, 2021 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-34230949

RESUMO

S100 calcium binding protein A9 (S100A9) is involved in a variety of biological processes such as inflammation and tumor cell migration and invasion regulation. The purpose of this study was to construct S100A9 gene-edited mice by using CRISPR/Cas9 technology, thereby providing an animal model for exploring the biological functions of this gene. According to the S100A9 gene sequence, the single-stranded small guide RNA (sgRNA) targeting exons 2 and 3 was transcribed in vitro, and a mixture of Cas9 mRNA and candidate sgRNA was injected into mouse fertilized eggs by microinjection. Early embryos were obtained and transferred to surrogate mice, and F0 mice were obtained and identified by PCR identification and gene sequencing. F0 mice were further mated with wild-type C57BL/6 mice to obtain F1 heterozygous mice, and then homozygous offspring were obtained through F1 mice self-crossing. Real-time PCR, Western blot and immunohistochemistry (IHC) were used to verify the expression and distribution of S100A9. In order to observe the pathological changes of mouse lung tissue using HE staining, an allergic asthma model was induced by ovalbumin from chicken egg white (OVA). The results showed that the 2 492 bp of exons 2, 3 of the S100A9 gene was successfully knocked out, and S100A9-/- mice with stable inheritance were obtained. Furthermore, it was found that S100A9 gene was highly expressed in the lung and spleen of wild-type mice. The expression of S100A9 mRNA and protein was not detected in the lung and spleen of S100A9-/- mice. However, compared with wild-type mice, the lungs of S100A9-/- mice showed a significantly worse inflammatory phenotype, and the proportion of eosinophils in bronchoalveolar lavage fluid (BALF) was significantly increased in response to the treatment of OVA. These results suggest we have successfully constructed a new strain of S100A9-/- mice, and preliminarily confirmed that the lack of S100A9 function can aggravate airway inflammation in asthmatic mice, providing a new mouse model for further study of S100A9 gene function.


Assuntos
Marcação de Genes , Animais , Líquido da Lavagem Broncoalveolar , Sistemas CRISPR-Cas/genética , Calgranulina B , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Pulmão , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina , Fenótipo
14.
Cancer Sci ; 112(9): 3846-3855, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34286904

RESUMO

Heterogeneous nuclear ribonucleoprotein L-like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial-mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT. The promoter activity was attributed to a region from -273 to -10 base pairs upstream of the transcription start site identified by 5'-RACE analysis, and the region contained potential binding sites for MYB and SP1. Luciferase reporter gene assays and knockdown or knockout experiments for genes encoding the MYB family proteins, MYB, MYBL1, and MYBL2, revealed that MYB was responsible for approximately half of the promoter activity. On the other hand, treatment with mithramycin A, an inhibitor for SP1 and SP3, suppressed the promoter activity and their additive contribution was confirmed by knockout experiments. The expression level of MYB was reduced on EMT while that of SP1 and SP3 was unchanged, suggesting that the downregulation of HNRNPLL during EMT was mediated by the decrease of MYB expression while SP1 and SP3 determine the basal transcription level of HNRNPLL. Histopathological analysis confirmed the accumulation of MYB-downregulated cancer cells at the invasion front of clinical CRC tissues. These results provide an insight into the molecular mechanism underlying CRC progression.


Assuntos
Neoplasias Colorretais/metabolismo , Transição Epitelial-Mesenquimal/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Sítios de Ligação , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Progressão da Doença , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HT29 , Humanos , Metástase Neoplásica , Plicamicina/análogos & derivados , Plicamicina/farmacologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myb/genética , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Transcrição Genética/genética , Transfecção
15.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208313

RESUMO

Intrahepatic cholangiocarcinoma (iCCA) is a heterogeneous bile duct cancer with a poor prognosis. Integrin αvß6 (ß6) has been shown to be upregulated in iCCA and is associated with its subclassification and clinicopathological features. In the present study, two ITGB6-knockout HuCCT1 CCA cell lines (ITGB6-ko cells) were established using the clustered regulatory interspaced short palindromic repeats (CRISPR), an associated nuclease 9 (Cas9) system, and single-cell cloning. RNA sequencing analysis, real-time polymerase chain reaction (PCR), and immunofluorescent methods were applied to explore possible downstream factors. ITGB6-ko cells showed significantly decreased expression of integrin ß6 on flow cytometric analysis. Both cell lines exhibited significant inhibition of cell migration and invasion, decreased wound-healing capability, decreased colony formation ability, and cell cycle dysregulation. RNA sequencing and real-time PCR analysis revealed a remarkable decrease in podocalyxin-like protein 2 (PODXL2) expression in ITGB6-ko cells. Colocalization of PODXL2 and integrin ß6 was also observed. S100 calcium-binding protein P and mucin 1, which are associated with CCA subclassification, were downregulated in ITGB6-ko cells. These results describe the successful generation of ITGB6-ko CCA cell clones with decreased migration and invasion and downregulation of PODXL2, suggesting the utility of integrin ß6 as a possible therapeutic target or diagnostic marker candidate.


Assuntos
Moléculas de Adesão Celular/metabolismo , Movimento Celular , Colangiocarcinoma/patologia , Técnicas de Inativação de Genes , Cadeias beta de Integrinas/genética , Sialoglicoproteínas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ligação ao Cálcio/metabolismo , Moléculas de Adesão Celular/genética , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Colangiocarcinoma/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Cadeias beta de Integrinas/metabolismo , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sialoglicoproteínas/genética , Ensaio Tumoral de Célula-Tronco , Regulação para Cima/genética
16.
Zhonghua Gan Zang Bing Za Zhi ; 29(6): 585-590, 2021 Jun 20.
Artigo em Chinês | MEDLINE | ID: mdl-34225436

RESUMO

Mdr2 knockout mice is a liver disease model, which causes cholestasis due to the lack of phospholipids in the bile. At present, it is not only used for the study of human homologous MDR3 gene, but also widely used as an animal model of liver diseases such as primary sclerosing cholangitis, liver fibrosis, progressive familial intrahepatic cholestasis, liver cancer. Herein, we review the Mdr2 knockout mice physiological characteristics and its application in liver disease research.


Assuntos
Colangite Esclerosante , Colestase Intra-Hepática , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Colangite Esclerosante/genética , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Fígado , Camundongos , Camundongos Knockout
17.
Viruses ; 13(6)2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205194

RESUMO

Mosquitoes are known as important vectors of many arthropod-borne (arbo)viruses causing disease in humans. These include dengue (DENV) and Zika (ZIKV) viruses. The exogenous small interfering (si)RNA (exo-siRNA) pathway is believed to be the main antiviral defense in arthropods, including mosquitoes. During infection, double-stranded RNAs that form during viral replication and infection are cleaved by the enzyme Dicer 2 (Dcr2) into virus-specific 21 nt vsiRNAs, which are subsequently loaded into Argonaute 2 (Ago2). Ago2 then targets and subsequently cleaves complementary RNA sequences, resulting in degradation of the target viral RNA. Although various studies using silencing approaches have supported the antiviral activity of the exo-siRNA pathway in mosquitoes, and despite strong similarities between the siRNA pathway in the Drosophila melanogaster model and mosquitoes, important questions remain unanswered. The antiviral activity of Ago2 against different arboviruses has been previously demonstrated. However, silencing of Ago2 had no effect on ZIKV replication, whereas Dcr2 knockout enhanced its replication. These findings raise the question as to the role of Ago2 and Dcr2 in the control of arboviruses from different viral families in mosquitoes. Using a newly established Ago2 knockout cell line, alongside the previously reported Dcr2 knockout cell line, we investigated the impact these proteins have on the modulation of different arboviral infections. Infection of Ago2 knockout cell line with alpha- and bunyaviruses resulted in an increase of viral replication, but not in the case of ZIKV. Analysis of small RNA sequencing data in the Ago2 knockout cells revealed a lack of methylated siRNAs from different sources, such as acute and persistently infecting viruses-, TE- and transcriptome-derived RNAs. The results confirmed the importance of the exo-siRNA pathway in the defense against arboviruses, but highlights variability in its response to different viruses and the impact the siRNA pathway proteins have in controlling viral replication. Moreover, this established Ago2 knockout cell line can be used for functional Ago2 studies, as well as research on the interplay between the RNAi pathways.


Assuntos
Aedes/genética , Aedes/virologia , Infecções por Arbovirus/transmissão , Infecções por Arbovirus/virologia , Arbovírus/fisiologia , Proteínas Argonauta/deficiência , Mosquitos Vetores/genética , Mosquitos Vetores/virologia , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno , Interferência de RNA , Replicação Viral
18.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298937

RESUMO

Trace amine-associated receptors (TAARs) are a group of G protein-coupled receptors that are expressed in the olfactory epithelium, central nervous system, and periphery. TAAR family generally consists of nine types of receptors (TAAR1-9), which can detect biogenic amines. During the last 5 years, the TAAR5 receptor became one of the most intriguing receptors in this subfamily. Recent studies revealed that TAAR5 is involved not only in sensing socially relevant odors but also in the regulation of dopamine and serotonin transmission, emotional regulation, and adult neurogenesis by providing significant input from the olfactory system to the limbic brain areas. Such results indicate that future antagonistic TAAR5-based therapies may have high pharmacological potential in the field of neuropsychiatric disorders. TAAR5 is known to be expressed in leucocytes as well. To evaluate potential hematological side effects of such future treatments we analyzed several hematological parameters in mice lacking TAAR5. In these mutants, we observed minor but significant changes in the osmotic fragility test of erythrocytes and hematocrit levels. At the same time, analysis of other parameters including complete blood count and reticulocyte levels showed no significant alterations in TAAR5 knockout mice. Thus, TAAR5 gene knockout leads to minor negative changes in the erythropoiesis or eryptosis processes, and further research in that field is needed. The impact of TAAR5 deficiency on other hematological parameters seems minimal. Such negative, albeit minor, effects of TAAR5 deficiency should be taken into account during future TAAR5-based therapy development.


Assuntos
Aminas Biogênicas/metabolismo , Eritrócitos/metabolismo , Fragilidade Osmótica/genética , Receptores Acoplados a Proteínas G/genética , Animais , Sistema Nervoso Central/metabolismo , Técnicas de Inativação de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa Olfatória/metabolismo
19.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299309

RESUMO

Rab GTPases are major coordinators of intracellular membrane trafficking, including vesicle transport, membrane fission, tethering, docking, and fusion events. Rab GTPases are roughly divided into two groups: conventional "small" Rab GTPases and atypical "large" Rab GTPases that have been recently reported. Some members of large Rab GTPases in mammals include Rab44, Rab45/RASEF, and Rab46. The genes of these large Rab GTPases commonly encode an amino-terminal EF-hand domain, coiled-coil domain, and the carboxyl-terminal Rab GTPase domain. A common feature of large Rab GTPases is that they express several isoforms in cells. For instance, Rab44's two isoforms have similar functions, but exhibit differential localization. The long form of Rab45 (Rab45-L) is abundantly distributed in epithelial cells. The short form of Rab45 (Rab45-S) is predominantly present in the testes. Both Rab46 (CRACR2A-L) and the short isoform lacking the Rab domain (CRACR2A-S) are expressed in T cells, whereas Rab46 is only distributed in endothelial cells. Although evidence regarding the function of large Rab GTPases has been accumulating recently, there are only a limited number of studies. Here, we report the recent findings on the large Rab GTPase family concerning their function in membrane trafficking, cell differentiation, related diseases, and knockout mouse phenotypes.


Assuntos
Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Feminino , Técnicas de Inativação de Genes , Humanos , Membranas Intracelulares/metabolismo , Masculino , Mastócitos/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Osteoclastos/citologia , Osteoclastos/metabolismo , Fenótipo , Domínios Proteicos , Linfócitos T/metabolismo , Proteínas rab de Ligação ao GTP/genética
20.
Front Cell Infect Microbiol ; 11: 706919, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34290994

RESUMO

Zinc finger and BTB domain containing 1(Zbtb1) is a transcriptional suppressor protein, and a member of the mammalian Zbtb gene family. Previous studies have shown that Zbtb1 is essential for T-cell development. However, the role of Zbtb1 in T-cell lymphoma is undetermined. In this study, an EL4 cell line with Zbtb1 deletion was constructed using the CRISPR-Cas9 technique. The expression profiles of microRNA and circRNA produced by the control and gene deletion groups were determined by RNA-seq. In general, 24 differentially expressed microRNA and 16 differentially expressed circRNA were found between normal group and gene deletion group. Through further analysis of differentially expressed genes, GO term histogram and KEGG scatter plot were drawn, and three pairs of miRNA and circRNA regulatory relationships were found. This study describes the differentially expressed microRNA and circRNA in normal and Zbtb1-deficient EL4 cell lines, thus providing potential targets for drug development and clinical treatment of T-cell lymphoma.


Assuntos
Linfoma de Células T/genética , MicroRNAs , RNA Circular , Proteínas Repressoras/genética , Animais , Diferenciação Celular , Linhagem Celular , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Camundongos , MicroRNAs/genética , RNA Circular/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...