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1.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361001

RESUMO

Epidermal progenitor cells divide symmetrically and asymmetrically to form stratified epidermis and hair follicles during late embryonic development. Flightless I (Flii), an actin remodelling protein, is implicated in Wnt/ß-cat and integrin signalling pathways that govern cell division. This study investigated the effect of altering Flii on the divisional orientation of epidermal progenitor cells (EpSCs) in the basal layer during late murine embryonic development and early adolescence. The effect of altering Flii expression on asymmetric vs. symmetric division was assessed in vitro in adult human primary keratinocytes and in vivo at late embryonic development stages (E16, E17 and E19) as well as adolescence (P21 day-old) in mice with altered Flii expression (Flii knockdown: Flii+/-, wild type: WT, transgenic Flii overexpressing: FliiTg/Tg) using Western blot and immunohistochemistry. Flii+/- embryonic skin showed increased asymmetrical cell division of EpSCs with an increase in epidermal stratification and elevated talin, activated-Itgb1 and Par3 expression. FliiTg/Tg led to increased symmetrical cell division of EpSCs with increased cell proliferation rate, an elevated epidermal SOX9, Flap1 and ß-cat expression, a thinner epidermis, but increased hair follicle number and depth. Flii promotes symmetric division of epidermal progenitor cells during murine embryonic development.


Assuntos
Divisão Celular , Proteínas dos Microfilamentos/genética , Células-Tronco Embrionárias Murinas/metabolismo , Pele/metabolismo , Transativadores/genética , Animais , Células Cultivadas , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Fatores de Transcrição SOX9/metabolismo , Pele/embriologia , Transativadores/metabolismo , beta Catenina/metabolismo
2.
Nat Commun ; 12(1): 4919, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389725

RESUMO

BRCA1 or BRCA2 germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and BRCA1/2 mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of ß-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed ß-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of CDKN1A gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, ß-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that ß-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Oncogenes/genética , Transcrição Genética/genética , beta Catenina/genética , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Feminino , Perfilação da Expressão Gênica/métodos , Células HeLa , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , RNA-Seq/métodos , beta Catenina/metabolismo
3.
Arch Oral Biol ; 130: 105219, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34364169

RESUMO

OBJECTIVE: The aim of this study was to investigate the role and molecular regulatory mechanisms of baicalin in oral squamous cell carcinoma (OSCC) progression. DESIGN: Gene expression in OSCC cells was detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR). OSCC cell viability, migration, invasion and stemness were measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), wound healing, Transwell, and sphere formation assays. The target genes of miR-106b-5p were predicted using bioinformatic tools. The interaction between microRNA-miR-106b-5p (miR-106b-5p) and disabled homolog 2 (DAB2) was confirmed by a luciferase reporter assay. TOP/FOP-Flash reporter assay and western blot analysis were used to analyze the activity of the Wnt/ß-catenin pathway. RESULTS: Baicalin inhibited OSCC cell viability, migration, invasion, and stemness. Baicalin downregulated miR-106b-5p expression. In addition, MiR-106b-5p upregulation reversed the effects of baicalin on OSCC cells. As a target gene of miR-106b-5p, DAB2 was negatively regulated by miR-106b-5p and upregulated by baicalin in OSCC cells. MiR-106b-5p activated Wnt/ß-catenin pathway in OSCC cells by inhibiting DAB2. Baicalin suppressed Wnt/ß-catenin pathway by upregulating DAB2. In rescue assays, miR-106b-5p overexpression-induced promotion of OSCC cellular processes was attenuated by DAB2 upregulation. CONCLUSIONS: Baicalin exerts anti-tumor effects in OSCC by inhibiting the miR-106b-5p-Wnt/ß-catenin pathway via upregulating DAB2.


Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , MicroRNAs , Neoplasias Bucais , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Flavonoides , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
4.
Arch Oral Biol ; 130: 105243, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34416564

RESUMO

OBJECTIVES: The aims of this study were to explore: (ⅰ) the effect of the polypeptide OP 3-4 on bone regeneration in vivo; (ⅱ) the effect of OP 3-4 on osteogenic differentiation of bone marrow mesenchymal stem cells in vitro; and (ⅲ) the potential mechanism of OP 3-4 in promoting osteogenic differentiation of bone marrow mesenchymal stem cells. DESIGNS: 30 Wistar rats (8-week, male) were randomly divided into Control group (n = 5), Hydrogel group (n = 5), and Hydrogel loaded OP 3-4 group (n = 5). Hematoxylin and eosin staining was used to evaluate the level of bone regeneration in mandibular defect. Immunohistochemistry staining was used to evaluate the expression of alkaline phosphatase, runt-related transcription factor 2, and type Ⅰ collagen. Flow cytometry was applied to identify the phenotype of bone marrow mesenchymal stem cells. Furthermore, LY294002, the inhibitor of protein kinase B, was applied to verify the role of OP 3-4 in promoting osteogenic differentiation via protein kinase B/glycogen synthase kinase 3ß/ß-catenin pathway through western blot. RESULTS: OP 3-4 promoted bone regeneration of rat mandibular defect. The expression of osteogenic differentiation related markers were increased after adding OP 3-4 to bone marrow mesenchymal stem cells. OP 3-4 promoted osteogenic differentiation of bone marrow mesenchymal stem cells via protein kinase B/glycogen synthase kinase 3ß/ß-catenin pathway. CONCLUSION: OP 3-4 could promote bone regeneration of mandibular defect and improve osteogenic differentiation through protein kinase B/glycogen synthase kinase 3ß/ß-catenin pathway.


Assuntos
Células-Tronco Mesenquimais , Osteogênese , Animais , Células da Medula Óssea/metabolismo , Regeneração Óssea , Cateninas , Diferenciação Celular , Células Cultivadas , Glicogênio Sintase Quinase 3 beta , Masculino , Células-Tronco Mesenquimais/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Wistar , Via de Sinalização Wnt , beta Catenina/metabolismo
5.
J Transl Med ; 19(1): 311, 2021 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-34281572

RESUMO

BACKGROUND: Colorectal cancer (CRC) is a common malignant tumour of the digestive tract that is characterized by high patient morbidity and mortality rates. Claudin-7 (Cldn7), a tight junction protein, was recently reported to function as a candidate tumour suppressor gene in CRC. Our previous study demonstrated that the large intestine of C57/BL6 mice showed intestinal adenomas and abnormal Ki67 expression and distribution in the intestinal crypt when Cldn7 was knocked out. The aim of this study was to further investigate whether Cldn7 deficiency has non-tight junction functions, affects intestinal stemness properties, promotes CRC and to determine the specific mechanism. METHODS: Cell proliferation assays, migration assays, apoptosis assays, tumour sphere formation assays in vitro, and subcutaneous xenograft models in vivo were used to determine the effects of Cldn7 knockdown on the biological characteristics of CRC stem cells. Western blotting, qPCR and immunofluorescence staining were performed to identify the epithelial-mesenchymal transition and the activation of Wnt/ß-catenin pathway in CRC stem cells. Cldn7 inducible conditional gene knockout mice and immunohistochemical staining further verified this hypothesis in vivo. The mechanism and target of Cldn7 were determined by performing a chromatin immunoprecipitation (ChIP) assay and coimmunoprecipitation (CoIP) assay. RESULTS: Cldn7 knock down in CRC stem cells promoted cell proliferation, migration, and globular growth in serum-free medium and the ability to form xenograft tumours; cell apoptosis was inhibited, while the cellular epithelial-mesenchymal transition was also observed. These changes in cell characteristics were achieved by activating the Wnt/ß-catenin pathway and promoting the expression of downstream target genes after ß-catenin entry into the nucleus, as observed in CRC cell lines and Cldn7 gene knockout mouse experiments. Using ChIP and CoIP experiments, we initially found that Cldn7 and Sox9 interacted at the protein level to activate the Wnt/ß-catenin pathway. CONCLUSIONS: Based on our research, Cldn7 deficiency confers stemness properties in CRC through Sox9-mediated Wnt/ß-catenin signalling. This result clarifies that Cldn7 plays an inhibitory role in CRC and reveals a possible molecular mechanism, which is conducive to further research on Cldn7 and cancer stem cells.


Assuntos
Neoplasias Colorretais , beta Catenina , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Claudinas/genética , Claudinas/metabolismo , Neoplasias Colorretais/genética , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Fatores de Transcrição SOX9 , Via de Sinalização Wnt , beta Catenina/metabolismo
7.
Development ; 148(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34195802

RESUMO

Tooth formation requires complex signaling interactions both within the oral epithelium and between the epithelium and the underlying mesenchyme. Previous studies of the Wnt/ß-catenin pathway have shown that tooth formation is partly inhibited in loss-of-function mutants, and gain-of-function mutants have perturbed tooth morphology. However, the stage at which Wnt signaling is first important in tooth formation remains unclear. Here, using an Fgf8-promoter-driven, and therefore early, deletion of ß-catenin in mouse molar epithelium, we found that loss of Wnt/ß-catenin signaling completely deletes the molar tooth, demonstrating that this pathway is central to the earliest stages of tooth formation. Early expression of a dominant-active ß-catenin protein also perturbs tooth formation, producing a large domed evagination at early stages and supernumerary teeth later on. The early evaginations are associated with premature mesenchymal condensation marker, and are reduced by inhibition of condensation-associated collagen synthesis. We propose that invagination versus evagination morphogenesis is regulated by the relative timing of epithelial versus mesenchymal cell convergence regulated by canonical Wnt signaling. Together, these studies reveal new aspects of Wnt/ß-catenin signaling in tooth formation and in epithelial morphogenesis more broadly.


Assuntos
Dente Molar/crescimento & desenvolvimento , Dente Molar/metabolismo , Odontogênese/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Proliferação de Células , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Mesoderma/metabolismo , Camundongos , Dente Molar/citologia , Morfogênese/fisiologia , Odontogênese/genética , beta Catenina/metabolismo
8.
Zhonghua Zhong Liu Za Zhi ; 43(7): 769-774, 2021 Jul 23.
Artigo em Chinês | MEDLINE | ID: mdl-34289571

RESUMO

Objective: To investigate the effect of long non-coding RNA HOTAIR (LncRNA HOTAIR) on the proliferation, apoptosis and drug resistance of Wilms tumor cells and its molecular mechanism. Methods: Collected nephroblastoma tissues and normal tumor side tissues in 32 children with renal syblastoma surgical treatment at Zhengzhou University Children's Hospital from 2015 to 2019. Real-time quantitative reverse transcription polymerase chain reaction, (qRT-PCR)was used to detect the expression of HOTAIR in Wilms tumor tissues and adjacent tissues. Small interfering RNA technology was used to delete the expression of HOTAIR in Wilms tumor cell SK-NEP-1. Cell counting kit-8 (CCK-8)was used to detect cell proliferation after transfection. Flow cytometry and terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) staining to detect the apoptosis. Western blot was used to detect the expression of Wnt/ß-catenin signaling pathway related proteins.CCK-8 was used to detect the proliferation inhibition of cells treated with different concentrations of cisplatin after transfection. Results: Compared with adjacent tissues, HOTAIR was highly expressed in Wilms tumor tissues (P<0.05). The expression levels of Wnt, ß-catenin, Cyclin D1, c-myc in the control group were (0.89±0.08), (0.94±0.10), (0.72±0.06), (1.10±0.11), and (1.06±0.11), (0.92±0.08), (0.66±0.07), (1.25±0.11) of the si-RNA group, while (0.54±0.05), (0.41±0.05), (0.25±0.03), (0.56±0.06) of the si-HOTAIR group. The expression levels of these protein were significantly down-regulated in the si-HOTAIR group when compared with the control group and the si-RNA group (P<0.05). The absorbance (A) values of SK-NEP-1 cells in the si-HOTAIR group at 24, 48 and 72 hours after transfection were (0.31±0.02), (0.37±0.04), (0.69±0.07), significantly lower than (0.49±0.05), (0.78±0.08), (1.22±0.14) in the control group and (0.57±0.06), (0.68±0.07), (0.94±0.09) in the si-RNA group (P<0.05). The apoptosis rate in the si-HOTAIR group was (13.81±1.25)%, significantly higher than (6.54±0.72)% in the control group and (4.35±0.40)% in the si-RNA group (P<0.05). The cell positive rate of TUNEL cells in the si-HOTAIR group was (35.14±3.50)%, significantly higher than (20.16±2.18)% in the control group and (21.09±2.35)% in the si-RNA group (P<0.05). The median inhibitory concentration (IC(50)) of the si-HOTAIR group was (62.48±5.97) µmol/L, significantly lower than (88.27±9.05) µmol/L of the control group and (92.50±9.11) µmol/L of the si-RNA group (P<0.05). Conclusions: Suppression of LncRNA HOTAIR can inhibit the proliferation of Wilms tumor cells, promote cell apoptosis, decrease cell resistance to cisplatin. The mechanism may be related to the inhibition of Wnt/ß-catenin signaling pathway activation.


Assuntos
Neoplasias Renais , RNA Longo não Codificante , Tumor de Wilms , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Criança , Resistência a Medicamentos , Humanos , Neoplasias Renais/genética , RNA Longo não Codificante/genética , Tumor de Wilms/genética , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
9.
Int J Mol Sci ; 22(13)2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206850

RESUMO

Treating postoperative (PO) pain is a clinical challenge. Inadequate PO pain management can lead to worse outcomes, for example chronic post-surgical pain. Therefore, acquiring new information on the PO pain mechanism would increase the therapeutic options available. In this paper, we evaluated the role of a natural substance, epigallocatechin-3-gallate (EGCG), on pain and neuroinflammation induced by a surgical procedure in an animal model of PO pain. We performed an incision of the hind paw and EGCG was administered for five days. Mechanical allodynia, thermal hyperalgesia, and motor dysfunction were assessed 24 h, and three and five days after surgery. At the same time points, animals were sacrificed, and sera and lumbar spinal cord tissues were harvested for molecular analysis. EGCG administration significantly alleviated hyperalgesia and allodynia, and reduced motor disfunction. From the molecular point of view, EGCG reduced the activation of the WNT pathway, reducing WNT3a, cysteine-rich domain frizzled (FZ)1 and FZ8 expressions, and both cytosolic and nuclear ß-catenin expression, and the noncanonical ß-catenin-independent signaling pathways, reducing the activation of the NMDA receptor subtype NR2B (pNR2B), pPKC and cAMP response element-binding protein (pCREB) expressions at all time points. Additionally, EGCG reduced spinal astrocytes and microglia activation, cytokines overexpression and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) pathway, downregulating inducible nitric oxide synthase (iNOS) activation, cyclooxygenase 2 (COX-2) expression, and prostaglandin E2 (PGE2) levels. Thus, EGCG administration managing the WNT/ß-catenin signaling pathways modulates PO pain related neurochemical and inflammatory alterations.


Assuntos
Analgésicos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Catequina/análogos & derivados , Dor Pós-Operatória/tratamento farmacológico , Analgésicos/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Catequina/farmacologia , Catequina/uso terapêutico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Masculino , NF-kappa B/genética , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
10.
Nat Commun ; 12(1): 4164, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230493

RESUMO

Spi-1 Proto-Oncogene (SPI1) fusion genes are recurrently found in T-cell acute lymphoblastic leukemia (T-ALL) cases but are insufficient to drive leukemogenesis. Here we show that SPI1 fusions in combination with activating NRAS mutations drive an immature T-ALL in vivo using a conditional bone marrow transplant mouse model. Addition of the oncogenic fusion to the NRAS mutation also results in a higher leukemic stem cell frequency. Mechanistically, genetic deletion of the ß-catenin binding domain within Transcription factor 7 (TCF7)-SPI1 or use of a TCF/ß-catenin interaction antagonist abolishes the oncogenic activity of the fusion. Targeting the TCF7-SPI1 fusion in vivo with a doxycycline-inducible knockdown results in increased differentiation. Moreover, both pharmacological and genetic inhibition lead to down-regulation of SPI1 targets. Together, our results reveal an example where TCF7-SPI1 leukemia is vulnerable to pharmacological targeting of the TCF/ß-catenin interaction.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator 1 de Transcrição de Linfócitos T/metabolismo , Transativadores/metabolismo , beta Catenina/metabolismo , Animais , Transplante de Medula Óssea , Carcinogênese/genética , Modelos Animais de Doenças , Feminino , GTP Fosfo-Hidrolases/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Oncogenes , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas/genética , Fator 1 de Transcrição de Linfócitos T/genética , Linfócitos T/metabolismo , Transativadores/genética , Transcriptoma , beta Catenina/genética
11.
Int J Mol Sci ; 22(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200709

RESUMO

Sepsis is characterized by multiple-organ dysfunction caused by the dysregulated host response to infection. Until now, however, the role of the Wnt signaling has not been fully characterized in multiple organs during sepsis. This study assessed the suppressive effect of a Wnt signaling inhibitor, Wnt-C59, in the kidney, lung, and liver of lipopolysaccharide-induced endotoxemic mice, serving as an animal model of sepsis. We found that Wnt-C59 elevated the survival rate of these mice and decreased their plasma levels of proinflammatory cytokines and organ-damage biomarkers, such as BUN, ALT, and AST. The Wnt/ß-catenin and NF-κB pathways were stimulated and proinflammatory cytokines were upregulated in the kidney, lung, and liver of endotoxemic mice. Wnt-C59, as a Wnt signaling inhibitor, inhibited the Wnt/ß-catenin pathway, and its interaction with the NF-κB pathway, which resulted in the inhibition of NF-κB activity and proinflammatory cytokine expression. In multiple organs of endotoxemic mice, Wnt-C59 significantly reduced the ß-catenin level and interaction with NF-κB. Our findings suggest that the anti-endotoxemic effect of Wnt-C59 is mediated via reducing the interaction between ß-catenin and NF-κB, consequently suppressing the associated cytokine upregulation in multiple organs. Thus, Wnt-C59 may be useful for the suppression of the multiple-organ dysfunction during sepsis.


Assuntos
Benzenoacetamidas/farmacologia , Citocinas/metabolismo , Endotoxemia/tratamento farmacológico , Lipopolissacarídeos/toxicidade , NF-kappa B/antagonistas & inibidores , Piridinas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/antagonistas & inibidores , Animais , Citocinas/genética , Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , Endotoxemia/patologia , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Domínios e Motivos de Interação entre Proteínas , beta Catenina/metabolismo
12.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209088

RESUMO

Breast cancer (BC) brain metastases is a life-threatening condition to which accounts the poor understanding of BC cells' (BCCs) extravasation into the brain, precluding the development of preventive strategies. Thus, we aimed to unravel the players involved in the interaction between BCCs and blood-brain barrier (BBB) endothelial cells underlying BBB alterations and the transendothelial migration of malignant cells. We used brain microvascular endothelial cells (BMECs) as a BBB in vitro model, under conditions mimicking shear stress to improve in vivo-like BBB features. Mixed cultures were performed by the addition of fluorescently labelled BCCs to distinguish individual cell populations. BCC-BMEC interaction compromised BBB integrity, as revealed by junctional proteins (ß-catenin and zonula occludens-1) disruption and caveolae (caveolin-1) increase, reflecting paracellular and transcellular hyperpermeability, respectively. Both BMECs and BCCs presented alterations in the expression pattern of connexin 43, suggesting the involvement of the gap junction protein. Myosin light chain kinase and phosphorylated myosin light chain were upregulated, revealing the involvement of the endothelial cytoskeleton in the extravasation process. ß4-Integrin and focal adhesion kinase were colocalised in malignant cells, reflecting molecular interaction. Moreover, BCCs exhibited invadopodia, attesting migratory properties. Collectively, hub players involved in BC brain metastases formation were unveiled, disclosing possible therapeutic targets for metastases prevention.


Assuntos
Barreira Hematoencefálica/citologia , Neoplasias Encefálicas/secundário , Neoplasias da Mama/metabolismo , Redes Reguladoras de Genes , Animais , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/metabolismo , Caveolina 1/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Conexina 43/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Fosforilação , Resistência ao Cisalhamento , Migração Transendotelial e Transepitelial , Proteína da Zônula de Oclusão-1/metabolismo , beta Catenina/metabolismo
13.
World J Gastroenterol ; 27(26): 4221-4235, 2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34326621

RESUMO

BACKGROUND: Ubiquitin-specific protease 15 (USP15) is an important member of the ubiquitin-specific protease family, the largest deubiquitinase subfamily, whose expression is dysregulated in many types of cancer. However, the biological function and the underlying mechanisms of USP15 in gastric cancer (GC) progression have not been elucidated. AIM: To explore the biological role and underlying mechanisms of USP15 in GC progression. METHODS: Bioinformatics databases and western blot analysis were utilized to determine the expression of USP15 in GC. Immunohistochemistry was performed to evaluate the correlation between USP15 expression and clinicopathological characteristics of patients with GC. A loss- and gain-of-function experiment was used to investigate the biological effects of USP15 on GC carcinogenesis. RNA sequencing, immunofluorescence, and western blotting were performed to explore the potential mechanism by which USP15 exerts its oncogenic functions. RESULTS: USP15 was up-regulated in GC tissue and cell lines. The expression level of USP15 was positively correlated with clinical characteristics (tumor size, depth of invasion, lymph node involvement, tumor-node-metastasis stage, perineural invasion, and vascular invasion), and was related to poor prognosis. USP15 knockdown significantly inhibited cell proliferation, invasion and epithelial-mesenchymal transition (EMT) of GC in vitro, while overexpression of USP15 promoted these processes. Knockdown of USP15 inhibited tumor growth in vivo. Mechanistically, RNA sequencing analysis showed that USP15 regulated the Wnt signaling pathway in GC. Western blotting confirmed that USP15 silencing led to significant down-regulation of ß-catenin and Wnt/ß-catenin downstream genes (c-myc and cyclin D1), while overexpression of USP15 yielded an opposite result and USP15 mutation had no change. Immunofluorescence indicated that USP15 promoted nuclear translocation of ß-catenin, suggesting activation of the Wnt/ß-catenin signaling pathway, which may be the critical mechanism promoting GC progression. Finally, rescue experiments showed that the effect of USP15 on gastric cancer progression was dependent on Wnt/ß-catenin pathway. CONCLUSION: USP15 promotes cell proliferation, invasion and EMT progression of GC via regulating the Wnt/ß-catenin pathway, which suggests that USP15 is a novel potential therapeutic target for GC.


Assuntos
Neoplasias Gástricas , Via de Sinalização Wnt , Linhagem Celular Tumoral , Proliferação de Células , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica/genética , Neoplasias Gástricas/genética , Proteases Específicas de Ubiquitina/genética , beta Catenina/metabolismo
14.
Stem Cell Res Ther ; 12(1): 415, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294121

RESUMO

BACKGROUND: Osteogenesis is tightly coupled with angiogenesis during bone repair and regeneration. However, the underlying mechanisms linking these processes remain largely undefined. The present study aimed to test the hypothesis that epidermal growth factor-like domain-containing protein 6 (EGFL6), an angiogenic factor, also functions in bone marrow mesenchymal stem cells (BMSCs), playing a key role in the interaction between osteogenesis and angiogenesis. METHODS: We evaluated how EGFL6 affects angiogenic activity of human umbilical cord vein endothelial cells (HUVECs) via proliferation, transwell migration, wound healing, and tube-formation assays. Alkaline phosphatase (ALP) and Alizarin Red S (AR-S) were used to assay the osteogenic potential of BMSCs. qRT-PCR, western blotting, and immunocytochemistry were used to evaluate angio- and osteo-specific markers and pathway-related genes and proteins. In order to determine how EGFL6 affects angiogenesis and osteogenesis in vivo, EGFL6 was injected into fracture gaps in a rat tibia distraction osteogenesis (DO) model. Radiography, histology, and histomorphometry were used to quantitatively evaluate angiogenesis and osteogenesis. RESULTS: EGFL6 stimulated both angiogenesis and osteogenic differentiation through Wnt/ß-catenin signaling in vitro. Administration of EGFL6 in the rat DO model promoted CD31hiEMCNhi type H-positive capillary formation associated with enhanced bone formation. Type H vessels were the referred subtype involved during DO stimulated by EGFL6. CONCLUSION: EGFL6 enhanced the osteogenic differentiation potential of BMSCs and accelerated bone regeneration by stimulating angiogenesis. Thus, increasing EGFL6 secretion appeared to underpin the therapeutic benefit by promoting angiogenesis-coupled bone formation. These results imply that boosting local concentrations of EGFL6 may represent a new strategy for the treatment of compromised fracture healing and bone defect restoration.


Assuntos
Osteogênese por Distração , Osteogênese , Animais , Diferenciação Celular , Células Cultivadas , Consolidação da Fratura , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Ratos , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
15.
Zhonghua Zhong Liu Za Zhi ; 43(6): 638-645, 2021 Jun 23.
Artigo em Chinês | MEDLINE | ID: mdl-34289555

RESUMO

Objective: To clarify the function and molecular mechanisms of serpin family E member 2 (SERPINE2) in cellular migration and invasion of esophageal squamous cell carcinoma (ESCC). Methods: The expression of SERPINE2 in ESCC was analyzed by using online databases TCGA (http: //gepia.cancer-pku.cn/detail.php and http: //ualcan.path.uab. edu/index.html). The expressions of SERPINE2 mRNA in normal human esophageal epithelial cell line NE2, human ESCC cell lines KYSE30 and KYSE150 were detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). SERPINE2-konckdown or SERPINE2-overexpressed plasmid was transfected into KYSE30 cells, and the efficiencies of the knockdown and overexpression system were tested by qRT-PCR. The relationships of SERPINE2 and ESCC migration and invasion were determined by migration and invasion assays in vitro. The associations between SERPINE2 expression and ß-catenin as well as its target genes including c-Myc, cyclin D1 and CD44 were analyzed by immunofluorescence, qRT-PCR and western blot, respectively. Results: The expressions of SERPINE2 were significantly upregulated in both esophageal cancer (ESCA) and ESCC tissues compared to normal tissues by analyzing 182 and 95 cases, respectively (P<0.01). SERPINE2 is highly expressed in both KYSE30 and KYSE150 cells (P<0.05). The number of migrating and invading cells in control group were (212.66±24.11)/field and (136.00±14.42)/field, while were (88.33±9.71)/field and (77.00±9.53)/field in SERPINE2-knockdown 1 group, and (66.00±8.00)/field and (45.66±3.78)/field in SERPINE2-knockdown 2 group, respectively, and the differences were dramatically significant compared with the control group (P<0.01). The number of migrating and invading cells in control group were (250.00±30.00)/field and (203.33±15.27)/field, while were (383.33±35.11)/field and (246.66±25.16)/field in SERPINE2-overpressed group, and the differences were strikingly significant compared with the control group (P<0.01). The protein expression of ß-catenin was upregulated while phosphorylated ß-catenin protein expression was downregulated in SERPINE2-overexpressed KYSE30 cells when compared to control cells.The transcription activity of ß-catenin was significantly upregulated and the mRNA expressions of its target genes including c-Myc, cyclin D1 and CD44 were all increased. After treated with 25 µM iCRT14, the number of migrated cells in the control and SERPINE2-overpressed groups were (200.00±36.05)/field and (258.33±22.54)/field, and the number of invaded cells were (160.00±17.32)/field and (188.33±25.65)/field, respectively, the differences were dramatically significant compared with the group without iCRT14 treatment (P<0.01). Conclusion: SERPINE2 is significantly upregulated in ESCC cells and can promote cellular migration and invasion by activating ß-catenin, which may provide a potential therapeutic target for patients with ESCC.


Assuntos
Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Neoplasias de Cabeça e Pescoço , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Piridinas , Pirróis , Serpina E2 , Tiazolidinedionas , beta Catenina/genética , beta Catenina/metabolismo
16.
Cancer Sci ; 112(9): 3732-3743, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34118099

RESUMO

Colorectal cancer (CRC) is a recurring cancer that is often resistant to conventional therapies and therefore requires the development of molecular-based therapeutic approaches. Dopamine receptor D2 (DRD2) is associated with the growth of many types of tumors, but its oncogenic role in CRC is unclear. Here, we observed that elevated DRD2 expression was associated with a poor survival rate among patients with CRC. Depletion of DRD2 suppressed CRC cell growth and motility by downregulating ß-catenin/ZEB signaling in vitro and in vivo, whereas overexpression of DRD2 promoted CRC cell progression. Inhibition of DRD2 by the antagonist pimozide inhibited tumor growth and lymph node metastasis in vivo and enhanced the cytotoxic effects of conventional agents in vitro. Taken together, our findings indicate that targeting the DRD2/ß-catenin/ZEB1 signaling axis is a potentially promising therapeutic strategy for patients with CRC.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Progressão da Doença , Receptores de Dopamina D2/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , beta Catenina/metabolismo , Idoso , Animais , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Antagonistas de Dopamina/farmacologia , Feminino , Células HCT116 , Células HT29 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Pimozida/farmacologia , Interferência de RNA , Receptores de Dopamina D2/genética , Transdução de Sinais , Taxa de Sobrevida , Transfecção , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Nat Commun ; 12(1): 3624, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131132

RESUMO

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/ß-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Junções Aderentes/metabolismo , Artrite/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Artrite/patologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Caderinas/metabolismo , Proteínas do Citoesqueleto/genética , Feminino , Proteínas de Homeodomínio , Proteínas com Domínio LIM/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos , beta Catenina/metabolismo
18.
Nan Fang Yi Ke Da Xue Xue Bao ; 41(5): 722-728, 2021 May 20.
Artigo em Chinês | MEDLINE | ID: mdl-34134960

RESUMO

OBJECTIVE: To investigate the effect of curcumin on cell cycle and apoptosis of human lens epithelial cells and the possible molecular mechanism. OBJECTIVE: Cultured human lens epithelial cell line HLEC-SRA01/04 was treated with 20, 40 and 60 µmol/L curcumin for 24 or 48 h. The cell proliferation inhibition rate was determined using MTT assay, and the changes in cell cycle, mitochondrial membrane potential and apoptosis rate were analyzed with flow cytometry. Western blotting was used to detect the expression levels of caspase-9, caspase-3, Bcl-2, Bax, cyclin B1, CDK1, ß-catenin, c-myc, and cyclin D1 in the cells. OBJECTIVE: Curcumin concentration- and time-dependently inhibited the proliferation of in HLEC-SRA01/04 cells as compared with the control cells (P < .05). Flow cytometric analysis showed that curcumin significantly increased apoptosis rate and cell percentage in G2/M phase and lowered mitochondrial membrane potential of HLEC-SRA01/04 cells in a concentrationdependent manner (P < 0.05). The results of Western blotting showed that curcumin also concentration-dependently increased the cellular expressions of caspase-3, caspase-9 and Bax and lowered the expressions of Bcl-2, cyclin B1, CDK1 and ß-catenin along with the downstream proteins cyclin D1 and c-myc in the Wnt/ß-catenin signaling pathway (P < 0.05). OBJECTIVE: Curcumin inhibits the proliferation of HLEC-SRA01/04 cells possibly by inhibiting the Wnt/ß-catenin signaling pathway and causing cell cycle arrest to induce cell apoptosis.


Assuntos
Curcumina , Via de Sinalização Wnt , Apoptose , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Curcumina/farmacologia , Células Epiteliais/metabolismo , Humanos , beta Catenina/metabolismo
19.
Life Sci ; 280: 119748, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34174322

RESUMO

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver. Long non-coding RNAs as master gene regulators play important roles in tumorigenesis and progression. However, the significance of lncRNAs and their regulatory mechanisms in HCC are largely unknown. Our study was to define the role of lncAY (long noncoding RNA AY927503) in HCC. METHODS: Methylated RNA immunoprecipitation qPCR combined with bioinformatics were used to identify the m6A modification of lncAY. qRT-PCR, western blotting and immunofluorescence were used to identify the expression of the lncAY/YTHDF2/BMI1/Wnt axis in HCC tissues and cell lines. Gain- and loss-of functions of lncAY and BMI1 were implemented to confirm their roles in the behaviors of HCC cells. RESULTS: Our findings suggested that m6A-modified lncAY expression relied on m6A "reader" protein YTHDF2. LncAY upregulated BMI1 expression in HCC cells and a notably positive relevance is evident between lncAY and BMI1 expression in TCGA HCC datasets. BMI1 was upregulated in HCC tissues and patients with higher BMI1 expression had a poor clinical prognosis. Besides, GSEA analysis showed remarkable enrichment of high BMI1 expression in gene sets associated with Wnt/ß-catenin signaling. Rescue results revealed that BMI1 reversed the suppressive effects of lncAY depletion in HCC cells. CONCLUSIONS: Our work suggested that lncAY might elevate BMI1 expression and further activate the Wnt/ß-catenin signaling. BMI1 reverses the suppressive effects of lncAY depletion in HCC cells. Collectively, our work uncovers a novel undefined regulatory signaling pathway, namely lncAY/BMI1/Wnt/ß-catenin axis, involved in liver cancer progression.


Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Complexo Repressor Polycomb 1/genética , RNA Longo não Codificante/genética , Via de Sinalização Wnt , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Complexo Repressor Polycomb 1/metabolismo , beta Catenina/metabolismo
20.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(7): 608-615, 2021 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-34140072

RESUMO

Objective To observe the effects of DNA methyltransferase 3B (DNMT3B) on the expression of secreted frizzled-related protein 1 (SFRP1) and regulation of Wnt/ß-catenin signaling pathway in renal tubular epithelial cells (RTECs) of mice under high glucose conditions. Methods in vitro cultured mouse RTECs were divided into normal glucose (NG) group and high glucose (HG) group. After DNMT3B short-hairclip RNA (sh-DNMT3B) and DNMT3B over-expression (DNMT3B-OE) plasmids were transfected separately into RTECs, mRNA expression of DNMT3B, SFRP1, collagen IV (Col4) and fibronectin (FN) were detected by reverse-transcription PCR. Protein expression of DNMT3B, SFRP1, glycogen synthase kinase 3ß (GSK3ß), phosphorylated glycogen synthase kinase 3ß (p-GSK3ß), ß-catenin, Col4 and FN were detected by Western blotting. The localization of DNMT3B and SFRP1 in RTECs was observed by immunofluorescence cytochemistry combined with confocal microscopy. Results Compared with the NG group, the protein expression of DNMT3B, ß-catenin, p-GSK3ß, Col4 and FN increased in the HG group, while SFRP1 protein expression was reduced in the HG group. Compared with the sh-vector group, SFRP1 mRNA and protein expression increased in the sh-DNMT3B group, while the expression of ß-catenin, p-GSK3ß and Col4 proteins decreased. FN mRNA and protein expression dropped in the sh-DNMT3B group, however, the expression of ß-catenin mRNA did not change significantly. Visually, DNMT3B over-expression reversed the above changes. Both DNMT3B and SFRP1 were expressed in the nucleus and cytoplasm of RTECs, and DNMT3B was aggregated in the nuclei of the cells in the HG group and the co-localization between DNMT3B and SFRP1 was also promoted in the HG group. Conclusion The expression of DNMT3B increases and the expression of SFRP1 decreases when the mouse RTECs were stimulated by HG. This subsequently leads to the activation of the Wnt/ß-catenin signaling pathway and promotes the formation of extracellular matrix.


Assuntos
DNA (Citosina-5-)-Metiltransferases , Células Epiteliais , Via de Sinalização Wnt , Animais , DNA (Citosina-5-)-Metiltransferases/metabolismo , Células Epiteliais/metabolismo , Fibrose , Glucose , Glicogênio Sintase Quinase 3 beta/genética , Camundongos , beta Catenina/genética , beta Catenina/metabolismo
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