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1.
Int J Mol Sci ; 22(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34502133

RESUMO

Keratin 8 and keratin 18 (K8/K18) are intermediate filament proteins that form the obligate heteropolymers in hepatocytes and protect the liver against toxins. The mechanisms of protection include the regulation of signaling pathway associated with cell survival. Previous studies show K8/K18 binding with Akt, which is a well-known protein kinase involved in the cell survival signaling pathway. However, the role of K8/K18 in the Akt signaling pathway is unclear. In this study, we found that K8/K18-Akt binding is downregulated by K8/K18 phosphorylation, specifically phosphorylation of K18 ser7/34/53 residues, whereas the binding is upregulated by K8 gly-62-cys mutation. K8/K18 expression in cultured cell system tends to enhance the stability of the Akt protein. A comparison of the Akt signaling pathway in a mouse system with liver damage shows that the pathway is downregulated in K18-null mice compared with nontransgenic mice. K18-null mice with Fas-induced liver damage show enhanced apoptosis combined with the downregulation of the Akt signaling pathway, i.e., lower phosphorylation levels of GSK3ß and NFκB, which are the downstream signaling factors in the Akt signaling pathway, in K18-null mice compared with the control mice. Our study indicates that K8/K18 expression protects mice from liver damage by participating in enhancing the Akt signaling pathway.


Assuntos
Queratina-18/metabolismo , Queratina-8/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Cricetinae , Cricetulus , Células HT29 , Humanos , Fígado/metabolismo , Camundongos , Fosforilação , Ligação Proteica , Estabilidade Proteica
2.
J Cell Sci ; 134(15)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34342355

RESUMO

Keratin 8 (K8) is the cytoskeletal intermediate filament protein of simple-type epithelia. Mutations in K8 predispose the affected individual and transgenic mouse to liver disease. However, the role of K8 in the lung has not been reported in mutant transgenic mouse models. Here, we investigated the susceptibility of two different transgenic mice expressing K8 Gly62-Cys (Gly62 replaced with Cys) or Ser74-Ala (Ser74 replaced with Ala) to lung injury. The mutant transgenic mice were highly susceptible to two independent acute and chronic lung injuries compared with control mice. Both K8 Gly62-Cys mice and K8 Ser74-Ala mice showed markedly increased mouse lethality (∼74% mutant mice versus ∼34% control mice) and more severe lung damage, with increased inflammation and apoptosis, under L-arginine-mediated acute lung injury. Moreover, the K8 Ser74-Ala mice had more severe lung damage, with extensive hemorrhage and prominent fibrosis, under bleomycin-induced chronic lung injury. Our study provides the first direct evidence that K8 mutations predispose to lung injury in transgenic mice.


Assuntos
Hepatopatias , Lesão Pulmonar , Animais , Queratina-18/genética , Queratina-8/genética , Queratinas/genética , Lesão Pulmonar/genética , Camundongos , Camundongos Transgênicos , Mutação/genética
3.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360548

RESUMO

Keratin (K) 7 is an intermediate filament protein expressed in ducts and glands of simple epithelial organs and in urothelial tissues. In the pancreas, K7 is expressed in exocrine ducts, and apico-laterally in acinar cells. Here, we report K7 expression with K8 and K18 in the endocrine islets of Langerhans in mice. K7 filament formation in islet and MIN6 ß-cells is dependent on the presence and levels of K18. K18-knockout (K18‒/‒) mice have undetectable islet K7 and K8 proteins, while K7 and K18 are downregulated in K8‒/‒ islets. K7, akin to F-actin, is concentrated at the apical vertex of ß-cells in wild-type mice and along the lateral membrane, in addition to forming a fine cytoplasmic network. In K8‒/‒ ß-cells, apical K7 remains, but lateral keratin bundles are displaced and cytoplasmic filaments are scarce. Islet K7, rather than K8, is increased in K18 over-expressing mice and the K18-R90C mutation disrupts K7 filaments in mouse ß-cells and in MIN6 cells. Notably, islet K7 filament networks significantly increase and expand in the perinuclear regions when examined in the streptozotocin diabetes model. Hence, K7 represents a significant component of the murine islet keratin network and becomes markedly upregulated during experimental diabetes.


Assuntos
Diabetes Mellitus Experimental/patologia , Células Secretoras de Insulina/patologia , Queratina-18/metabolismo , Queratina-7/metabolismo , Queratina-8/metabolismo , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Regulação da Expressão Gênica , Células Secretoras de Insulina/metabolismo , Queratina-18/genética , Queratina-7/genética , Queratina-8/genética , Camundongos , Camundongos Knockout , Regulação para Cima
4.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203895

RESUMO

Although hepatocellular carcinoma (HCC) is developed with various etiologies, protection of hepatocytes seems basically essential to prevent the incidence of HCC. Keratin 8 and keratin 18 (K8/K18) are cytoskeletal intermediate filament proteins that are expressed in hepatocytes. They maintain the cell shape and protect cells under stress conditions. Their protective roles in liver damage have been described in studies of mouse models, and K8/K18 mutation frequency in liver patients. Interestingly, K8/K18 bind to signaling proteins such as transcription factors and protein kinases involved in HCC development. Since K8/K18 are abundant cytoskeletal proteins, K8/K18 binding with the signaling factors can alter the availability of the factors. Herein, we discuss the potential roles of K8/K18 in HCC development.


Assuntos
Carcinogênese/metabolismo , Carcinoma Hepatocelular/metabolismo , Queratina-18/metabolismo , Queratina-8/metabolismo , Neoplasias Hepáticas/metabolismo , Transdução de Sinais , Carcinogênese/patologia , Ensaios Clínicos como Assunto , Humanos
5.
Cells ; 10(5)2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069578

RESUMO

Corneal injuries are among the leading causes of blindness and vision impairment. Trauma, infectious keratitis, thermal and chemical (acids and alkali burn) injuries may lead to irreversible corneal scarring, neovascularization, conjunctivalization, and limbal stem cell deficiency. Bilateral blindness constitutes 12% of total global blindness and corneal transplantation remains a stand-alone treatment modality for the majority of end-stage corneal diseases. However, global shortage of donor corneas, the potential risk of graft rejection, and severe side effects arising from long-term use of immunosuppressive medications, demands alternative therapeutic approaches. Umbilical cord-derived mesenchymal stem cells can be isolated in large numbers using a relatively less invasive procedure. However, their role in injury induced corneal repair is largely unexplored. Here, we isolated, cultured and characterized mesenchymal stem cells from human umbilical cord, and studied the expression of mesenchymal (CD73, CD90, CD105, and CD34), ocular surface and epithelial (PAX6, WNT7A, and CK-8/18) lineage markers through immunofluorescence. The cultured human limbal and corneal epithelial cells were used as controls. Scratch assay was used to study the corneal epithelial repair potential of umbilical cord-derived mesenchymal stem cells, in vitro. The in vitro cultured umbilical cord-derived mesenchymal stem cells were plastic adherent, showed trilineage differentiation and expressed: mesenchymal markers CD90, CD105, CD73; epithelial marker CK-8/18, and ocular lineage developmental markers PAX6 and WNT-7A. Our findings suggest that umbilical cord-derived mesenchymal stem cells promote repair of the injured corneal epithelium by stimulating the proliferation of corneal epithelial cells, in vitro. They may serve as a potential non-ocular source of stem cells for treating injury induced bilateral corneal diseases.


Assuntos
Células Epiteliais/metabolismo , Epitélio Corneano/metabolismo , Células-Tronco Mesenquimais/metabolismo , Comunicação Parácrina , Cordão Umbilical/citologia , Cicatrização , Adulto , Animais , Linhagem da Célula , Movimento Celular , Proliferação de Células , Células Cultivadas , Cricetinae , Meios de Cultivo Condicionados/metabolismo , Células Epiteliais/patologia , Epitélio Corneano/patologia , Feminino , Humanos , Queratina-18/metabolismo , Queratina-8/metabolismo , Pessoa de Meia-Idade , Fator de Transcrição PAX6/metabolismo , Reepitelização , Transdução de Sinais , Proteínas Wnt/metabolismo , Adulto Jovem
6.
Sci Rep ; 11(1): 8340, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863935

RESUMO

Invasive ductal carcinoma (IDC) constitutes the most frequent malignant cancer endangering women's health. In this study, a new spontaneously immortalized breast cancer cell line, DHSF-BR16 cells, was isolated from the primary IDC of a 74-years old female patient, treated with neoadjuvant chemotherapy and disease-free 5-years after adjuvant chemotherapy. Primary breast cancer tissue surgically removed was classified as ER-/PR-/HER2+, and the same phenotype was maintained by DHSF-BR16 cells. We examined DHSF-BR16 cell morphology and relevant biological and molecular markers, as well as their response to anticancer drugs commonly used for breast cancer treatment. MCF-7 cells were used for comparison purposes. The DHSF-BR16 cells showed the ability to form spheroids and migrate. Furthermore, DHSF-BR16 cells showed a mixed stemness phenotype (i.e. CD44+/CD24-/low), high levels of cytokeratin 7, moderate levels of cytokeratin 8 and 18, EpCAM and E-Cadh. Transcriptome analysis showed 2071 differentially expressed genes between DHSF-BR16 and MCF-7 cells (logFC > 2, p-adj < 0.01). Several genes were highly upregulated or downregulated in the new cell line (log2 scale fold change magnitude within - 9.6 to + 12.13). A spontaneous immortalization signature, mainly represented by extracellular exosomes-, plasma membrane- and endoplasmic reticulum membrane pathways (GO database) as well as by metabolic pathways (KEGG database) was observed in DHSF-BR16 cells. Also, these cells were more resistant to anthracyclines compared with MCF-7 cells. Overall, DHSF-BR16 cell line represents a relevant model useful to investigate cancer biology, to identify both novel prognostic and drug response predictive biomarkers as well as to assess new therapeutic strategies.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Ductal/genética , Carcinoma Ductal/patologia , Receptor ErbB-2 , Receptores de Estrogênio , Receptores de Progesterona , Idoso , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/cirurgia , Antígeno CD24/genética , Antígeno CD24/metabolismo , Carcinoma Ductal/tratamento farmacológico , Carcinoma Ductal/cirurgia , Linhagem Celular Tumoral , Movimento Celular , Quimioterapia Adjuvante , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Feminino , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Membranas Intracelulares/metabolismo , Queratina-7/genética , Queratina-7/metabolismo , Queratina-8/genética , Queratina-8/metabolismo , Terapia Neoadjuvante , Esferoides Celulares/patologia
7.
Pac Symp Biocomput ; 26: 297-308, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33691026

RESUMO

An early biomarker would transform our ability to screen and treat patients with cancer. The large amount of multi-scale molecular data in public repositories from various cancers provide unprecedented opportunities to find such a biomarker. However, despite identification of numerous molecular biomarkers using these public data, fewer than 1% have proven robust enough to translate into clinical practice. One of the most important factors affecting the successful translation to clinical practice is lack of real-world patient population heterogeneity in the discovery process. Almost all biomarker studies analyze only a single cohort of patients with the same cancer using a single modality. Recent studies in other diseases have demonstrated the advantage of leveraging biological and technical heterogeneity across multiple independent cohorts to identify robust disease biomarkers. Here we analyzed 17149 samples from patients with one of 23 cancers that were profiled using either DNA methylation, bulk and single-cell gene expression, or protein expression in tumor and serum. First, we analyzed DNA methylation profiles of 9855 samples across 23 cancers from The Cancer Genome Atlas (TCGA). We then examined the gene expression profile of the most significantly hypomethylated gene, KRT8, in 6781 samples from 57 independent microarray datasets from NCBI GEO. KRT8 was significantly over-expressed across cancers except colon cancer (summary effect size=1.05; p < 0.0001). Further, single-cell RNAseq analysis of 7447 single cells from lung tumors showed that genes that significantly correlated with KRT8 (p < 0.05) were involved in p53-related pathways. Immunohistochemistry in tumor biopsies from 294 patients with lung cancer showed that high protein expression of KRT8 is a prognostic marker of poor survival (HR = 1.73, p = 0.01). Finally, detectable KRT8 in serum as measured by ELISA distinguished patients with pancreatic cancer from healthy controls with an AUROC=0.94. In summary, our analysis demonstrates that KRT8 is (1) differentially expressed in several cancers across all molecular modalities and (2) may be useful as a biomarker to identify patients that should be further tested for cancer.


Assuntos
Biomarcadores Tumorais , Neoplasias Pulmonares , Biomarcadores Tumorais/genética , Estudos de Coortes , Biologia Computacional , Metilação de DNA , Humanos , Queratina-8/genética , Queratina-8/metabolismo , Neoplasias Pulmonares/genética , Análise de Sobrevida
8.
Artigo em Inglês | MEDLINE | ID: mdl-33472210

RESUMO

OBJECTIVE: This study aimed to evaluate the association between the single-nucleotide polymorphism (SNP) and tissue protein level of keratin-8/18 and the occurrence and progression of vocal leukoplakia. METHODS: The case-control study enrolled 158 patients with vocal leukoplakia, 326 patients with laryngeal squamous cell carcinoma (LSCC), and 268 healthy controls, which were tested for genotype analysis with keratin-8 and keratin-18 gene polymorphisms using pyrosequencing. The tissue protein expression levels of keratin-8 and keratin-18 were evaluated using immunohistochemistry. RESULTS: The keratin-8 SNP RS1907671 showed an obvious increased risk for vocal leukoplakia (OR 1.56, p = 0.002), while the other SNPs (RS2035875, RS2035878, RS4300473) were tested as protective factors for vocal leukoplakia and LSCC (OR <1, p < 0.05). In keratin-18 SNP test, both RS2070876 and RS2638526 polymorphisms demonstrated decreased risks for vocal leukoplakia and LSCC (OR <1, p < 0.05). The protein levels of keratin-8 and keratin-18 in vocal leukoplakia group were significantly higher than those of the LSCC group (p < 0.05). CONCLUSIONS: Keratin-8 and keratin-18 polymorphisms and protein levels are associated with the occurrence and progression of vocal leukoplakia.


Assuntos
Queratina-18/genética , Queratina-8/genética , Leucoplasia/genética , Carcinoma de Células Escamosas/genética , Estudos de Casos e Controles , Neoplasias de Cabeça e Pescoço , Humanos , Neoplasias Laríngeas , Prega Vocal
9.
Int J Gynecol Pathol ; 40(4): 324-332, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32897971

RESUMO

Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation and/or environment recognized by the mouse monoclonal antibody H. O-GlcNAcH is present in several types of cells and in several polypeptides, including cytokeratin 8 and vimentin, on the latter in cells under stress. In the present work, we examined the expression of the O-GlcNAcH in 60 cases of endometrial curettings from missed miscarriage cases containing normal and simple hydropic degenerated chorionic villi in each case, using monoclonal antibody H and indirect immunoperoxidase and Western blot immunoblot. In all cases examined the expression of the O-GlcNAcH was cytoplasmic as follows: (1) syncytiotrophoblastic cells showed very low expression in chorionic villi (CV) with nonhydropic degeneration (NHD) and high expression in hydropic degenerated (HD) CV; (2) cytotrophoblastic cells showed low expression in CV with NHD and high expression in HD CV; (3) fibroblastic cells showed high expression in CV with NHD and very low expression in HD CV; (4) histiocytes showed very low expression in both types of CV; (5) endothelial cells showed high expression in both types of CV. An immunoblot of CV from one case of a legal abortion from a normal first-trimester pregnancy showed 5 polypeptides with 118.5, 106.3, 85, 53, and 36.7 kD bearing the epitope H and the 53 kD corresponded to cytokeratin 8. The expression of the O-GlcNAcH is upregulated in the trophoblastic cells and downregulated in the fibroblastic cells in the HD CV in comparison to the NHD CV.


Assuntos
Aborto Espontâneo/metabolismo , Acetilglucosamina/metabolismo , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Epitopos/metabolismo , Queratina-8/metabolismo , Vimentina/metabolismo , Aborto Espontâneo/imunologia , Acetilglucosamina/imunologia , Vilosidades Coriônicas/imunologia , Vilosidades Coriônicas/metabolismo , Citoplasma/metabolismo , Regulação para Baixo , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Feminino , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , Gravidez , Primeiro Trimestre da Gravidez/imunologia , Primeiro Trimestre da Gravidez/metabolismo , Trofoblastos/imunologia , Trofoblastos/metabolismo , Regulação para Cima
10.
BMC Vet Res ; 16(1): 461, 2020 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-33243240

RESUMO

BACKGROUND: Squamous Cell Carcinoma of horn, also known as horn cancer, is a prevailing type of cancer in cattles especially Bos indicus. It is one of the most prevalent disease in Indian bullocks often resulting in death and huge economic losses to farmers. Here, we have reported the use of targeted exome sequencing to identify variants present in horn cancer affected horn mucosa tissue and blood of the same animal to identify some of the prevalent markers of horn cancer. RESULTS: We have observed higher number of variants present in tissue as compared to blood as well as among cancer samples compared to samples from normal animals. Eighty six and 1437 cancer-specific variants were identified among the predicted variants in blood and tissue samples, respectively. Total 25 missense variants were observed distributed over 18 genes. KRT8 gene coding for Keratin8, one of the key constituents of horn, displayed 5 missense variants. Additionally, three other genes involved in apoptosis pathway and two genes involved in antigen presentation and processing also contained variants. CONCLUSIONS: Several genes involved in various apoptotic pathways were found to contain non-synonymous mutations. Keratin8 coding for Keratin, a chief constituent of horn was observed to have the highest number of mutations. In all, we present a preliminary report of mutations observed in horn cancer.


Assuntos
Carcinoma de Células Escamosas/veterinária , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Cornos/patologia , Animais , Apoptose/genética , Carcinoma de Células Escamosas/genética , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/genética , Doenças dos Bovinos/patologia , Índia , Queratina-8/genética , Masculino , Mutação
11.
Nat Commun ; 11(1): 3559, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678092

RESUMO

The cell type specific sequences of transcriptional programs during lung regeneration have remained elusive. Using time-series single cell RNA-seq of the bleomycin lung injury model, we resolved transcriptional dynamics for 28 cell types. Trajectory modeling together with lineage tracing revealed that airway and alveolar stem cells converge on a unique Krt8 + transitional stem cell state during alveolar regeneration. These cells have squamous morphology, feature p53 and NFkB activation and display transcriptional features of cellular senescence. The Krt8+ state appears in several independent models of lung injury and persists in human lung fibrosis, creating a distinct cell-cell communication network with mesenchyme and macrophages during repair. We generated a model of gene regulatory programs leading to Krt8+ transitional cells and their terminal differentiation to alveolar type-1 cells. We propose that in lung fibrosis, perturbed molecular checkpoints on the way to terminal differentiation can cause aberrant persistence of regenerative intermediate stem cell states.


Assuntos
Células Epiteliais Alveolares/metabolismo , Queratina-8/metabolismo , Alvéolos Pulmonares/fisiologia , Fibrose Pulmonar/patologia , Regeneração , Células-Tronco/metabolismo , Células Epiteliais Alveolares/citologia , Animais , Comunicação Celular , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Humanos , Queratina-8/genética , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Camundongos , Camundongos Endogâmicos C57BL , Alvéolos Pulmonares/citologia , Fibrose Pulmonar/metabolismo , Análise de Célula Única , Células-Tronco/citologia
12.
Sci Rep ; 10(1): 9227, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32513981

RESUMO

Aurea helianthus extract is associated with various properties including anti-melanogenesis, anti-oxidation, tumorigenic suppression, and immunoregulation; however, the mechanism by which it executes the immunomodulation of human vaginal epithelial cells (HVECs) remains elusive. We established three immunological functions of the extract. First, it mediated tumorigenic suppression in HVECs. Expression of cytokeratin 8, cancer antigen-125, and vimentin was dramatically downregulated in HVECs exposed to the extract under oxidative and fungal stresses. Second, the extract activated dendritic cells and macrophages. On exposing progenitor dendritic cells to the extract, the number of CD304+ cells increased by 40%; further, under oxidative and fungal stresses, this number was approximately 1.8 and 1.3 times lower, respectively, compared to that in the stressed cells. In monocytic differentiation, the number of dendritic cells and macrophages increased 9 and 6 times, respectively, compared to that in the control. Additionally, the extract enhanced and recovered polarisation by approximately 1.5 and 2 times, respectively, than that under stressed conditions. Third, the phagocytic activity of macrophages, against HPV16, 18, and 33 peptides, was enhanced by 12-35 times compared with that under stressed conditions. Thus, A. helianthus extract is a strong stimulator of the immune system and tumorigenic suppression under stress conditions.


Assuntos
Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/metabolismo , Extratos Vegetais/farmacologia , Substâncias Protetoras/farmacologia , Rosa/química , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Peróxido de Hidrogênio/toxicidade , Queratina-8/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Fagocitose/efeitos dos fármacos , Extratos Vegetais/química , Substâncias Protetoras/química , Rosa/metabolismo , Vagina/citologia , Vimentina/metabolismo
13.
Biosci Rep ; 40(7)2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32519739

RESUMO

BACKGROUND: Lung adenocarcinoma (LUAD) is the most common histological type of lung cancer. To date, the prognosis of patients with LUAD remains dismal. METHODS: Three datasets were downloaded from the GEO database. Differentially expressed genes (DEGs) were obtained. FunRich was used to perform pathway enrichment analysis. Protein-protein interaction (PPI) networks were established and hub genes were obtained by Cytoscape software. GEPIA was utilized to conduct correlation and survival analysis. Upstream miRNAs of DEGs were predicted via miRNet database, and methylation status of promoters of DEGs was determined through UALCAN database. RESULTS: A total of 375 DEGs, including 105 and 270 up-regulated and down-regulated genes in LUAD, were commonly appeared in three datasets. These DEGs were significantly enriched in mesenchymal-to-epithelial transition (MET) and epithelial-to-mesenchymal transition (EMT). About 8 up-regulated and 5 down-regulated DEGs were commonly appeared in EMT/MET-related gene set and the top 50 hub gene set. Among the 13 genes, increased expression of KRT8 and KRT19 indicated unfavorable prognosis whereas high expression of DCN and CXCL12 suggested favorable prognosis in LUAD. Correlation analysis showed that KRT8 (DCN) expression was linked to KRT19 (CXCL12) expression. Further analysis displayed that KRT8 and KRT19 could jointly forecast poor prognosis in LUAD. About 42 and 2 potential miRNAs were predicted to target KRT8 and KRT19, respectively. Moreover, methylation level analysis demonstrated that KRT8 and KRT19 were significantly hypomethylated in LUAD compared with normal controls. CONCLUSIONS: All these findings suggest that KRT8 and KRT19 are hypomethylated and overexpressed in LUAD and associated with unfavorable prognosis.


Assuntos
Adenocarcinoma de Pulmão/mortalidade , Biomarcadores Tumorais/genética , Queratina-19/genética , Queratina-8/genética , Neoplasias Pulmonares/mortalidade , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Estudos de Casos e Controles , Quimiocina CXCL12/genética , Metilação de DNA , Conjuntos de Dados como Assunto , Decorina/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Prognóstico , Análise de Sobrevida , Regulação para Cima
14.
In Vitro Cell Dev Biol Anim ; 56(5): 386-398, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32472301

RESUMO

This study aimed to establish an in vitro model for lipid synthesis in primary bovine mammary epithelial cells (pbMECs) extracted from milk and cultured on Transwell permeable supports (TW culture). The suitability of these cells as a functional model for lactation was assessed by measuring κ-casein (CSN3) and diacylglycerol acyl transferase 1 (DGAT1) gene expression, the presence of intracellular lipid droplets, and the concentration of triacylglycerol in the cell lysates. The functionality of the milk-derived pbMECs cultured under lactogenic conditions, with and without oleic acid supplementation, was evaluated by comparing the cells grown on Transwell supports to cells grown on an extracellular matrix (ECM) gel (3D culture) or a plastic surface (2D culture). Furthermore, the functionality of milk-derived cells was compared to pbMECs obtained from bovine mammary tissue. Here, we show that in both tissue and milk-derived pbMECs, 3D culture offered the most suitable in vitro environment and led to increased levels of CSN3 and DGAT1 gene expression along with increased intracellular triacylglycerol content. The TW culture conditions also resulted in increased DGAT1 gene expression compared to the 2D conditions and milk-derived pbMECs cultured on TW inserts showed the highest viability compared to cells grown under 2D or 3D treatments. However, this was not observed for tissue-derived pbMECs, suggesting that TW culture may offer a beneficial environment specifically for milk-derived cells. We suggest that with further optimization of the culture conditions, TW culture may present a suitable model for the study of milk lipid synthesis in pbMECs.


Assuntos
Células Epiteliais/citologia , Lactação , Glândulas Mamárias Animais/citologia , Membranas Artificiais , Leite/citologia , Modelos Biológicos , Animais , Caseínas/genética , Caseínas/metabolismo , Bovinos , Polaridade Celular , Proliferação de Células , Sobrevivência Celular , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Impedância Elétrica , Feminino , Queratina-8/genética , Queratina-8/metabolismo , Permeabilidade , Triglicerídeos/metabolismo
15.
Int J Mol Sci ; 21(9)2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32365802

RESUMO

Leukamenin E is a natural ent-kaurane diterpenoid isolated from Isodon racemosa (Hemsl) Hara that has been found to be a novel and potential keratin filament inhibitor, but its underlying mechanisms remain largely unknown. Here, we show that leukamenin E induces keratin filaments (KFs) depolymerization, largely independently of microfilament (MFs) and microtubules (MTs) in well-spread cells and inhibition of KFs assembly in spreading cells. These effects are accompanied by keratin phosphorylation at K8-Ser73/Ser431 and K18-Ser52 via the by extracellular signal-regulated kinases (ERK) pathway in primary liver carcinoma cells (PLC) and human umbilical vein endothelial cells (HUVECs). Moreover, leukamenin E increases soluble pK8-Ser73/Ser431, pK18-Ser52, and pan-keratin in the cytoplasmic supernatant by immunofluorescence imaging and Western blotting assay. Accordingly, leukamenin E inhibits the spreading and migration of cells. We propose that leukamenin E-induced keratin phosphorylation may interfere with the initiation of KFs assembly and block the formation of a new KFs network, leading to the inhibition of cell spreading. Leukamenin E is a potential target drug for inhibition of KFs assembly.


Assuntos
Diterpenos do Tipo Caurano/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Queratina-18/metabolismo , Queratina-8/metabolismo , Queratinas/metabolismo , Movimento Celular , Sobrevivência Celular , Citoplasma , Citoesqueleto/patologia , Diterpenos do Tipo Caurano/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Fosforilação/efeitos dos fármacos , Ligação Proteica , Solubilidade
16.
J Tradit Chin Med ; 40(2): 245-252, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32242390

RESUMO

OBJECTIVE: To explore the underlying mechanism of action of Tongxieyaofang decoction in rats with visceral hypersensitivity using proteomics technology. METHODS: Twenty-four female Sprague-Dawley rats were randomly divided into three groups: control group, irritable bowel syndrome (IBS) group and Tongxieyaofang treatment group. An IBS model, characterized as visceral hypersensitivity, was established using the odour of mothballs as conditional stimulation and colorectal distension combined with classic physical restraint as non-conditional stimulation. Rats were intragastrically treated with Tongxieyaofang (2 or 4 mL·kg-1·d-1) for 4 weeks. On the 45th day, the rats were dissected and the colonic mucosal proteins were extracted. Differential protein spots were screened by fluorescent two-dimensional differential gel electrophoresis (2D-DIGE), and identified by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS). Western blotting experiments were performed to verify the changes observed in 2D-DIGE and MALDI-TOF-MS. RESULTS: It was found that the visceral sensitivity of rats in the Tongxieyaofang treatment group (4 mL/kg) was lower than that in the IBS group (P < 0.01). Sixty-one protein spots were differentially expressed between the IBS group and the Tongxieyaofang treatment group. Of these, 23 spots were upregulated in the Tongxieyaofang treatment group, while 38 spots were downregulated. Three specific proteins were successfully identified from the five protein spots with the most obvious changes. The two upregulated proteins were transgelin (TAGLN) and acetaldehyde dehydrogenase 2 (Aldh2) and the downregulated protein was cytokeratin 8 (CK8). CONCLUSION: Tongxieyaofang can dose-dependently ameliorate visceral hypersensitivity in rats and the mechanism of action may involve the upregulation of TAGLN and Aldh2 and the downregulation of CK8.


Assuntos
Colo/imunologia , Medicamentos de Ervas Chinesas/administração & dosagem , Síndrome do Intestino Irritável/tratamento farmacológico , Vísceras/imunologia , Aldeído-Desidrogenase Mitocondrial/genética , Aldeído-Desidrogenase Mitocondrial/imunologia , Animais , Colo/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Síndrome do Intestino Irritável/genética , Síndrome do Intestino Irritável/imunologia , Queratina-8/genética , Queratina-8/imunologia , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/imunologia , Proteínas Musculares/genética , Proteínas Musculares/imunologia , Ratos , Ratos Sprague-Dawley , Vísceras/efeitos dos fármacos
17.
Biomed Res Int ; 2020: 5058928, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32337254

RESUMO

Protein phosphorylation is a posttranslational modification that is essential for normal cellular processes; however, abnormal phosphorylation is one of the prime causes for alteration of many structural, functional, and regulatory proteins in disease conditions. In cancer, changes in the states of protein phosphorylation in tyrosine residues have been more studied than phosphorylation in threonine or serine residues, which also undergo alterations with greater predominance. In general, serine phosphorylation leads to the formation of multimolecular signaling complexes that regulate diverse biological processes, but in pathological conditions such as tumorigenesis, anomalous phosphorylation may result in the deregulation of some signaling pathways. Cervical cancer (CC), the main neoplasm associated with human papillomavirus (HPV) infection, is the fourth most frequent cancer worldwide. Persistent infection of the cervix with high-risk human papillomaviruses produces precancerous lesions starting with low-grade squamous intraepithelial lesions (LSIL), progressing to high-grade squamous intraepithelial lesions (HSIL) until CC is generated. Here, we compared the proteomic profile of phosphorylated proteins in serine residues from healthy, LSIL, HSIL, and CC samples. Our data show an increase in the number of phosphorylated proteins in serine residues as the grade of injury rises. These results provide a support for future studies focused on phosphorylated proteins and their possible correlation with the progression of cervical lesions.


Assuntos
Progressão da Doença , Proteômica , Neoplasias do Colo do Útero/fisiopatologia , Adulto , Colo do Útero/fisiopatologia , Colo do Útero/virologia , Clusterina/metabolismo , Feminino , Proteínas de Choque Térmico/metabolismo , Humanos , Queratina-19/metabolismo , Queratina-8/metabolismo , México , Pessoa de Meia-Idade , Chaperonas Moleculares/metabolismo , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/fisiopatologia , Infecções por Papillomavirus/virologia , Fosforilação , Lesões Pré-Cancerosas/virologia , Serina/metabolismo , Lesões Intraepiteliais Escamosas Cervicais/complicações , Lesões Intraepiteliais Escamosas Cervicais/fisiopatologia , Lesões Intraepiteliais Escamosas Cervicais/virologia , Treonina/metabolismo , Neoplasias do Colo do Útero/complicações , Neoplasias do Colo do Útero/virologia , Adulto Jovem
19.
Stem Cells Dev ; 29(10): 638-647, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32098606

RESUMO

Taste bud cells are specialized epithelial cells that undergo continuous turnover, and thus require active progenitors for their renewal and an intact taste function. Our previous studies suggested that a population of taste bud cells originates from outside of the surrounding tongue epithelium-previously regarded sole source of taste bud progenitors. In this study, we demonstrated that SOX10 (SRY-related HMG-box gene 10)-expressing cells, known to be in the migrating neural crest, were also distributed in taste bud-surrounding tissue compartments under the tongue epithelium, that is, the connective tissue core of taste papillae and von Ebner's glands. By lineage tracing of SOX10-expressing cells using SOX10-Cre, a Cre model driven by the endogenous SOX10 promoter, crossing with a Cre reporter line R26-tdTomato (tdT), we found SOX10-Cre-labeled tdT+ cells within taste buds in all three types of taste papillae (fungiform, circumvallate, and foliate) as well as in the soft palate in postnatal mice. The tdT+ taste bud cells were progressively more abundant along the developmental stages, from virtually zero at birth to over 35% in adults. Most of tdT+ taste bud cells had a low intensity of immunosignals of Keratin 8 (a widely used taste bud cell marker). In circumvallate taste buds, tdT signals were co-localized principally with a type III taste bud cell marker, less so with type I and II cell makers. Together, our data demonstrate a novel progenitor source for taste buds of postnatal mice-SOX10-Cre-labeled cells in the connective tissue core and/or von Ebner's glands.


Assuntos
Epitélio/metabolismo , Integrases/metabolismo , Queratina-8/metabolismo , Fatores de Transcrição SOXE/metabolismo , Animais , Células Epiteliais/metabolismo , Camundongos , Crista Neural/metabolismo , Língua/metabolismo
20.
J Cell Mol Med ; 24(5): 3217-3228, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32022439

RESUMO

Proliferative vitreoretinopathy (PVR) is a severe ocular disease which results in complex retinal detachment and irreversible vision loss. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is considered to be critical in the pathogenesis of PVR. In this study, we focused on the potential impact of keratin 8 (KRT8) phosphorylation and autophagy on TGF-ß2-induced EMT of RPE cells and explored the relationship between them. Using immunofluorescence and Western blot analysis, the co-localization of KRT8 and autophagy marker, as well as the abundance of phosphorylated KRT8 (p-KRT8) expression, was observed within subretinal and epiretinal membranes from PVR patients. Moreover, during TGF-ß2-induced EMT process, we found that p-KRT8 was enhanced in RPE cells, which accompanied by an increase in autophagic flux. Inhibition of autophagy with pharmacological inhibitors or specific siRNAs was associated with a reduction in cell migration and the synthesis of several EMT markers. In the meantime, we demonstrated that p-KRT8 was correlated with the autophagy progression during the EMT of RPE cells. Knockdown the expression or mutagenesis of the critical phosphorylated site of KRT8 would induce autophagy impairment, through affecting the fusion of autophagosomes and lysosomes. Therefore, this study may provide a new insight into the pathogenesis of PVR and suggests the potential therapeutic value of p-KRT8 in the prevention and treatment of PVR.


Assuntos
Queratina-8/genética , Descolamento Retiniano/genética , Fator de Crescimento Transformador beta2/genética , Vitreorretinopatia Proliferativa/genética , Adulto , Idoso , Autofagia/genética , Linhagem Celular , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação/genética , Descolamento Retiniano/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Vitreorretinopatia Proliferativa/patologia
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