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1.
Int J Biol Sci ; 16(2): 251-263, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31929753

RESUMO

Long noncoding RNAs (lncRNAs) play multiple key roles during inflammatory processes. In this study, a novel lncRNA identified by the high-throughput sequencing analysis was found significantly down-regulated in Escherichia coli-introduced cell model of bovine mastitis. Given that this lncRNA consists of the antisense of leucine-rich repeat-containing protein 75A (LRRC75A), it was named LRRC75A antisense lncRNA1 (LRRC75A-AS1). The expression of LRRC75A-AS1 was down-regulated in bovine mammary epithelial cells and mammary tissues under inflammatory condition. Knockout (KO) of LRRC75A-AS1 by CRISPR-Cas9 system in bovine mammary alveolar cell-T (MAC-T) cell line could enhance expressions of tight junction (TJ) proteins Claudin-1, Occludin and ZO-1, reduce cell monolayer permeability, and inhibit Staphylococcus aureus adhesion and invasion. Meanwhile, it also down-regulated expressions of inflammatory factors and attenuated activation of NF-κB pathway. Similarly, knockdown of LRRC75A caused the changes as LRRC75A-AS1 KO did, while overexpression of LRRC75A enabled the opposite effects. TJ of epithelioid cells barriers the pathogenic microorganisms outside during inflammation, in which LRRC75A-AS1 can regulate the expression of TJ proteins through LRRC75A, affecting the development of inflammation.

2.
PeerJ ; 7: e6715, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30984483

RESUMO

The function of long non-coding RNA H19 (H19) on cell proliferation has been observed in various cell types, and the increased expression of H19 was also found in the lipopolysaccharide (LPS)-induced inflammatory bovine mammary epithelial cells (MAC-T). However, the roles of H19 in the inflammatory response and physiological functions of bovine mammary epithelial cell are not clear. In the present study, we found that overexpression of H19 in MAC-T cells significantly promoted cell proliferation, increased the protein and mRNA level of ß-casein, and enhanced the expression of tight junction (TJ)-related proteins while inhibited staphylococcus aureus adhesion to cells. In addition, results demonstrated that overexpression of H19 affected the LPS-induced immune response of MAC-T cells by promoting expressions of inflammatory factors, including TNF-α, IL-6, CXCL2 and CCL5, and activating the NF-κB signal pathway. Our findings indicate that H19 is likely to play an important role in maintaining normal functions and regulating immune response of bovine mammary epithelial cells.

3.
Res Vet Sci ; 124: 158-165, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30901668

RESUMO

The expression of N-myc downstream-regulated gene 1 (NDRG1) was significantly correlated with diverse processes such as cell growth and differentiation, lipid synthesis, stress and immune responses. Here we explored the role of NDRG1 expression in bovine mammary tissue and epithelial cells under an inflammatory condition. Results showed that NDRG1 expression was elevated in bovine mammary tissue with mastitis and mammary epithelial cells treated by heat inactivated Escherichia coli and Staphylococcus aureus compared to normal tissue and untreated cells. Overexpression of NDRG1 significantly inhibited cell proliferation and migration, ß-casein secretion, gene expressions of inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-6 and IL-8, and activation of mTOR signal pathway of mammary epithelial cells, and vice versa by NDRG1 knockdown. These findings suggest that NDRG1 has immense potential in the regulation of properties in bovine mammary epithelial cells under an inflammatory condition.


Assuntos
Proteínas de Ciclo Celular/genética , Citocinas/metabolismo , Células Epiteliais/imunologia , Regulação da Expressão Gênica/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Glândulas Mamárias Animais/imunologia , Leite/metabolismo , Animais , Caseínas/metabolismo , Bovinos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Escherichia coli/fisiologia , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Staphylococcus aureus/fisiologia
4.
Microb Pathog ; 130: 169-177, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30878619

RESUMO

Lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are key virulence factors of Escherichia coli and Staphylococcus aureus respectively, and both of them could cause inflammatory reaction in bovine mammary glands. In this study, we used bovine mammary epithelial cells (BMECs) as pattern recognition receptors and stimulated them with LPS or LTA to investigate the global transcriptional response variations of BMECs to these two different virulent factors through RNA-Seq analysis. We found 100 differentially expressed genes (DEGs) with 95 up-regulated and 5 down-regulated genes in LPS-treated group, whereas 24 DEGs with 12 up-regulated and 12 down-regulated genes in LTA-treated group compared to control. Although the number and expression changes of DEGs are significantly different between LPS vs Control and LTA vs Control, KEGG pathway enrichment analysis showed the majorities of DEGs in each pair were enriched on cytokine-cytokine receptor interaction, NF-κB signaling pathway, and NOD-like receptor signaling pathway, especially cytokines and chemokines. These results provided a comprehensive analysis of gene expression profiles elicited by LPS and LTA in BMECs, contributing to the understanding of early "pathogen-host" interactions during intramammary infections.


Assuntos
Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Lipopolissacarídeos/metabolismo , Estresse Fisiológico , Ácidos Teicoicos/metabolismo , Fatores de Virulência/metabolismo , Animais , Bovinos , Células Cultivadas , Células Epiteliais/fisiologia , Perfilação da Expressão Gênica , Análise de Sequência de RNA
5.
FEBS J ; 286(9): 1780-1795, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30771271

RESUMO

The long non-coding RNAs (lncRNAs) are known to transcriptionally regulate a wide spectrum of diseases. Here, we screened for potentially functional lncRNAs in a mammary epithelial cell model of bovine mastitis by RNA-Seq technology and identified a class of previously undetected mastitis-related lncRNAs. A novel lncRNA was widely expressed in a variety of bovine tissues with diverse relative abundance and had a relatively low expression in mammary tissue. Given its predicted target gene is TUBA1C, we name it lncRNA-TUB. We found a higher expression of lncRNA-TUB in mammary epithelial cells that received a proinflammatory stimulus compared to normal cells. Knockout of lncRNA-TUB by the CRISPR/Cas9 system revealed that it plays crucial roles in the morphological shape, proliferation, migration and ß-casein secretion of mammary epithelial cells. In addition, lncRNA-TUB mediates Escherichia coli-induced inflammatory factor secretion and Staphylococcus aureus adhesion to epithelial cells. Our results suggest that the lncRNAs identified here function in bovine mastitis, and that lncRNA-TUB affects the basic biological characteristics and functions of bovine mammary epithelial cells in inflammatory conditions, providing valuable insights into the mechanisms of bovine mastitis.

6.
Cell Prolif ; 52(1): e12525, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30362186

RESUMO

OBJECTIVES: The correlations between long non-coding RNAs (lncRNAs) and diverse mammal diseases have been clarified by many researches, but the cognition about bovine mastitis-related lncRNAs remains limited. This study aimed to investigate the potential role of lncRNA X-inactive specific transcript (XIST) in the inflammatory response of bovine mammary epithelial cells. MATERIALS AND METHODS: Two inflammatory bovine mammary alveolar cell-T (MAC-T) models were established by infecting the cells with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The expressions of pro-inflammatory cytokines were measured, and the proliferation, viability and apoptosis of the inflammatory cells were evaluated after XIST was knocked down by an siRNA. The relationship among XIST, NF-κB pathway and NOD-like receptor protein 3 (NLRP3) inflammasome was investigated using an inhibitor of NF-κB signal pathway. RESULTS: The expression of XIST was abnormally increased in bovine mastitic tissues and inflammatory MAC-T cells. Silencing of XIST significantly increased the expression of E. coli or S. aureus-induced pro-inflammatory cytokines. Additionally, knockdown of XIST could inhibit cell proliferation, suppress cell viability and promote cell apoptosis under inflammatory conditions. Furthermore, XIST inhibited E. coli or S. aureus-induced NF-κB phosphorylation and the production of NLRP3 inflammasome. CONCLUSIONS: The expression of XIST was promoted by activated NF-κB pathway and, in turn, XIST generated a negative feedback loop to regulate NF-κB/NLRP3 inflammasome pathway for mediating the process of inflammation.


Assuntos
Infecções por Escherichia coli/patologia , Glândulas Mamárias Animais/patologia , Mastite Bovina/patologia , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , RNA Longo não Codificante/metabolismo , Infecções Estafilocócicas/patologia , Animais , Apoptose/genética , Bovinos , Proliferação de Células , Sobrevivência Celular , Células Epiteliais/patologia , Escherichia coli , Feminino , Inflamação/genética , Inflamação/imunologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/imunologia , Mastite Bovina/microbiologia , NF-kappa B/antagonistas & inibidores , Interferência de RNA , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética , Staphylococcus aureus
7.
Acta Biochim Biophys Sin (Shanghai) ; 51(2): 150-158, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30590418

RESUMO

Monocyte chemoattractant protein-induced protein 1 (MCPIP1) is a kind of zinc finger RNA binding protein, which exerts immune responses in a variety of cell types. However, the role of MCPIP1 in bovine mammary epithelial cells during mastitis has not been studied. In this study, we explored the functions of MCPIP1 in the inflammatory process induced by virulence factors of pathogens in bovine mammary alveolar cell-T (MAC-T) cell line. Our results showed that MCPIP1 was significantly highly expressed both in the mammary tissue of dairy cows with mastitis and in inflammatory MAC-T cells induced by lipopolysaccharide (LPS) or lipoteichoic acid (LTA). Furthermore, we found that overexpression of MCPIP1 in MAC-T cells abated the LPS-induced increase at the gene expression levels of inflammatory mediators tumor necrosis factor-α-α, interleukin (IL)-1ß, IL-6 and IL-8, enhanced the LPS- and LTA-induced inhibition of epithelial proliferation and promoted the LPS- and LTA-induced oxidative and DNA damage. These findings indicated that MCPIP1 has an enormous potential in regulating the inflammatory response of bovine mammary epithelial cells during infection and may provide an effective therapeutic target for bovine mastitis to reduce the damage caused by inflammatory reactions.


Assuntos
Citocinas/imunologia , Células Epiteliais/imunologia , Mediadores da Inflamação/imunologia , Mastite Bovina/imunologia , Proteínas de Ligação a RNA/metabolismo , Dedos de Zinco , Animais , Bovinos , Linhagem Celular , Proliferação de Células/genética , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos , Glândulas Mamárias Animais/citologia , Mastite Bovina/genética , Mastite Bovina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia , Proteínas de Ligação a RNA/genética , Ácidos Teicoicos
8.
J Cell Biochem ; 119(11): 9028-9041, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30011095

RESUMO

Bovine mastitis occurs frequently in dairy cows and is often caused by various aetiological organisms, for example, Escherichia coli. Lipopolysaccharide (LPS) is a key virulence factor of E. coli. In this study, we stimulated bovine mammary epithelial cells (BMECs) with LPS to investigate the global transcriptional response and identify specific proinflammatory factors that play important roles in blood-milk barrier damage during mastitis caused by E. coli. By performing RNA-seq, we identified a large number of significantly differentially expressed genes (DEGs) between the LPS-treated BMECs and the control cells. Among the DEGs, interleukin-1ß (IL-1ß) was selected because its messenger RNA expression was induced by LPS and its enrichment is involved in multiple inflammatory signal pathways, and its roles in blood-milk barrier damage during the process of mastitis were investigated. Exogenous IL-1ß treatment damaged the integrity of the blood-milk barrier, as indicated by the increased BMEC tight junction (TJ) permeability and confirmed by in vitro and in vivo experiments. Furthermore, the IL-1ß-induced increase in the BMEC TJ permeability was mediated by the IL-1ß-ERK1/2-MLCK axis pathway. Our data provide insights into the functions of IL-1ß in blood-milk barrier damage caused by mastitis in dairy cows.


Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Interleucina-1beta/farmacologia , Glândulas Mamárias Animais/citologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Junções Íntimas/metabolismo , Animais , Western Blotting , Bovinos , Proliferação de Células/efeitos dos fármacos , Claudina-1/metabolismo , Feminino , Camundongos , Ocludina/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Junções Íntimas/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo
9.
Mediators Inflamm ; 2018: 4093285, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29686530

RESUMO

The expression of cytochrome P4501A1 (CYP1A1) enzyme is changed in various organs during the host response to inflammation or infection, leading to alterations in the metabolism of endogenous and exogenous compounds. Results of this study showed that CYP1A1 expression was significantly downregulated in the mammary tissue of bovine with mastitis, in inflammatory epithelial cells (INEs) extracted from the tissue, and in lipopolysaccharide- (LPS-) induced INEs compared with their corresponding counterparts. Overexpression of CYP1A1 in bovine mammary epithelial cells alleviated the LPS-induced inhibition of epithelial proliferation, abated the LPS-induced increase of gene expression and protein secretion of inflammatory cytokine tumor necrosis factor-α and interleukin-6, and attenuated the LPS-induced activation of NF-κB signaling. These findings suggest that CYP1A1 has immense potential in the regulation of inflammatory responses in bovine mammary epithelial cells during mastitis and may serve as a useful therapeutic target in mitigating injuries caused by inflammatory overreaction.


Assuntos
Citocromo P-450 CYP1A1/metabolismo , Células Epiteliais/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Lipopolissacarídeos/toxicidade , Glândulas Mamárias Animais/citologia , Animais , Bovinos , Linhagem Celular , Citocromo P-450 CYP1A1/genética , Células Epiteliais/efeitos dos fármacos , Feminino , Inflamação/imunologia , Interleucina-6/metabolismo , Mastite/imunologia , Mastite/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
10.
Food Chem Toxicol ; 115: 198-204, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29530639

RESUMO

Bovine mastitis is an infectious disease of the mammary gland which has been generally treated by antibiotic delivery. While the increasing drug-resistant bacteria and the high consumption of the antibiotic had become a noticeable concern. In a previous study, a mammary special vector expressing human ß-defensin 3 (hBD3) was transfected into bovine fetal fibroblasts to produce mastitis-resistant bovine. This investigation focused on potential unintended effects of transgenic milk containing hBD3 produced by these mastitis-resistant bovine on the reproductive system of C57BL/6J mice. Mice were fed with diets containing transgenic milk or conventional milk, nutritionally balanced to an AIN93G diet for 90 days, and non-milk diet was selected as the negative group. The reproductive system was given special attention including reproductive organ/body ratios, necropsy and histopathology, serum sex hormone, sperm parameters, estrus cycle and the expression level of some specific genes which could indicate the development and function of reproductive system. No diet-related significant differences were observed among three groups in this 90-day feeding study. The results indicated that hBD3 milk does not appear to exert any effect on the reproductive system in C57BL/6J rats compared with conventional milk or the control diet.


Assuntos
Animais Geneticamente Modificados/metabolismo , Bovinos/genética , Bovinos/metabolismo , Inocuidade dos Alimentos , Genitália/fisiologia , Leite/química , beta-Defensinas/genética , Animais , Animais Geneticamente Modificados/genética , Feminino , Alimentos Geneticamente Modificados , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Leite/metabolismo , beta-Defensinas/metabolismo
11.
PeerJ ; 5: e3950, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29062612

RESUMO

Increased levels of long noncoding RNA H19 (H19) have been observed in many inflammatory and organ fibrosis diseases including ulcerative colitis, osteoarthritis, liver fibrosis, renal fibrosis and pulmonary fibrosis. However, the role of H19 in bovine mastitis and mastitis-caused fibrosis is still unclear. In our study, H19 was characterized as a novel regulator of EMT induced by transforming growth factor-ß1 (TGF-ß1) in bovine mammary alveolar cell-T (MAC-T) cell line. We found that H19 was highly expressed in bovine mastitis tissues and inflammatory MAC-T cells induced by virulence factors of pathogens. TGF-ß1 was also highly expressed in inflammatory MAC-T cells, and exogenous TGF-ß1 could induce EMT, enhance extracellular matrix protein expression, and upregulate H19 expression in epithelial cells. Stable expression of H19 significantly promotes EMT progression and expression of ECM protein induced by TGF-ß1 in MAC-T cells. Furthermore, by using a specific inhibitor of the PI3K/AKT pathway, we demonstrated that TGF-ß1 upregulated H19 expression through PI3K/AKT pathway. All these observations imply that the lncRNA H19 modulated TGF-ß1-induced epithelial to mesenchymal transition in bovine epithelial cells through PI3K/AKT signaling pathway, which suggests that mammary epithelial cells might be one source for myofibroblasts in vivo in the mammary glands under an inflammatory condition, thereby contributing to mammary gland fibrosis.

12.
Cell Physiol Biochem ; 43(1): 82-93, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28848180

RESUMO

BACKGROUND/AIMS: Transforming growth factor-ß1 (TGF-ß1) plays a crucial role in chronic inflammation in various tissues, and is related to inflammation-caused organ fibrogenesis associated with the epithelial-mesenchymal transition (EMT) and the deposition of the extracellular matrix (ECM). However, the effect of TGF-ß1 on bovine mammary epithelial cells (BMECs) with mastitis, and its mechanism, remain unknown. METHODS: We analyzed the level of TGF-ß1 in inflamed mammary tissues and cells using western blotting. BMECs were treated with TGF-ß1, and EMT-related gene and protein expression changes were evaluated using quantitative real-time polymerase chain reaction (qPCR), western blotting, and immunofluorescence. We also inhibited the TGF/Smad signaling pathway using a receptor inhibitor, and analyzed EMT-related protein expression by western blotting. In addition, we injected TGF-ß1 into mice mammary glands to investigate whether it can cause mammary fibrosis in vivo. RESULTS: The TGF-ß1 level was up-regulated in mammary tissues with mastitis and in inducible inflammatory BMECs. TGF-ß1 treatment activated the TGF/ Smad signaling pathway in BMECs during their transition to the EMT phenotype, as indicated by morphological changes from a cobblestone-like shape to a spindle-like one. TGF-ß1 treatment also up-regulated the expression of α-smooth muscle actin, vimentin, and collagen I, albumin, and down-regulated the expression of E-cadherin both in mRNA level and protein level. Furthermore, TGF-ß1 enhanced the gene expressions of MMP2, MMP7, and fibronectin in BMECs. TGF-ß1 injection induced mice mammary infection and fibrosis. CONCLUSION: These findings suggested that aberrant up-regulation of TGF-ß1 in bovine mastitic mammary glands might play an important role in bovine mammary fibrosis caused by unresolved inflammation.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Actinas/metabolismo , Animais , Caderinas/metabolismo , Bovinos , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Glândulas Mamárias Animais/citologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 7 da Matriz/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Pirazóis/farmacologia , Pirróis/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima/efeitos dos fármacos
13.
Int J Biol Sci ; 13(5): 604-614, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28539833

RESUMO

Fibroblasts constitute the majority of the stromal cells within bovine mammary gland, yet the functional contributions of these cells to mastitis and fibrosis and the mechanism are poorly understood. In this study, we demonstrate that inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis had different expression pattern regarding to several extracellular matrix (ECM) proteins, chemokines and cytokines compared to normal fibroblasts (NFs) from dairy cows during lactation. The INFs induced epithelial-mesenchymal transition (EMT) and inflammatory responses of mammary epithelial cells in a vitro co-culture model. These functional contributions of INFs to normal epithelial cells were mediated through their ability to secrete stromal cell-derived factor 1 (SDF-1). SDF-1 was highly secreted/expressed by INFs, lipopolysaccharide (LPS) -treated NFs, lipoteichoic acid (LTA) -treated NFs, as well as mastitic tissue compared to their counterparts. Exogenous SDF-1 promoted EMT on epithelial cells through activating NF-κB pathway, induced inflammation response and inhibited proliferation of epithelial cells. In addition, SDF-1 was able to induce mastitis and slight fibrosis of mouse mammary gland, which was attenuated by a specific inhibitor of the receptor of SDF-1. Our findings indicate that stromal fibroblasts within mammary glands with mastitis contribute to EMT and inflammatory responses of epithelial cells through the secretion of SDF-1, which could result in the inflammation spread and fibrosis within mammary gland.


Assuntos
Quimiocina CXCL12/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Mastite Bovina/metabolismo , Animais , Bovinos , Células Epiteliais/citologia , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Lipopolissacarídeos/farmacologia , Glândulas Mamárias Animais/citologia , Mastite Bovina/patologia , Transdução de Sinais/efeitos dos fármacos , Ácidos Teicoicos/farmacologia
14.
Cell Death Dis ; 8(3): e2646, 2017 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-28252641

RESUMO

Tumor growth generates mechanical compression, which may trigger mechanotransduction in cancer and stromal cells and promote tumor progression. However, very little is known about how compression stimulates signal transduction and contributes to tumor progression. In the present study, we demonstrated that compression enhances a tumor progression phenotype using an in vitro compression model, and validated the results from the in vitro model with high- and low-compressed breast cancer tissues. Mechanical compression induced miR-9 downregulation by DNMT3A-dependent promoter methylation in the MDA-MB-231 and BT-474 breast cancer cell lines and in cancer-associated fibroblasts. The overexpression of miR-9 target genes (LAMC2, ITGA6, and EIF4E) was induced by miR-9 downregulation, which eventually enhanced vascular endothelial growth factors production. Demethylation and decompression could reverse compression-induced miR-9 downregulation and following overexpression of miR-9 target genes and VEGFA.


Assuntos
Neoplasias da Mama/genética , DNA (Citosina-5-)-Metiltransferases/genética , Regulação para Baixo/genética , MicroRNAs/genética , Fator A de Crescimento do Endotélio Vascular/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Metilação de DNA/genética , Progressão da Doença , Feminino , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , Mecanotransdução Celular/genética , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética
15.
Food Chem Toxicol ; 100: 34-41, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27979776

RESUMO

In recent years, transgenic technology has been widely applied in many fields. There is concern about the safety of genetically modified (GM) products with the increased prevalence of GM products. In order to prevent mastitis in dairy cows, our group produced transgenic cattle expressing human beta-defensin-3 (HBD3) in their mammary glands, which confers resistance to the bacteria that cause mastitis. The milk derived from these transgenic cattle thus contained HBD3. The objective of the present study was to analyze the nutritional composition of HBD3 milk and conduct a 90-day feeding study on rats. Rats were divided into 5 groups which consumed either an AIN93G diet (growth purified diet for rodents recommended by the American Institute of Nutrition) with the addition of 10% or 30% HBD3 milk, an AIN93G diet with the addition of 10% or 30% conventional milk, or an AIN93G diet alone. The results showed that there was no difference in the nutritional composition of HBD3 and conventional milk. Furthermore, body weight, food consumption, blood biochemistry, relative organ weight, and histopathology were normal in those rats that consumed diets containing HBD3. No adverse effects were observed between groups that could be attributed to varying diets or gender.


Assuntos
Peso Corporal/efeitos dos fármacos , Alimentos Geneticamente Modificados/toxicidade , Leite/química , Tamanho do Órgão/efeitos dos fármacos , beta-Defensinas/farmacologia , Animais , Animais Geneticamente Modificados , Bovinos , Qualidade de Produtos para o Consumidor , Dieta , Feminino , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Medição de Risco
16.
Exp Cell Res ; 349(1): 45-52, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27680776

RESUMO

Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis.


Assuntos
Células Epiteliais/patologia , Fibroblastos/patologia , Inflamação/patologia , Mastite Bovina/patologia , Animais , Bovinos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Inflamação/genética , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Ácidos Teicoicos/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
17.
Environ Entomol ; 45(4): 1090-6, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27389683

RESUMO

The effects of three transgenic Bacillus thuringiensis (Bt) rice lines, KMD1, KMD2, and G8-7, on biological parameters and population dynamics of nontarget insect, Rhopalosiphum maidis (Fitch) (Homoptera: Aphididae), were investigated in the laboratory and field. No significant differences were found between Bt and non-Bt rice lines for aphid survival. The developmental time of R. maidis that fed on KMD1 and KMD2 did not differ significantly from those of the individuals feeding on the parental variety Xiushui11, but significantly prolonged developmental time was observed on G8-7 as compared with its parental variety Xiushui110. Aphid fecundity was significantly higher on Bt than on parental rice. A 2-yr field survey indicated that Bt rice did not significantly affect the population dynamics of R. maidis in comparison with non-Bt rice. Additionally, guttation droplets of Bt rice and aphids feeding on Bt rice were analyzed for presence of Cry1Ab using ELISA. No Cry1Ab protein was found in aphid adults feeding on Bt rice lines both in the laboratory and field. By using the guttation droplets from the top of rice seedlings, we designed a novel method to collect phloem sap, and found that relatively low concentrations were detected in the guttation droplets from Bt rice lines. In conclusion, although the Bt rice lines tested in this study stimulate the fecundity of R. maidis, the aphid population density did not increase in Bt rice fields.


Assuntos
Afídeos/fisiologia , Oryza/genética , Plantas Geneticamente Modificadas , Animais , Afídeos/efeitos dos fármacos , Afídeos/crescimento & desenvolvimento , Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Ensaio de Imunoadsorção Enzimática , Fertilidade/efeitos dos fármacos , Proteínas Hemolisinas/farmacologia , Longevidade/efeitos dos fármacos , Ninfa/efeitos dos fármacos , Ninfa/crescimento & desenvolvimento , Ninfa/fisiologia , Dinâmica Populacional
18.
PLoS One ; 11(7): e0159700, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27438026

RESUMO

This study was performed to investigate the effects of genetically modified (GM) milk containing human beta-defensin-3 (HBD3) on mice by a 90-day feeding study. The examined parameters included the digestibility of GM milk, general physical examination, gastric emptying function, intestinal permeability, intestinal microflora composition of mice, and the possibility of horizontal gene transfer (HGT). The emphasis was placed on the effects on gastrointestinal (GI) tract due to the fact that GI tract was the first site contacting with food and played crucial roles in metabolic reactions, nutrition absorption and immunity regulation in the host. However, the traditional methods for analyzing the potential toxicological risk of GM product pay little attention on GI health. In this study, the results showed GM milk was easy to be digested in simulated gastric fluid, and it did not have adverse effects on general and GI health compared to conventional milk. And there is little possibility of HGT. This study may enrich the safety assessment of GM product on GI health.


Assuntos
Alimentos Geneticamente Modificados/efeitos adversos , Leite/efeitos adversos , beta-Defensinas/genética , Animais , Qualidade de Produtos para o Consumidor , Digestão , Trato Gastrointestinal/efeitos dos fármacos , Humanos , Camundongos , Leite/metabolismo
19.
Sci Rep ; 6: 27462, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27272504

RESUMO

Fibroblasts are predominant components of mammary stromal cells and play crucial roles in the development and involution of bovine mammary gland; however, whether these cells contribute to mastitis has not been demonstrated. Thus, we have undertaken biological and molecular characterization of inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis and normal fibroblasts (NFs) from slaughtered dairy cows because of fractured legs during lactation. The functional contributions of INFs to normal epithelial cells were also investigated by using an in vitro co-culture model. We present evidence that the INFs were activated fibroblasts and showed inflammation-related features. Moreover, INFs significantly inhibited the proliferation and ß-casein secretion of epithelial cells, as well as upregulated the expression of tumor necrosis factor-α and interleukin-8 in epithelial cells. These findings indicate that functional alterations can occur in stromal fibroblasts within the bovine mammary gland during mastitis, demonstrating the importance of stromal fibroblasts in bovine mastitis and its treatment.


Assuntos
Inflamação/patologia , Mastite Bovina/patologia , Células Estromais/citologia , Animais , Bovinos , Feminino , Perfilação da Expressão Gênica , Mastite Bovina/genética , Células Estromais/metabolismo
20.
Int J Cancer ; 135(9): 2024-33, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24643985

RESUMO

Brain metastases are associated with high morbidity as well as with poor prognosis and survival in breast cancer patients. Despite its clinical importance, metastasis of breast cancer cells through the blood-brain barrier (BBB) is poorly understood. The objective of our study was to investigate whether cancer-associated fibroblasts (CAFs) play crucial roles in breast cancer brain metastasis. Using a cell adhesion assays, in vitro BBB permeability and transmigration assays and soft agar colony formation assays, we investigated the physical roles of CAFs in breast cancer brain metastasis. We also performed immunofluorescence, flow cytometric analysis, Droplet Digital PCR and Simon™ Simple Western System to confirm changes in expression levels. We established two novel three-dimensional (3D) culture systems using a perpendicular slide chamber and applying 3D embedded culture method to reflect brain metastasis conditions. With a newly developed device, CAFs was proven to promote cell adhesion to human brain microvascular endothelial cells, in vitro BBB permeability and transmigration and colony formation of breast cancer cells. Furthermore, CAFs enhanced the invasive migration of breast cancer cells in two kinds of 3D cultures. These 3D models also reliably recapitulate the initial steps of BBB transmigration, micro-metastasis and colonization. Expression of integrin α5ß1 and αvß3, c-MET and α2,6-siayltransferase was increased in breast cancer cells that migrated through the BBB. In conclusion, based on our in vitro BBB and co-culture models, our data suggest that CAFs may play a role in breast cancer brain metastasis.


Assuntos
Neoplasias Encefálicas/secundário , Encéfalo/patologia , Neoplasias da Mama/patologia , Movimento Celular , Endotélio Vascular/patologia , Fibroblastos/patologia , Células Estromais/patologia , Transporte Biológico , Barreira Hematoencefálica , Western Blotting , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Adesão Celular , Proliferação de Células , Técnicas de Cocultura , Endotélio Vascular/metabolismo , Feminino , Fibroblastos/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/metabolismo , Células Tumorais Cultivadas
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