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1.
J Agric Food Chem ; 67(34): 9477-9491, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31429552

RESUMO

Lipopolysaccharide (LPS) is a bacterial endotoxin that induces intestine inflammation. Milk exosomes improve the intestine and immune system development of newborns. This study aims to establish the protective mechanisms of porcine milk exosomes on the attenuation of LPS-induced intestinal inflammation and apoptosis. In vivo, exosomes prevented LPS-induced intestine damage and inhibited (p < 0.05) LPS-induced inflammation. In vitro, exosomes inhibited (p < 0.05) LPS-induced intestinal epithelial cells apoptosis (23% ± 0.4% to 12% ± 0.2%). Porcine milk exosomes also decreased (p < 0.05) the LPS-induced TLR4/NF-κB signaling pathway activation. Furthermore, exosome miR-4334 and miR-219 reduced (p < 0.05) LPS-induced inflammation through the NF-κB pathway and miR-338 inhibited (p < 0.05) the LPS-induced apoptosis via the p53 pathway. Cotransfection with these three miRNAs more effectively prevented (p < 0.05) LPS-induced cell apoptosis than these miRNAs individual transfection. The apoptosis percentage in the group cotransfected with the three miRNAs (14% ± 0.4%) was lower (p < 0.05) than that in the NC miRNA group (28% ± 0.5%), and also lower than that in each individual miRNA group. In conclusion, porcine milk exosomes protect the intestine epithelial cells against LPS-induced injury by inhibiting cell inflammation and protecting against apoptosis through the action of exosome miRNAs. The presented results suggest that the physiological amounts of miRNAs-enriched exosomes addition to infant formula could be used as a novel preventative measure for necrotizing enterocolitis.

2.
EMBO Rep ; 20(9): e47892, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31318145

RESUMO

The conversion of skeletal muscle fiber from fast twitch to slow-twitch is important for sustained and tonic contractile events, maintenance of energy homeostasis, and the alleviation of fatigue. Skeletal muscle remodeling is effectively induced by endurance or aerobic exercise, which also generates several tricarboxylic acid (TCA) cycle intermediates, including succinate. However, whether succinate regulates muscle fiber-type transitions remains unclear. Here, we found that dietary succinate supplementation increased endurance exercise ability, myosin heavy chain I expression, aerobic enzyme activity, oxygen consumption, and mitochondrial biogenesis in mouse skeletal muscle. By contrast, succinate decreased lactate dehydrogenase activity, lactate production, and myosin heavy chain IIb expression. Further, by using pharmacological or genetic loss-of-function models generated by phospholipase Cß antagonists, SUNCR1 global knockout, or SUNCR1 gastrocnemius-specific knockdown, we found that the effects of succinate on skeletal muscle fiber-type remodeling are mediated by SUNCR1 and its downstream calcium/NFAT signaling pathway. In summary, our results demonstrate succinate induces transition of skeletal muscle fiber via SUNCR1 signaling pathway. These findings suggest the potential beneficial use of succinate-based compounds in both athletic and sedentary populations.

3.
J Dairy Sci ; 102(8): 6726-6737, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31155266

RESUMO

Previous studies have demonstrated that bovine milk contains mRNA and microRNA that are largely encapsulated in milk-derived exosomes. However, little information is available about long noncoding RNAs (lncRNA) in bovine milk. Increasing evidence suggests that lncRNA are of particular interest given their key role in gene expression and development. We performed a comprehensive analysis of lncRNA in bovine milk exosomes by RNA sequencing. We used a validated human in vitro digestion model to investigate the stability of lncRNA encapsulated in bovine milk exosomes during the digestion process. We identified 3,475 novel lncRNA and 6 annotated lncRNA. The lncRNA shared characteristics with those of other mammals in terms of length, exon number, and open reading frames. However, lncRNA showed higher expression than mRNAs. We selected 12 lncRNA of high-expression abundance and identified them by PCR. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that lncRNA regulate immune function, osteoblastogenesis, neurodevelopment, reproduction, cell proliferation, and cell-cell communication. We also investigated the 12 lncRNA using quantitative real-time PCR to reveal their expression profiles in milk exosomes during different stages of lactation (colostrum 2 d, 30 d, 150 d, and 270 d); their resulting expression levels in milk exosomes showed variations across the stages. A digestion experiment showed that bovine milk exosome lncRNA was resistant to in vitro digestion with different digestive juices, including saliva, gastric juice, pancreatic juice, and bile juice. Taken together, these results show for the first time that cow milk contains lncRNA, and that their abundance varied at different stages of lactation. As expected, bovine milk exosomal lncRNA were stable during in vitro digestion. These findings provide a basis for further understanding of the physiological role of milk lncRNA.

4.
Am J Physiol Cell Physiol ; 317(3): C434-C448, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31166713

RESUMO

MicroRNAs (miRNAs) are important negative regulators of genes involved in physiological and pathological processes in plants and animals. Recent studies have shown that miRNAs might regulate gene expression among different species in a cross-kingdom manner. However, the specific roles of plant miRNAs in animals remain poorly understood and somewhat. Herein, we found that plant MIR156 regulates proliferation of intestinal cells both in vitro and in vivo. Continuous administration of a high plant miRNA diet or synthetic MIR156 elevated MIR156 levels and inhibited the Wnt/ß-catenin signaling pathway in mouse intestine. Bioinformatics predictions and luciferase reporter assays indicated that MIR156 targets Wnt10b. In vitro, MIR156 suppressed proliferation by downregulating the Wnt10b protein and upregulating ß-catenin phosphorylation in the porcine jejunum epithelial (IPEC-J2) cell line. Lithium chloride and an MIR156 inhibitor relieved this inhibition. This research is the first to demonstrate that plant MIR156 inhibits intestinal cell proliferation by targeting Wnt10b. More importantly, plant miRNAs may represent a new class of bioactive molecules that act as epigenetic regulators in humans and other animals.

5.
Transgenic Res ; 28(2): 237-246, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30697646

RESUMO

Producing heterologous enzymes in the animal digestive tract to improve feed utilization rate is a new research strategy by transgenic technology. In this study, transgenic pigs specifically expressing ß-glucanase gene in the intestine were successfully produced by somatic cell nuclear transfer technology in order to improve digestibility of dietary ß-glucan and absorption of nutrients. The ß-glucanase activity in the intestinal juice of 4 transgenic pigs was found to be 8.59 ± 2.49 U/mL. The feeding trial results showed that the crude protein digestion of 4 transgenic pigs was significantly increased compared with that of the non-transgenic pigs. In order to investigate the inheritance of the transgene, 7 G1 transgenic pigs were successfully obtained. The ß-glucanase activity in the intestinal juice of 7 G1 transgenic pigs was found to be 2.35 ± 0.72 U/mL. The feeding trial results showed the crude protein digestion and crude fat digestion were significantly higher in 7 G1 transgenic pigs than in non-transgenic pigs. Taken together, our study demonstrated that the foreign ß-glucanase expressing in the intestine of the transgenic pigs could reduce the anti-nutritional effect of ß-glucans in feed. In addition, ß-glucanase gene could be inherited to the offsprings and maintain its physiological function. It is a promising approach to improve feed utilization by producing transgenic animals.


Assuntos
Ração Animal/análise , Animais Geneticamente Modificados/metabolismo , Glucanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Intestinos/enzimologia , Paenibacillus polymyxa/enzimologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Glicosídeo Hidrolases/genética , Suínos
6.
Int J Mol Sci ; 19(3)2018 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-29522441

RESUMO

Adipose tissue plays an important role in energy metabolism. Adipose dysfunction is closely related to obesity and type II diabetes. Glucose uptake is the key step for fat synthesis in adipocyte. miRNAs have been proven to play a crucial role in adipocyte differentiation, adipogenesis and glucose homeostasis. In this paper, we firstly reported that miR-146b decreased glucose consumption by up-regulating miR-146b in a porcine primary adipocyte model, while the inhibitor of endogenous miR-146b rescued the reduction. Then, miR-146b was predicated to target IRS1 by bioinformatics analysis, and a dual-luciferase reporter assay validated this predication. Western blot analyses indicated both IRS1 and glucose transporter type 4 (GLUT4) were down-regulated by miR-146b overexpression. Our study demonstrated that miR-146b regulated glucose homeostasis in porcine primary pre-adipocyte by targeting IRS1, and provided new understandings on regulations of lipogenesis by miRNAs.


Assuntos
Adipócitos/metabolismo , Glucose/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , MicroRNAs/metabolismo , Suínos/metabolismo , Adipogenia/genética , Tecido Adiposo , Animais , Sequência de Bases , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Lipogênese/genética , Cultura Primária de Células , Suínos/genética , Regulação para Cima
7.
Gen Comp Endocrinol ; 259: 104-114, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29174487

RESUMO

Tibetan minipig is an important animal model for human diseases. The anterior pituitary is the master gland responsible for growth, reproduction, and metabolism and is regulated by thousands of miRNAs/mRNAs molecules. However, little is known about miRNAs and their relationships with mRNAs in Tibetan minipig anterior pituitary. Using microarray and mRNA-Sequencing, we identified 203 miRNAs and 12,040 mRNA transcripts from the anterior pituitary of Tibetan minipigs. These miRNAs were corresponding to 194 hairpin precursors, 25 miRNA clusters and 24 miRNA families. In addition, 64 intragenic miRNAs were annotated. Using three bioinformatic algorithms (TargetScan, miRanda and RNAhybrid), 359,184 possible miRNA-mRNA interactions were predicted, and an integrated network of miRNAs and pituitary-specific mRNA transcripts was established. To validate the predicted results, the degradome sequencing was employed to confirm miRNA-mRNA interactions, totally, 30 miRNA-mRNA pairs were identified. The present study provided a general overview of miRNA and mRNA annotation in Tibetan minipig anterior pituitary and established a miRNA-mRNA interactions database at the whole genome scale, which helps shed light on the molecular mechanisms in the anterior pituitary of pigs even other mammals.


Assuntos
MicroRNAs/genética , Adeno-Hipófise/crescimento & desenvolvimento , Porco Miniatura , Animais , Modelos Animais de Doenças , Feminino , Suínos , Tibet
8.
BMC Vet Res ; 13(1): 101, 2017 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-28407805

RESUMO

BACKGROUND: Milk is a complex liquid that provides nutrition to newborns. Recent reports demonstrated that milk is enriched in maternal-derived exosomes that are involved in fetal physiological and pathological conditions by transmission of exosomal mRNAs, miRNAs and proteins. Until now, there is no such research relevant to exosomal mRNAs and proteins in porcine milk, therefore, we have attempted to investigate porcine milk exosomal mRNAs and proteins using RNA-sequencing and proteomic analysis. RESULTS: A total of 16,304 (13,895 known and 2,409 novel mRNAs) mRNAs and 639 (571 known, 66 candidate and 2 putative proteins) proteins were identified. GO and KEGG annotation indicated that most proteins were located in the cytoplasm and participated in many immunity and disease-related pathways, and some mRNAs were closely related to metabolisms, degradation and signaling pathways. Interestingly, 19 categories of proteins were tissue-specific and detected in placenta, liver, milk, plasma and mammary. COG analysis divided the identified mRNAs and proteins into 6 and 23 categories, respectively, 18 mRNAs and 10 proteins appeared to be involved in cell cycle control, cell division and chromosome partitioning. Additionally, 14 selected mRNAs were identified by qPCR, meanwhile, 10 proteins related to immunity and cell proliferation were detected by Western blot. CONCLUSIONS: These results provide the first insight into porcine milk exosomal mRNA and proteins, and will facilitate further research into the physiological significance of milk exosomes for infants.


Assuntos
Exossomos/química , Exossomos/genética , Leite/química , Proteoma/análise , Sus scrofa/genética , Transcriptoma , Animais , MicroRNAs/genética , RNA Mensageiro/genética , Análise de Sequência de RNA
9.
Anim Sci J ; 88(6): 863-872, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27758021

RESUMO

There are many reports that dietary supplementation with plant polysaccharides in pigs might promote their growth, but little is known about the maternal effect of ginseng polysaccharides (GPS) on piglets' growth by dietary supplementation to pregnant and lactating sows. In the current study, the effects of dietary supplementation with GPS on the immunity of sows and growth of their piglets were investigated. Results showed no significant difference among the four groups in the total number of piglets, live piglets, weak piglets and birth weight of piglets, indicating the GPS-treatment has no adverse effect on reproduction. Furthermore, the weaning weight of the GPS-treated groups was higher than that of control group (P < 0.05); among them, the addition of 200 mg/kg dose has the best effect. Interestingly, GPS increased the total immunoglobulin G concentration in milk and serum of sows (P < 0.05). The concentrations of interleukin (IL)-2, IL-6, tumor necrosis factor (TNF)-α, and interferon-γ in milk and serum of sows were also increased in the experimental groups relative to the control (P < 0.05). Meanwhile, maternal supplementation of GPS significantly increased IL-2 and TNF-α concentration in the piglets' serum of the experimental groups relative to control (P < 0.05). GPS (200 mg/kg) significantly increased the glutathione peroxidase activity in milk and serum (P < 0.05), while the concentrations of malondialdehyde were significantly reduced (P < 0.05). The present results indicated that GPS supplementation during late pregnancy and lactation improved immunity-related bio-molecular levels in sow serum and milk, which may be further beneficial to piglet health and growth through biological transmission effects.


Assuntos
Ração Animal , Dieta/veterinária , Suplementos Nutricionais , Lactação/fisiologia , Troca Materno-Fetal/fisiologia , Panax/química , Polissacarídeos/administração & dosagem , Prenhez/fisiologia , Suínos/crescimento & desenvolvimento , Suínos/imunologia , Animais , Citocinas/sangue , Citocinas/metabolismo , Feminino , Glutationa Peroxidase/sangue , Glutationa Peroxidase/metabolismo , Imunoglobulina G/sangue , Imunoglobulina G/metabolismo , Masculino , Malondialdeído/sangue , Malondialdeído/metabolismo , Leite/metabolismo , Polissacarídeos/efeitos adversos , Polissacarídeos/farmacologia , Gravidez , Reprodução/efeitos dos fármacos
10.
Transgenic Res ; 26(1): 1-11, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27995503

RESUMO

ß-Glucan is the predominant anti-nutritional factors in monogastric animal feed. Although ß-glucanase supplementation in diet can help to eliminate the adverse effects, enzyme stability is substantially modified during the feed manufacturing process. To determine whether the expression of endogenous ß-glucanase gene (GLU) in vivo can improve digestibility of dietary ß-glucan and absorption of nutrients, we successfully produced transgenic pigs via nuclear transfer which express the GLU from Paenibacillus polymyxa CP7 in the parotid gland. In three live transgenic founders, ß-glucanase activities in the saliva were 3.2, 0.07 and 0.03 U/mL, respectively, and interestingly the enzyme activities increased in the pigs from 178 days old to 789 days old. From the feed the amount of gross energy, crude protein and crude fat absorbed by the transgenic pigs was significantly higher than the non-transgenic pigs. Meanwhile the moisture content of the feces was significantly reduced in transgenic pigs compared with the non-transgenic pigs. Furthermore, in all positive G1 pigs, ß-glucanase activity was detectable and the highest enzyme activity reached 3.5 U/mL in saliva. Also, crude protein digestion was significantly higher in G1 transgenic pigs than in control pigs. Taken together, our data showed that the transgenic ß-glucanase exerted its biological catalytic function in vivo in the saliva, and the improved performance of the transgenic pigs could be accurately passed on to the offspring, indicating a promising alternative approach to improving nutrient availability was established to improve utilization of livestock feed through transgenic animals.


Assuntos
Animais Geneticamente Modificados/metabolismo , Suplementos Nutricionais , Glicosídeo Hidrolases/genética , Paenibacillus polymyxa/genética , Ração Animal , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Fezes/química , Glicosídeo Hidrolases/metabolismo , Paenibacillus polymyxa/enzimologia , Glândula Parótida/metabolismo , Suínos/genética , Suínos/crescimento & desenvolvimento
11.
Reproduction ; 153(3): 341-349, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27998941

RESUMO

FSH plays an essential role in processes involved in human reproduction, including spermatogenesis and the ovarian cycle. While the transcriptional regulatory mechanisms underlying its synthesis and secretion have been extensively studied, little is known about its posttranscriptional regulation. A bioinformatics analysis from our group indicated that a microRNA (miRNA; miR-361-3p) could regulate FSH secretion by potentially targeting the FSHB subunit. Herein, we sought to confirm these findings by investigating the miR-361-3p-mediated regulation of FSH production in primary pig anterior pituitary cells. Gonadotropin-releasing hormone (GnRH) treatment resulted in an increase in FSHB synthesis at both the mRNA, protein/hormone level, along with a significant decrease in miR-361-3p and its precursor (pre-miR-361) levels in time- and dose-dependent manner. Using the Dual-Luciferase Assay, we confirmed that miR-361-3p directly targets FSHB. Additionally, overexpression of miR-361-3p using mimics significantly decreased the FSHB production at both the mRNA and protein levels, with a reduction in both protein synthesis and secretion. Conversely, both synthesis and secretion were significantly increased following miR-361-3p blockade. To confirm that miR-361-3p targets FSHB, we designed FSH-targeted siRNAs, and co-transfected anterior pituitary cells with both the siRNA and miR-361-3p inhibitors. Our results indicated that the siRNA blocked the miR-361-3p inhibitor-mediated upregulation of FSH, while no significant effect on non-target expression. Taken together, our results demonstrate that miR-361-3p negatively regulates FSH synthesis and secretion by targeting FSHB, which provides more functional evidence that a miRNA is involved in the direct regulation of FSH.


Assuntos
Hormônio Foliculoestimulante/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , Modelos Biológicos , Adeno-Hipófise/metabolismo , Receptores do FSH/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Hormônio Foliculoestimulante/genética , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/metabolismo , Masculino , Adeno-Hipófise/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do FSH/genética , Suínos , Regulação para Cima
12.
Sci Rep ; 6: 33291, 2016 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-27686746

RESUMO

Ammonia detoxification, which takes place via the hepatic urea cycle, is essential for nitrogen homeostasis and physiological well-being. It has been reported that a reduction in dietary protein reduces urea nitrogen. MicroRNAs (miRNAs) are major regulatory non-coding RNAs that have significant effects on several metabolic pathways; however, little is known on whether miRNAs regulate hepatic urea synthesis. The objective of this study was to assess the miRNA expression profile in a low protein diet and identify miRNAs involved in the regulation of the hepatic urea cycle using a porcine model. Weaned 28-days old piglets were fed a corn-soybean normal protein diet (NP) or a corn-soybean low protein diet (LP) for 30 d. Hepatic and blood samples were collected, and the miRNA expression profile was assessed by sequencing and qRT-PCR. Furthermore, we evaluated the possible role of miR-19b in urea synthesis regulation. There were 25 differentially expressed miRNAs between the NP and LP groups. Six of these miRNAs were predicted to be involved in urea cycle metabolism. MiR-19b negatively regulated urea synthesis by targeting SIRT5, which is a positive regulator of CPS1, the rate limiting enzyme in the urea cycle. Our study presented a novel explanation of ureagenesis regulation by miRNAs.

13.
Sci Rep ; 6: 33862, 2016 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-27646050

RESUMO

Milk-derived exosomes were identified as a novel mechanism of mother-to-child transmission of regulatory molecules, but their functions in intestinal tissues of neonates are not well-studied. Here, we characterized potential roles of porcine milk-derived exosomes in the intestinal tract. In vitro, treatment with milk-derived exosomes (27 ± 3 ng and 55 ± 5 ng total RNA) significantly promoted IPEC-J2 cell proliferation by MTT, CCK8, EdU fluorescence and EdU flow cytometry assays. The qRT-PCR and Western blot analyses indicated milk-derived exosomes (0.27 ± 0.03 µg total RNA) significantly promoted expression of CDX2, IGF-1R and PCNA, and inhibited p53 gene expression involved in intestinal proliferation. Additionally, six detected miRNAs were significantly increased in IPEC-J2 cell, while FAS and SERPINE were significantly down-regulated relative to that in control. In vivo, treated groups (0.125 µg and 0.25 µg total RNA) significantly raised mice' villus height, crypt depth and ratio of villus length to crypt depth of intestinal tissues, significantly increased CDX2, PCNA and IGF-1R' expression and significantly inhibited p53' expression. Our study demonstrated that milk-derived exosomes can facilitate intestinal cell proliferation and intestinal tract development, thus giving a new insight for milk nutrition and newborn development and health.


Assuntos
Proliferação de Células , Células Epiteliais/metabolismo , Exossomos/química , Regulação da Expressão Gênica , Mucosa Intestinal/metabolismo , Leite/química , Animais , Linhagem Celular , Células Epiteliais/citologia , Mucosa Intestinal/citologia , Suínos
14.
J Lipid Res ; 57(8): 1360-72, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27324794

RESUMO

TNF-α is a multifunctional cytokine participating in immune disorders, inflammation, and tumor development with regulatory effects on energy metabolism. Our work focused on the function of TNF-α in adipogenesis of primary porcine adipocytes. TNF-α could suppress the insulin receptor (IR) at the mRNA and protein levels. Microarray analysis of TNF-α-treated porcine adipocytes was used to screen out 29 differentially expressed microRNAs (miRNAs), 13 of which were remarkably upregulated and 16 were intensely downregulated. These 29 differentially expressed miRNAs were predicted to mainly participate in the insulin signaling pathway, adipocytokine signaling pathway, and type 2 diabetes mellitus pathway by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. miR-146a-5p, reportedly involved in immunity and cancer relevant processes, was one of the most highly differentially expressed miRNAs after TNF-α treatment. Red Oil O staining and TG assay revealed that miR-146a-5p suppressed adipogenesis. A dual-luciferase reporter and siRNA assay verified that miR-146a-5p targeted IR and could inhibit its protein expression. miR-146a-5p was also validated to be involved in the insulin signaling pathway by reducing tyrosine phosphorylation of insulin receptor substrate-1. Our study provides the first evidence of miR-146a-5p targeting IR, which facilitates future studies related to obesity and diabetes using pig models.


Assuntos
Adipogenia , MicroRNAs/genética , Receptor de Insulina/genética , Fator de Necrose Tumoral alfa/fisiologia , Regiões 3' não Traduzidas , Adipócitos/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Expressão Gênica , Ontologia Genética , Fosforilação , Cultura Primária de Células , Processamento de Proteína Pós-Traducional , Interferência de RNA , Receptor de Insulina/metabolismo , Sus scrofa
15.
Oncotarget ; 7(21): 30597-609, 2016 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-27121315

RESUMO

Nutrient absorption mediated by nutrient transporters expressed in the intestinal epithelium supplies substrates to support intestinal processes, including epithelial cell proliferation. We evaluated the role of Caudal type homeobox 2 (CDX2), an intestine-specific transcription factor, in the proliferation of pig intestinal epithelial cells (IPEC-1) and searched for novel intestinal nutrient transporter genes activated by CDX2. Our cloned pig CDX2 cDNA contains a "homeobox" DNA binding motif, suggesting it is a transcriptional activator. CDX2 overexpression in IPEC-1 cells increased cell proliferation, the percentage of cells in S/G2 phase, and the abundance of transcripts of the cell cycle-related genes Cyclin A2; Cyclin B; Cyclin D2; proliferating cell nuclear antigen; and cell cycle cyclin-dependent kinases 1, 2 and 4, as well as the predicted CDX2 target genes SLC1A1, SLC5A1 and SLC7A7. In addition, luciferase reporter and chromatin immunoprecipitation assays revealed that CDX2 binds directly to the SLC7A7 promoter. This is the first report of CDX2 function in pig intestinal epithelial cells and identifies SLC7A7 as a novel CDX2 target gene. Our findings show that nutrient transporters are activated during CDX2-induced proliferation of normal intestinal epithelial cells.


Assuntos
Sistema y+ de Transporte de Aminoácidos/genética , Fator de Transcrição CDX2/genética , Proliferação de Células/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Sequência de Aminoácidos , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Fator de Transcrição CDX2/metabolismo , Linhagem Celular , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Feminino , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Masculino , Regiões Promotoras Genéticas/genética , Ligação Proteica , Homologia de Sequência de Aminoácidos , Suínos
16.
PLoS One ; 10(7): e0131987, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26134288

RESUMO

The anterior pituitary is the most important endocrine organ modulating animal postnatal growth, mainly by controlling growth hormone (GH) gene transcription, synthesis, and secretion. As an ideal model for animal postnatal growth studies, the Bama minipig is characterized as having a lower growth performance and fewer individual differences compared with larger pig breeds. In this study, anterior pituitaries from Bama minipig and Landrace pig were used for miRNA and mRNA expression profile analysis using miRNA microarrays and mRNA-seq. Consequently, a total of 222 miRNAs and 12,909 transcripts were detected, and both miRNAs and mRNAs in the two breeds showed high correlation (r > 0.97). Additionally, 41 differentially expressed miRNAs and 2,254 transcripts were identified. Pathways analysis indicated that 32 pathways significantly differed in the two breeds. Importantly, two GH-regulation-signalling pathways, cAMP and inositol 1, 4, 5-triphosphate (IP3), and multiple GH-secretion-related transcripts were significantly down-regulated in Bama minipigs. Moreover, TargetScan and RNAHybrid algorithms were used for predicting differentially expressed miRNAs (DE miRNAs) and differentially expressed mRNAs (DE mRNAs) interaction. By examining their fold-changes, interestingly, most DE miRNA-DE mRNA target pairs (63.68-71.33%) presented negatively correlated expression pattern. A possible network among miRNAs, mRNAs, and GH-regulation pathways was also proposed. Among them, two miRNA-mRNA interactions (Y-47 targets FSHB; ssc-miR-133a-3p targets GNAI3) were validated by dual-luciferase assay. These data will be helpful in understanding the possible molecular mechanisms involved in animal postnatal growth.


Assuntos
MicroRNAs/genética , Adeno-Hipófise/metabolismo , RNA Mensageiro/genética , Porco Miniatura/genética , Suínos/genética , Transcriptoma , Algoritmos , Animais , Células CHO , Biologia Computacional , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hormônio do Crescimento/metabolismo , Inositol 1,4,5-Trifosfato/química , MicroRNAs/metabolismo , Análise em Microsséries , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Especificidade da Espécie
17.
Growth Horm IGF Res ; 25(2): 66-74, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25613666

RESUMO

OBJECTIVE: Growth hormone releasing hormone (GHRH) is a major positive regulator of growth hormone (GH) in the anterior pituitary gland, while cortistatin's (CST) role is negative. miRNAs (microRNAs or miRs) are small RNA molecules modulating gene expression at the post-transcriptional level. However, little is known about the function of miRNAs in the regulation of GH synthesis and/or secretion. This study investigated potential functional miRNAs involved in GH secretion in the normal porcine pituitary. DESIGN: Primary porcine anterior pituitary cells were cultivated and then treated with 10 nmol/L GHRH and 100 nmol/L CST, respectively. The effects of GHRH and CST on GH secretion were determined using RIA. miRNA microarrays were employed to analyze miRNA expression after treatment and then differentially expressed miRNAs were screened. Bioinformatics analysis was used to analyze the potential targets in growth hormone regulation of altered miRNAs. Furthermore, functional experiments were conducted to study the function of ssc-let-7c. RESULTS: GHRH significantly promoted GH secretion, while CST suppressed GH secretion. 19 and 35 differentially expressed miRNAs were identified in response to GHRH and CST treatments respectively. Verification of 5 randomly selected miRNAs by quantitative real-time PCR (qRT-PCR) showed similar changes with microarray analysis. Target analysis showed that some miRNAs may be involved in GH secretion-related pathways. Importantly, ssc-let-7c was predicted to target GH1 and GHRHR mRNA 3'untranslated regions (3'UTRs), which was supported by luciferase reporter assay. Furthermore, functional experimental results showed that ssc-let-7c was involved in GH secretion regulation, and overexpression of ssc-let-7c inhibited GH secretion in porcine anterior pituitary cells. CONCLUSIONS: GHRH and CST modulated porcine pituitary cell miRNA expression. Bioinformatics analysis revealed a complicated network among differentially expressed miRNAs, GH regulation-related genes and hormones. More interestingly, ssc-let-7c inhibited both GH1 and GHRHR mRNA 3'UTR reporter vectors' luciferase activity and overexpression of ssc-let-7c led to a decrease of GH secretion.


Assuntos
Hormônio Liberador de Hormônio do Crescimento/farmacologia , Hormônio do Crescimento/genética , MicroRNAs/genética , Neuropeptídeos/farmacologia , Adeno-Hipófise/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/metabolismo , Masculino , MicroRNAs/metabolismo , Análise em Microsséries , Adeno-Hipófise/metabolismo , Suínos
18.
Transgenic Res ; 24(3): 489-96, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25603989

RESUMO

The aflatoxin-detoxifizyme (ADTZ) gene derived from Armillariella tabescens was cloned into parotid gland-specific expression vector (pPSPBGPneo) to construct the parotid gland-specific vector expressing ADTZ (pPSPBGPneo-ADTZ). Transgenic mice were generated by microinjection and identified by using PCR and Southern blotting analysis. PCR and Southern blotting analysis showed that total six transgenic mice carried the ADTZ gene were generated. RT-PCR analysis indicated that the expression of ADTZ mRNA could be detected only in parotid glands of the transgenic mice. The ADTZ activity in the saliva was found to be 3.72 ± 1.64 U/mL. After feeding a diet containing aflatoxin B1 (AFB1) for 14 days, the effect of ADTZ on serum biochemical indexes and AFB1 residues in serum and liver of mice were evaluated. The results showed that total protein and globulin contents in the test treatment (transgenic mice) produced ADTZ were significantly higher than that of the positive control, while alanine aminotransferase and aspartate aminotransferase activity in serum of the test treatment (transgenic mice) were remarkably lower compared to that of the positive control (P < 0.05). Moreover, AFB1 residues in serum and liver of the test treatment (transgenic mice) were significantly lower compared with that of the positive control (P < 0.05). These results in the study confirmed that ADTZ produced in transgenic mice could reduce, even eliminate the negative effects of AFB1 on mice.


Assuntos
Inativação Metabólica/genética , Complexos Multienzimáticos/genética , Glândula Parótida/fisiologia , Aflatoxina B1/sangue , Aflatoxina B1/metabolismo , Aflatoxina B1/farmacocinética , Animais , Feminino , Fígado/metabolismo , Masculino , Camundongos Transgênicos , Complexos Multienzimáticos/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Saliva/fisiologia , Suínos/genética
19.
Mol Biol Rep ; 42(1): 61-9, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25227525

RESUMO

Of late years, a large amount of conserved and species-specific microRNAs (miRNAs) have been performed on identification from species which are economically important but lack a full genome sequence. In this study, Solexa deep sequencing and cross-species miRNA microarray were used to detect miRNAs in white shrimp. We identified 239 conserved miRNAs, 14 miRNA* sequences and 20 novel miRNAs by bioinformatics analysis from 7,561,406 high-quality reads representing 325,370 distinct sequences. The all 20 novel miRNAs were species-specific in white shrimp and not homologous in other species. Using the conserved miRNAs from the miRBase database as a query set to search for homologs from shrimp expressed sequence tags (ESTs), 32 conserved computationally predicted miRNAs were discovered in shrimp. In addition, using microarray analysis in the shrimp fed with Panax ginseng polysaccharide complex, 151 conserved miRNAs were identified, 18 of which were significant up-expression, while 49 miRNAs were significant down-expression. In particular, qRT-PCR analysis was also performed for nine miRNAs in three shrimp tissues such as muscle, gill and hepatopancreas. Results showed that these miRNAs expression are tissue specific. Combining results of the three methods, we detected 20 novel and 394 conserved miRNAs. Verification with quantitative reverse transcription (qRT-PCR) and Northern blot showed a high confidentiality of data. The study provides the first comprehensive specific miRNA profile of white shrimp, which includes useful information for future investigations into the function of miRNAs in regulation of shrimp development and immunology.


Assuntos
Sequência Conservada/genética , Genoma , MicroRNAs/genética , Penaeidae/genética , Animais , Biologia Computacional , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Polissacarídeos , Reprodutibilidade dos Testes
20.
J Nutr Biochem ; 25(12): 1296-308, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25283330

RESUMO

Adipocyte dysfunction is associated with many metabolic diseases such as obesity, insulin resistance and diabetes. Previous studies found that phloretin promotes 3T3-L1 cells differentiation, but the underlying mechanisms for phloretin's effects on adipogenesis remain unclear. In this study, we demonstrated that phloretin enhanced the lipid accumulation in porcine primary adipocytes in a time-dependent manner. Furthermore, phloretin increased the utilization of glucose and nonesterified fatty acid, while it decreased the lactate output. Microarray analysis revealed that genes associated with peroxisome proliferator-activated receptor-γ (PPARγ), mitogen-activated protein kinase and insulin signaling pathways were altered in response to phloretin. We further confirmed that phloretin enhanced expression of PPARγ, CAAT enhancer binding protein-α (C/EBPα) and adipose-related genes, such as fatty acids translocase and fatty acid synthase. In addition, phloretin activated the Akt (Thr308) and extracellular signal-regulated kinase, and therefore, inactivated Akt targets protein. Wortmannin effectively blocked the effect of phloretin on Akt activity and the protein levels of PPARγ, C/EBPα and fatty acid binding protein-4 (FABP4/aP2). Oral administration of 5 or 10 mg/kg phloretin to C57BL BKS-DB mice significantly decreased the serum glucose level and improved glucose tolerance. In conclusion, phloretin promotes the adipogenesis of porcine primary preadipocytes through Akt-associated signaling pathway. These findings suggested that phloretin might be able to increase insulin sensitivity and alleviate the metabolic diseases.


Assuntos
Adipócitos/citologia , Adipogenia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Floretina/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Animais , Glicemia/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Insulina/sangue , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , PPAR gama/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Suínos
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