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1.
Ecotoxicol Environ Saf ; 188: 109918, 2020 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-31753310

RESUMEN

Hormonal regulation controls mammary gland (MG) development. Therefore some hormone-related factors can disrupt the early phases of MGs development, making the gland more susceptible to long term modifications in its response to circulating hormones. Endocrine disruptors, such as bisphenol A (BPA), are able to cause alterations in hormone receptor expression, leading to changes in the cell proliferation index, which may expose the tissue to neoplastic alterations. Thus, we evaluated the variations in hormone receptor expression in the MG of 6-month old Mongolian gerbils exposed to BPA and 17ß estradiol during the perinatal period. Receptors for estrogen alpha (ERα), beta (ERß), progesterone (PGR), prolactin (PRL-R), and co-localization of connexin 43 (Cx43) and ERα in gerbils were analyzed, and serum concentrations of estradiol and progesterone were assessed. No alterations in body, liver, and ovary-uterus complex weights were observed. However, there was an increase in epithelial ERα expression in the 17ß estradiol (E2) group and in PGR in the BPA group. Although immunohistochemistry did not show alterations in ERß expression, western blotting revealed a decrease in this protein in the BPA group. PRL-R was more present in epithelial cells in the vehicle control (VC), E2, and BPA groups in comparison to the intact control group. Cx43 was more frequent in E2 and BPA groups, suggesting a protective response from the gland against possible malignancy. Serum concentration of estradiol reduced in VC, E2, and BPA groups, confirming that alterations also impacts steroid levels. Consequently, perinatal exposure to BPA and the reference endogenous estrogen, 17ß estradiol, are able to increase the tendency of endocrine disruption in MG in a long term manner, since repercussions are observed even 6 months after exposure.


Asunto(s)
Compuestos de Bencidrilo/toxicidad , Disruptores Endocrinos/toxicidad , Estradiol/toxicidad , Glándulas Mamarias Animales/efectos de los fármacos , Fenoles/toxicidad , Efectos Tardíos de la Exposición Prenatal/metabolismo , Receptores Estrogénicos/metabolismo , Receptores de Progesterona/metabolismo , Animales , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Femenino , Gerbillinae , Glándulas Mamarias Animales/embriología , Glándulas Mamarias Animales/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente
2.
DNA Cell Biol ; 38(11): 1188-1196, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31603699

RESUMEN

The mammary gland is an important organ for lactation in dairy goats. Mammary gland development and lactation functions are primarily regulated by natural hormones and certain crucial regulatory factors. Nedd4 family-interacting protein 1 (Ndfip1) can specifically bind to neural precursor cell-expressed, developmentally downregulated protein 4 (Nedd4) family members to participate in ubiquitination, which in turn regulates a range of biological processes in the body. However, the effects of Ndfip1 expression regulation at the post-transcriptional level on the development of mammary gland cells have not been previously reported. To study the regulation of Ndfip1 at post-transcriptional level, the overexpression and interference vectors of Ndfip1 were constructed, and co-transfected into the primary mammary gland epithelial cells cultured in vitro with miR-143 mimics and inhibitor. Dual luciferase reporter gene system, real-time quantitative polymerase chain reaction, western blotting, cholecystokinin octapeptide assays, and flow cytometry were used to identify their regulation and function. As a result, Ndfip1 was targeted and regulated by miR-143, which influences the development of mammary gland epithelial cells in dairy goats cultured in vitro. This study will lay an experimental foundation for further understanding the functions of Ndfip1 and miR-143.


Asunto(s)
Apoptosis/genética , Células Epiteliales/fisiología , Cabras , Lactancia/genética , Glándulas Mamarias Animales/fisiología , Proteínas de la Membrana/genética , MicroARNs/fisiología , Animales , Células Cultivadas , Industria Lechera , Células Epiteliales/metabolismo , Femenino , Regulación de la Expresión Génica , Cabras/genética , Cabras/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo
3.
J Agric Food Chem ; 67(40): 11167-11178, 2019 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-31542928

RESUMEN

Milk contains a number of beneficial fatty acids including short and medium chain and unsaturated conjugated and nonconjugated fatty acids. In this study, microRNA sequencing of mammary tissue collected in early-, peak-, mid-, and late-lactation periods was performed to determine the miRNA expression profiles. miR-16a was one of the differentially expressed miRNA and was selected for in-depth functional studies pertaining to fatty acid metabolism. The mimic of miR-16a impaired fat metabolism [triacylglycerol (TAG) and cholesterol] while knock-down of miR-16a promoted fat metabolism in vitro in bovine mammary epithelial cells (BMECs). In addition, the in vitro work with BMECs also revealed that miR-16a had a negative effect on the cellular concentration of cis 9-C18:1, total C18:1, C20:1, and C22:1 and long-chain polyunsaturated fatty acids. Therefore, these data suggesting a negative effect on fatty acid metabolism extend the discovery of the key role of miR-16a in mediating adipocyte differentiation. Through a combination of bioinformatics analysis, target gene 3' UTR luciferase reporter assays, and western blotting, we identified large tumor suppressor kinase 1 (LATS1) as a target of miR-16a. Transfection of siRNA-LATS1 into BMECs led to increases in TAG, cholesterol, and cellular fatty acid concentrations, suggesting a positive role of LATS1 in mammary cell fatty acid metabolism. In summary, data suggest that miR-16a regulates biological processes associated with intracellular TAG, cholesterol, and unsaturated fatty acid synthesis through LATS1. These data provide a theoretical and experimental framework for further clarifying the regulation of lipid metabolism in mammary cells of dairy cows.


Asunto(s)
Bovinos/metabolismo , Células Epiteliales/enzimología , Metabolismo de los Lípidos , Glándulas Mamarias Animales/enzimología , MicroARNs/metabolismo , Leche/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Bovinos/genética , Colesterol/metabolismo , Células Epiteliales/metabolismo , Ácidos Grasos/metabolismo , Femenino , Regulación de la Expresión Génica , Glándulas Mamarias Animales/metabolismo , MicroARNs/genética , Proteínas Serina-Treonina Quinasas/genética , Triglicéridos/metabolismo
4.
Int J Mol Sci ; 20(18)2019 Sep 09.
Artículo en Inglés | MEDLINE | ID: mdl-31505876

RESUMEN

: Cancer-associated fibroblasts (CAFs) in the tumor microenvironment contribute to all stages of tumorigenesis and are usually considered to be tumor-promoting cells. CAFs show a remarkable degree of heterogeneity, which is attributed to developmental origin or to local environmental niches, resulting in distinct CAF subsets within individual tumors. While CAF heterogeneity is frequently investigated in late-stage tumors, data on longitudinal CAF development in tumors are lacking. To this end, we used the transgenic polyoma middle T oncogene-induced mouse mammary carcinoma model and performed whole transcriptome analysis in FACS-sorted fibroblasts from early- and late-stage tumors. We observed a shift in fibroblast populations over time towards a subset previously shown to negatively correlate with patient survival, which was confirmed by multispectral immunofluorescence analysis. Moreover, we identified a transcriptomic signature distinguishing CAFs from early- and late-stage tumors. Importantly, the signature of early-stage CAFs correlated well with tumor stage and survival in human mammary carcinoma patients. A random forest analysis suggested predictive value of the complete set of differentially expressed genes between early- and late-stage CAFs on bulk tumor patient samples, supporting the clinical relevance of our findings. In conclusion, our data show transcriptome alterations in CAFs during tumorigenesis in the mammary gland, which suggest that CAFs are educated by the tumor over time to promote tumor development. Moreover, we show that murine CAF gene signatures can harbor predictive value for human cancer.


Asunto(s)
Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/metabolismo , Transcripción Genética , Animales , Femenino , Fibroblastos/patología , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Ratones , Ratones Transgénicos
5.
J Agric Food Chem ; 67(40): 11137-11147, 2019 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-31532202

RESUMEN

MicroRNA-mediated gene regulation is important for the development of the mammary gland and the lactating process. A previous study has shown that the expression of microRNA-21 (miR-21) is different in the dry and early lactation period of the dairy cow mammary gland, but the molecular mechanisms underlying the lactation cycle are not fully understood. Here, the function of miR-21-3p on bovine mammary gland epithelial cells (BMECs) was detected by MTT assay and flow cytometry analysis, which showed that miR-21-3p significantly promoted the cell viability and proliferation. Then, the regulating mechanism of miR-21-3p on cell viability and proliferation was elucidated. Dual luciferase assay, RT-qPCR, and Western blot results revealed that IGFBP5 was a target gene of miR-21-3p. It was known that lncRNA could act as a competing endogenous RNA to sequester miRNAs and reduce the regulatory effect of miRNA-targeted genes. Based on our previous lncRNA-seq data and bioinformatics analysis, lncRNA NONBTAT017009.2 was potentially associated with miR-21-3p, and its expression was specifically inhibited with the transfection of miR-21-3p mimic into BMECs. Inversely, the overexpression of NONBTAT017009.2 significantly decreased the expression level of miR-21-3p in BMECs, while the expression of IGFBP5, the target gene of miR-21-3p, was significantly upregulated. In addition, the promoter region of miR-21 contained two STAT3 binding sites, and the dual luciferase reporter assays revealed that the overexpression of STAT3 significantly reduced the promoter activity of miR-21, implying that the transcription factor STAT3 may act as an upstream regulator affecting the regulation process of miR-21-3p. The overexpression of STAT3 significantly inhibited the expression of miR-21-3p, while the mRNA expression of IGFBP5 was significantly increased compared with the control group. Besides, there are no STAT3 binding sites in the promoter region of IGFBP5 as we predicted by gene-regulation and JASPAR software. Therefore, it could infer that STAT3 might regulate the expression of IGFBP5 by miR-21-3p. Taken together, these results established a regulatory network of miR-21-3p to illustrate the regulating mechanism on promoting cow mammary epithelial cell proliferation.


Asunto(s)
Bovinos/genética , Proliferación Celular , Células Epiteliales/citología , Redes Reguladoras de Genes , Glándulas Mamarias Animales/citología , MicroARNs/metabolismo , Animales , Bovinos/crecimiento & desarrollo , Bovinos/metabolismo , Supervivencia Celular , Células Epiteliales/metabolismo , Femenino , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , MicroARNs/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo
6.
J Agric Food Chem ; 67(37): 10513-10520, 2019 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-31475823

RESUMEN

Amino acids can stimulate milk fat synthesis, but the underlying molecular mechanism is still largely unknown. In this study, we studied the regulatory role and corresponding molecular mechanism of cAMP response element-binding protein-regulated transcription coactivator 2 (CRTC2) in amino acid-induced milk fat synthesis in mammary epithelial cells. We showed that leucine and methionine stimulated CRTC2 but not p-CRTC2(Ser171) expression and nuclear localization in cow mammary epithelial cells. Knockdown of CRTC2 decreased milk fat synthesis and sterol regulatory element binding protein 1c (SREBP-1c) expression and activation, whereas its overexpression had the opposite effects. Neither knockdown nor overexpression of CRTC2 affected ß-casein synthesis and phosphorylation of the machanistic target of rapamycin (mTOR), suggesting that CRTC2 only regulates milk fat synthesis. CRTC2 knockdown abolished the stimulation of leucine and methionine on SREBP-1c expression and activation. Knockdown or overexpression of CRTC2 did not affect the protein level of cAMP-response element-binding protein (CREB) and its phosphorylation but decreased or increased the binding of p-CREB to the promoter of SREBP-1c gene and its mRNA expression, respectively. Mutation of Ser171 of CRTC2 did not alter the stimulation of CRTC2 on SREBP-1c expression and activation, further suggesting that CRTC2 functions in the nucleus. mTOR inhibition by rapamycin totally blocked the stimulation of leucine and methionine on CRTC2 expression. The expression of CRTC2 was dramatically higher in the mouse mammary gland of lactation period, compared with that of the dry and puberty periods, whereas p-CRTC2(Ser171) was not changed, further supporting that CRTC2 is a key transcription coactivator for milk fat synthesis. These results uncover that CRTC2 is a key transcription coactivator of amino acid-stimulated mTOR-mediated milk fat synthesis in mammary epithelial cells.


Asunto(s)
Aminoácidos/metabolismo , Bovinos/metabolismo , Células Epiteliales/metabolismo , Grasas/metabolismo , Glándulas Mamarias Animales/citología , Leche/metabolismo , Factores de Transcripción/metabolismo , Animales , Bovinos/genética , Femenino , Glándulas Mamarias Animales/metabolismo , Ratones , Fosforilación , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/genética
7.
J Dairy Res ; 86(3): 307-314, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31451129

RESUMEN

This study aimed to evaluate the transcriptional changes occurring in isolated perfused mammary alveolar tissue in response to inoculation with S. agalactiae and to identify the most affected biological functions and pathways after 3 h. Four udders taken at slaughter from cows with healthy mammary gland were perfused ex situ with warmed and gassed Tyrode's solution. Mammary alveolar tissue samples were taken from the left fore and rear quarters (IQ-inoculated quarters) before inoculation (hour 0) and at 3 h post inoculation (hpi) and at the same times from control right fore and rear quarters (not inoculated: NIQ). A total of 1756 differentially expressed genes (DEGs) were identified between IQ and NIQ at 3 hpi using edgeR package. Within this set of DEGs, 952 were up regulated and mainly involved with innate immune response and inflammatory response, e.g., CD14, CCL5, TLR2, IL-8, SAA3, as well as in transcriptional regulation such as FOS, STAT3 and NFKBIA. Genes down-regulated (804) included those involved with lipid synthesis e.g., APOC2, SCD, FABP3 and FABP4. The most affected pathways were chemokine signaling, Wnt signaling and complement and coagulation cascades, which likely reflects the early stage response of mammary tissue to S. agalactiae infection. No significant gene expression changes were detected by RNA-Seq in the others contrasts. Real time-PCR confirmed the increase in mRNA abundance of immune-related genes: TLR2, TLR4, IL-1ß, and IL-10 at 3 hpi between IQ and NIQ. The expression profiles of Casp1 and Bax for any contrasts were unaffected whereas Bcl2 was increased in IQ, which suggests no induction of apoptosis during the first hours after infection. Results provided novel information regarding the early functional pathways and gene network that orchestrate innate immune responses to S. agalactiae infection. This knowledge could contribute to new strategies to enhance resistance to this disease, such as genomic selection.


Asunto(s)
Perfilación de la Expresión Génica/veterinaria , Glándulas Mamarias Animales/metabolismo , Infecciones Estreptocócicas/veterinaria , Streptococcus agalactiae , Animales , Bovinos , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Inmunidad/genética , Inflamación/genética , Mastitis Bovina/genética , Mastitis Bovina/inmunología , Mastitis Bovina/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Infecciones Estreptocócicas/genética , Infecciones Estreptocócicas/inmunología
8.
Biomed Res Int ; 2019: 2596914, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31467878

RESUMEN

According to our previous studies, bta-miR-124a was differentially expressed in breast tissue between high-fat and low-fat dairy cows. However, the function of bta-miR-124a in lipid metabolism of dairy cows and the identification of its target genes have not been reported. Therefore, this study will identify the target gene of bta-miR-124a and explore its role in the regulation of milk lipid metabolism. First, preliminary bioinformatics prediction of bta-miR-124a candidate target genes was performed, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze relative expression changes of bta-miR-124a and its candidate target genes and the expression level of the downstream gene of the target gene in the lipid metabolism signaling pathway in dairy mammary epithelial cell lines (Mac-T), using the dual luciferase reporter system for the identification of the targeting relationship between bta-miR-124a and the candidate target gene. Then, the effect of transfection of bta-miR-124a mimics and inhibitors on triglyceride (TG) and free fatty acid (FFA) levels was analyzed. The results indicate that bta-miR-124a directly interacts with the 3'-untranslated region of peroxisomal trans-2-enoyl-CoA reductase (PECR) to downregulate its expression in Mac-T cells. Further, bta-mir-124a regulates the expression of PECR and the downstream gene extension of very long chain fatty acid protein 2 (ELOVL2) through an unsaturated fatty acid biosynthesis signaling pathway. In conclusion, bta-miR-124a is involved in lipid metabolism by directly downregulating the PECR gene and affecting the expression of the downstream gene ELOVL2 and regulates the content of some key secretory elements such as TG and FFA. The function of bta-miR-124a has a certain effect on the synthesis and secretion of milk fat in the mammary epithelial cells of dairy cows.


Asunto(s)
Metabolismo de los Lípidos/genética , Glándulas Mamarias Animales/metabolismo , MicroARNs/genética , Leche/metabolismo , Animales , Bovinos , Línea Celular , Ácidos Grasos no Esterificados/genética , Ácidos Grasos no Esterificados/metabolismo , Femenino , Regulación de la Expresión Génica/genética , Humanos , Glándulas Mamarias Animales/citología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Transfección , Triglicéridos/genética , Triglicéridos/metabolismo
9.
J Dairy Sci ; 102(10): 8977-8985, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31421874

RESUMEN

Extracellular amino acid profiles affect intracellular AA concentrations and profile as well as signaling proteins that regulate protein translation rates. The objective of this study was to assess whether various extracellular AA profiles and varied ratios of Lys to Met would increase the phosphorylation of signaling proteins related to protein metabolism. Six AA profiles, reflecting Dulbecco's modified Eagle's medium (DMEM), blood meal (BM), corn gluten meal (CGM), casein (CAS), plasma of lactating cows (PLA), and a negative control (NEG) represented the first factor (F1), and the ratio of Lys to Met (unaltered or set to 3:1) was the second factor (F2). Treatments were arranged in a 6 × 2 factorial manner, resulting in 12 treatments that were replicated 4 times. The total AA masses for all treatments were set to 659 mg/L (63% of DMEM) except NEG (0 mg/L). Confluent mammary epithelial cells were exposed to treatment media for 80 min (SD = 7.4). Intracellular concentrations of 17 AA were changed according to F1. The Met and Leu percent of total intracellular AA mass, as an example, varied from 0.58 (PLA) to 6.94 (NEG, +F2) for Met and 0.05 (NEG, -F2) to 4.63 (CGM, +F2) for Leu. Overall, balancing for Lys and Met at a 3:1 ratio increased intracellular concentrations of Lys and Met by 54 and 71%, respectively. Within the mechanistic target of rapamycin (mTOR) pathway, phosphorylation of mTOR (Ser2448), ribosomal protein S6 (Ser235/236), and eukaryotic initiation factor 4E binding protein 1 (Thr37/46) (4EBP1) were increased by all 5 AA profiles compared with the NEG control. We found no differences in phosphorylation state among the 5 AA profiles, indicating lack of sensitivity to various AA profiles. This lack of sensitivity between AA profiles might also be due to assay imprecision or other experimental limitations. Only phosphorylation of 4EBP1 was increased for F2. Phosphorylation of eukaryotic initiation factor 2 α subunit (Ser51) was unaffected by either F1 or F2 factors. Regression analyses indicated that intracellular concentrations of Met, Thr, Ile, and Leu predicted phosphorylation of mTOR-related proteins with adequate precision and accuracy, suggesting that multiple EAA dictate regulation, regardless of AA ratios. Changes in extracellular AA profiles translated to modified intracellular AA profiles, and no single profile uniquely stimulated phosphorylation of the mTOR pathway-related proteins.


Asunto(s)
Aminoácidos/metabolismo , Bovinos/fisiología , Biosíntesis de Proteínas , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Animales , Caseínas/metabolismo , Células Epiteliales/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Espacio Extracelular/metabolismo , Femenino , Lactancia , Lisina/metabolismo , Glándulas Mamarias Animales/metabolismo , Metionina/metabolismo , Fosforilación , Proteína S6 Ribosómica/metabolismo
10.
DNA Cell Biol ; 38(10): 1125-1133, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31408364

RESUMEN

Mammary gland involution is a regressive process for the gland to return to its prepregnancy state after lactation and comprises an initial reversible and second remodeling stage. Although many genes and the multiple expression profiles of their mRNAs have been found in this process, the mechanisms controlling the profiles are largely unknown. In this study, we identified and analyzed transcription factor Sox4 in mammary gland involution. Elevated expression of Sox4 gene in the first stage (48 h after weaning) was observed at the mRNA and protein levels in the mouse mammary gland. Immunohistochemistry of the involuting gland indicated that Sox4 was located in the nuclei of epithelial cells. Nuclear Sox4 was also detected in the second stage, but unlikely to be involved in cell death, one of the characteristic events of involution. To clarify the functional roles of Sox4 in involution, we introduced a model, including a normal mammary epithelial cell line, for finding candidate target genes of this transcription factor and examined its effect on tenascin C mRNA expression.


Asunto(s)
Núcleo Celular/genética , Glándulas Mamarias Animales/metabolismo , ARN Mensajero/genética , Factores de Transcripción SOXC/genética , Tenascina/genética , Animales , Animales Recién Nacidos , Muerte Celular , Línea Celular , Núcleo Celular/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Regulación de la Expresión Génica , Lactancia/fisiología , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Ratones , Embarazo , Unión Proteica , ARN Mensajero/metabolismo , Factores de Transcripción SOXC/metabolismo , Tenascina/metabolismo , Transcripción Genética , Destete
11.
J Agric Food Chem ; 67(34): 9532-9542, 2019 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-31369265

RESUMEN

Murine mammary gland is an ideal model for studying the development and milk synthesis in dairy animals. MicroRNAs play an important role in milk synthesis and mammary gland development; however, the molecular mechanism of miR-142-3p continues to be poorly understood. Here, we knocked down miR-142-3p expression in vitro and vivo, increased the prolactin receptor expression and activated many downstream cellular proteins, such as mammalian target of rapamycin, sterol regulatory element-binding transcription factor 1, cyclin D1, and signal transducer and activator of transcription 5. Additionally, miR-142-3p knockdown in mouse mammary gland epithelial cells increased proliferation but not viability, induced cell cycle progression, decreased apoptosis, and increased the expression of triglycerides and ß-casein. Moreover, miR-142-3p knockdown in murine mammary gland tissue in vivo affected the structure and function of the mammary gland, which showed an increased number of lobules and ducts and was more capable of producing milk. However, overexpression of miR-142-3p had the opposite effects. In summary, these data reveal that miR-142-3p regulates milk synthesis and the structure of murine mammary glands via PRLR-mediated multiple signaling pathways.


Asunto(s)
Glándulas Mamarias Animales/metabolismo , MicroARNs/metabolismo , Leche/metabolismo , Receptores de Prolactina/metabolismo , Animales , Caseínas/metabolismo , Células Epiteliales/metabolismo , Femenino , Glándulas Mamarias Animales/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , MicroARNs/genética , Receptores de Prolactina/genética , Transducción de Señal , Triglicéridos/metabolismo
12.
BMC Genomics ; 20(1): 621, 2019 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-31362707

RESUMEN

BACKGROUND: Genetic polymorphisms are known to influence milk production and composition. However, the genomic mechanisms involved in the genetic regulation of milk component synthesis are not completely understood. MicroRNAs (miRNAs) regulate gene expression. Previous research suggests that the high developmental potential of the mammary gland may depend in part on a specific miRNA expression pattern. The objective of the present study was to compare the mammary gland miRNomes of two dairy cow breeds, Holstein and Montbéliarde, which have different mammogenic potentials that are related to differences in dairy performance. RESULTS: Milk, fat, protein, and lactose yields were lower in Montbéliarde cows than in Holstein cows. We detected 754 distinct miRNAs in the mammary glands of Holstein (n = 5) and Montbéliarde (n = 6) midlactating cows using RNA-Seq technology, among which 738 were known and 16 were predicted miRNAs. The 25 most abundant miRNAs accounted for 90.6% of the total reads. The comparison of their abundances in the mammary glands of Holstein versus Montbéliarde cows identified 22 differentially expressed miRNAs (Padj ≤ 0.05). Among them, 11 presented a fold change ≥2, and 2 (miR-100 and miR-146b) were highly expressed. Among the most abundant miRNAs, miR-186 is known to inhibit cell proliferation and epithelial-to-mesenchymal transition. Data mining showed that 17 differentially expressed miRNAs with more than 20 reads were involved in the regulation of mammary gland plasticity. Several of them may potentially target mRNAs involved in signaling pathways (such as mTOR) and lipid metabolism, thereby indicating that they could influence milk composition. CONCLUSION: We found differences in the mammary gland miRNomes of two dairy cattle breeds. These differences suggest a potential role for miRNAs in mammary gland plasticity and milk component synthesis, both of which are related to milk production and composition. Further research is warranted on the genetic regulation of miRNAs and their role in milk synthesis.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Lactancia/genética , Glándulas Mamarias Animales/metabolismo , MicroARNs/genética , Animales , Bovinos , Femenino , Perfilación de la Expresión Génica , Leche/química , Leche/metabolismo
13.
BMC Genomics ; 20(1): 623, 2019 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-31366318

RESUMEN

BACKGROUND: Epigenetic changes such as cytosine (CpG) DNA methylations regulate gene expression patterns in response to environmental cues including infections. Microbial infections induce DNA methylations that play a potential role in modulating host-immune response. In the present study, we sought to determine DNA methylation changes induced by the mastitis causing Escherichia coli (E. coli) in porcine mammary epithelial cells (PMEC). Two time points (3 h and 24 h) were selected based on specific transcriptomic changes during the early and late immune responses, respectively. RESULTS: DNA methylation analysis revealed 561 and 898 significant (P < 0.01) differentially methylated CpG sites at 3 h and 24 h after E. coli challenge in PMEC respectively. These CpG sites mapped to genes that have functional roles in innate and adaptive immune responses. Significantly, hypomethylated CpG sites were found in the promoter regions of immune response genes such as SDF4, SRXN1, CSF1 and CXCL14. The quantitative transcript estimation indicated higher expression associated with the DNA CpG methylation observed in these immune response genes. Further, E. coli challenge significantly reduced the expression levels of DNMT3a, a subtype of de novo DNA methylation enzyme, in PMEC indicating the probable reason for the hypomethylation observed in the immune response genes. CONCLUSIONS: Our study revealed E. coli infection induced DNA methylation loci in the porcine genome. The differentially methylated CpGs were identified in the regulatory regions of genes that play important role in immune response. These results will help to understand epigenetic mechanisms for immune regulation during coliform mastitis in pigs.


Asunto(s)
Metilación de ADN/inmunología , Escherichia coli/fisiología , Sitios Genéticos/genética , Glándulas Mamarias Animales/inmunología , Glándulas Mamarias Animales/metabolismo , Animales , Epigenómica , Femenino , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/microbiología , Mastitis/genética , Mastitis/inmunología , Mastitis/microbiología , Porcinos
14.
J Dairy Sci ; 102(10): 9017-9027, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31351725

RESUMEN

The supply and profile of absorbed AA may affect milk protein synthesis through hormonal changes and mammalian target of rapamycin (mTOR) signaling pathways; and Ile, Leu, Met, and Thr (ILMT) are the 4 AA that have been reported to have the greatest effect on mammary mTOR signaling. The extent to which ILMT and the other remaining AA (RAA) differ in their effects on milk protein synthesis needs to be systematically investigated. In this study, 5 lactating goats, averaging 120 ± 10 d in milk, fitted with jugular vein and carotid artery catheters, were fasted for 24 h, followed by intravenous infusions of a mixture containing AA and glucose for 8 h in a 5 × 5 Latin square design. The AA mixtures were formulated according to the profile of casein. The amounts of AA infused were calculated based on supplies of AA when metabolizable protein (MP) was at requirement (MR). Treatments were an infusate containing glucose without AA (NTAA); an infusate containing 3 × the MR of Ile, Leu, Met and Thr (3F0R); and infusates containing 3F0R plus 1, 2, or 3 × MR of RAA (3F1R, 3F2R, and 3F3R, respectively) according to amounts provided when fed to meet MP requirements for maintenance and lactation for each goat. Milk, arterial blood, and mammary tissue samples were collected immediately after halting the infusion. Relative to NTAA, supplementation of ILMT tended to increase milk protein production and plasma glucose concentrations, and increased milk and lactose production, but had no effects on production or content of milk fat. Graded supplementation of RAA tended to quadratically affect production of milk and lactose. Arterial glucose and glucagon concentrations decreased linearly, and plasma insulin concentrations decreased quadratically with increased RAA. Mammary p70-S6K1 phosphorylation was decreased by addition of ILMT compared with NTAA but increased linearly with increased RAA infusion. Furthermore, EIF4EBP1 gene expression was much lower for 3F-treated goats than for the NTAA treatment. Both MTOR and RPS6KB1 gene expressions were decreased quadratically with increased RAA supply. These results suggested that short-term milk protein yield tended to be increased by elevated ILMT availability, and this trend was not explained by variations in mammary mTOR signaling or pancreatic hormone secretions, whereas graded increase of RAA in combination with ILMT appeared to regulate the efficiency of conversion of glucose to lactose in a manner not involving milk protein production.


Asunto(s)
Aminoácidos/administración & dosificación , Cabras/fisiología , Insulina/administración & dosificación , Proteínas de la Leche/análisis , Leche/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Caseínas/análisis , Femenino , Glucagón/administración & dosificación , Glucosa/metabolismo , Isoleucina/administración & dosificación , Lactancia , Lactosa/análisis , Leucina/administración & dosificación , Glándulas Mamarias Animales/metabolismo , Metionina/administración & dosificación , Leche/química , Fosforilación/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Treonina/administración & dosificación
15.
Genome Res ; 29(8): 1223-1234, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31337711

RESUMEN

Most transcription factors, including nuclear receptors, are widely modeled as binding regulatory elements as monomers, homodimers, or heterodimers. Recent findings in live cells show that the glucocorticoid receptor NR3C1 (also known as GR) forms tetramers on enhancers, owing to an allosteric alteration induced by DNA binding, and suggest that higher oligomerization states are important for the gene regulatory responses of GR. By using a variant (GRtetra) that mimics this allosteric transition, we performed genome-wide studies using a GR knockout cell line with reintroduced wild-type GR or reintroduced GRtetra. GRtetra acts as a super receptor by binding to response elements not accessible to the wild-type receptor and both induces and represses more genes than GRwt. These results argue that DNA binding induces a structural transition to the tetrameric state, forming a transient higher-order structure that drives both the activating and repressive actions of glucocorticoids.


Asunto(s)
Cromatina/ultraestructura , Células Epiteliales/efectos de los fármacos , Genoma , Glucocorticoides/farmacología , ARN Mensajero/genética , Receptores de Glucocorticoides/química , Animales , Secuencia de Bases , Sistemas CRISPR-Cas , Línea Celular Tumoral , Cromatina/química , ADN/genética , ADN/metabolismo , Elementos de Facilitación Genéticos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Edición Génica/métodos , Glucocorticoides/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Ratones , Unión Proteica , Estructura Cuaternaria de Proteína , ARN Mensajero/metabolismo , Ratas , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Activación Transcripcional
16.
J Agric Food Chem ; 67(32): 8884-8895, 2019 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-31345029

RESUMEN

Leucine is an essential amino acid in the milk production of bovine mammary glands, but the regulatory roles and molecular mechanisms of leucine are still not known well. This study investigated the roles of leucine on milk synthesis and explored the corresponding mechanism in bovine mammary epithelial cells (BMECs). Leucine (0, 0.25, 0.5, 0.75, 1.0, and 1.25 mM) was added to BMECs that were cultured in FBS-free OPTI-MEM medium. Leucine significantly promoted milk protein and milk fat synthesis and also increased phosphorylation of mTOR signaling protein and the protein expression levels of SREBP-1c, with the most significant effects at 0.75 mM concentration. Leucine increased the expression and nuclear localization of DDX59, and loss and gain of gene function experiments further reveal that DDX59 mediates the stimulation of leucine on the mRNA expression variation of mTOR and SREBP-1c genes. PI3K inhibition experiment further detected that leucine upregulated expression of DDX59 and its downstream signaling via PI3K activation. ChIP-qPCR analysis further proved the binding of DDX59 to the promoter regions of mTOR and SREBP-1c. In summary, these data prove that DDX59 positively regulates the mTOR and SREBP-1c signaling pathways leading to synthesis of milk, and leucine regulates these two signaling pathways through the PI3K-DDX59 signaling.


Asunto(s)
Bovinos/metabolismo , Células Epiteliales/metabolismo , Leucina/metabolismo , Glándulas Mamarias Animales/metabolismo , Leche/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Helicasas/metabolismo , Animales , Bovinos/genética , Femenino , Fosfatidilinositol 3-Quinasas/genética , ARN Helicasas/genética , Transducción de Señal , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo
17.
J Agric Food Chem ; 67(31): 8485-8492, 2019 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-31304752

RESUMEN

How short-chain fatty acids (FAs) affect cell membrane morphology and milk fat biosynthesis in mammary epithelial cells (MECs) is yet unclear. This study investigated the primary bovine MEC response to different FAs. We observed that the cell surface ultrastructures were influenced by chain length and degree of saturability of FAs. The CD36, FATP1, and FABP3 gene expression was affected independent of the type of FA. FASN, LPIN1, PPARα, and PPARγ transcripts were more sensitive to the short-chain FAs (acetic and ß-hydroxybutyric acids). Furthermore, short-chain FAs inclined to regulate FA degradation-, elongation-, and metabolism-associated pathways, while long-chain FAs (stearic and trans-10,cis-12 conjugated linolenic acids) modulated extracellular matrix-receptor interaction-, transcriptional misregulation-, microRNA-, and ribosome biogenesis-related pathways. However, triacylglycerol accumulation in the cytoplasm was not changed by all of the FAs. Overall, FAs with different chain lengths and degrees of saturability could differentially alter primary bovine MEC cell morphology and influence protein profiles involved in milk fat synthesis pathways.


Asunto(s)
Bovinos/metabolismo , Células Epiteliales/metabolismo , Grasas/metabolismo , Ácidos Grasos no Esterificados/química , Ácidos Grasos no Esterificados/metabolismo , Glándulas Mamarias Animales/metabolismo , Leche/metabolismo , Animales , Antígenos CD36/genética , Antígenos CD36/metabolismo , Bovinos/genética , Grasas/química , Proteínas de Transporte de Ácidos Grasos/genética , Proteínas de Transporte de Ácidos Grasos/metabolismo , Femenino , Glándulas Mamarias Animales/citología , Leche/química , PPAR alfa/genética , PPAR alfa/metabolismo , Triglicéridos/metabolismo
18.
Molecules ; 24(14)2019 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-31311137

RESUMEN

In this study, we prepared stabilized vitamin A and C nanoemulsions, and investigated their efficacy on milk-specific proteins in bovine mammary epithelial cells (MAC-T). Emulsions of vitamin A (vit-A) and C (vit-C) were prepared using Lipoid S 75 and microfluidization. The particle size and polydispersity index (PDI) of nanoemulsified vit-A and vit-C were studied. The cytotoxic effect of nanoemulsion-free and nanoemulsified vit-A and vit-C was determined by an MTT assay. In addition, the efficacy of nanoemulsified vit-A and vit-C on the in vitro expression pattern of milk-specific proteins in MAC-T cells was investigated by quantitative RT-PCR. The results showed that the efficacies of stabilized nanoemulsions of vit-A and vit-C were 100% and 92.7%, respectively. The particle sizes were around 475.7 and 225.4 nm, and the zeta potentials were around -33.5 and -21.3 mV, respectively. The expression changes of αs2-, ß- and κ-casein were higher in the presence of a stabilized nanoemulsion of vit-A, compared with nanoemulsion-free vit-A. Furthermore, the expression changes of αs2- and ß-casein were lower and that of κ-casein was higher in the presence of a stabilized nanoemulsion of vit-C, compared with nanoemulsion-free vit-C. Thus, our findings demonstrate the efficacy of nanoemulsified vit-A and vit-C in changing the expression of milk-specific proteins in MAC-T cells.


Asunto(s)
Ácido Ascórbico/farmacología , Glándulas Mamarias Animales/metabolismo , Proteínas de la Leche/metabolismo , Vitamina A/farmacología , Animales , Ácido Ascórbico/química , Bovinos , Línea Celular , Estabilidad de Medicamentos , Emulsiones , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/efectos de los fármacos , Técnicas Analíticas Microfluídicas , Proteínas de la Leche/efectos de los fármacos , Nanopartículas , Tamaño de la Partícula , Vitamina A/química
19.
J Anim Sci ; 97(9): 3823-3831, 2019 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-31278739

RESUMEN

Milk fat is a main nutritional component of milk, and it has become one of the important traits of dairy cow breeding. Recently, there is increasing evidence that microRNAs (miRNA) play significant roles in the process of milk fat synthesis in the mammary gland. Primary bovine mammary epithelial cells (BMEC) were harvested from midlactation cows and cultured in DMEM/F-12 medium with 10% fetal bovine serum, 100 units/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL bovine insulin, 1 µg/mL hydrocortisone, and 2 µg/mL bovine prolactin. We found that miR-34b mimic transfection in BMEC reduced the content of intracellular triacylglycerol (TAG) and lipid droplet accumulation via triacylglycerol assay and Oil Red O staining; meanwhile, overexpression of miR-34b inhibited mRNA expression of lipid metabolism-related genes such as peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FASN), fatty acid binding protein 4 (FABP4), and CCAAT enhancer binding protein alpha (C/EBPα). Whereas miR-34b inhibitor resulted in completely opposite results. Furthermore, q-PCR and western blot analysis revealed the mRNA and protein expression levels of DCP1A were downregulated in miR-34b mimic transfection group and upregulated in miR-34b inhibitor group. Moreover, luciferase reporter assays verified that DCP1A was the direct target of miR-34b and DCP1A gene silencing in BMEC-inhibited TAG accumulation and suppressed lipid droplet formation. In conclusion, these findings revealed a novel miR-34b-DCP1A axis that has a significant role in regulating milk fat synthesis and suggested that miR-34b may be used to improve the beneficial ingredients in milk.


Asunto(s)
Bovinos/genética , Endorribonucleasas/metabolismo , Glucolípidos/metabolismo , Glicoproteínas/metabolismo , Metabolismo de los Lípidos/genética , MicroARNs/genética , Leche/química , Animales , Bovinos/fisiología , Células Cultivadas , Endorribonucleasas/genética , Células Epiteliales/metabolismo , Femenino , Silenciador del Gen , Gotas Lipídicas/metabolismo , Glándulas Mamarias Animales/metabolismo , ARN Mensajero/metabolismo , Triglicéridos/metabolismo
20.
EMBO J ; 38(14): e100852, 2019 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-31267556

RESUMEN

Breast cancer prevention is daunting, yet not an unsurmountable goal. Mammary stem and progenitors have been proposed as the cells-of-origin in breast cancer. Here, we present the concept of limiting these breast cancer precursors as a risk reduction approach in high-risk women. A wealth of information now exists for phenotypic and functional characterization of mammary stem and progenitor cells in mouse and human. Recent work has also revealed the hormonal regulation of stem/progenitor dynamics as well as intrinsic lineage distinctions between mammary epithelial populations. Leveraging these insights, molecular marker-guided chemoprevention is an achievable reality.


Asunto(s)
Neoplasias de la Mama/patología , Glándulas Mamarias Humanas/citología , Células Madre/citología , Animales , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Femenino , Humanos , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Ratones , Transducción de Señal , Células Madre/metabolismo , Células Madre/patología
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