Project description:In order to investigate the transcriptomic responses of bovine mammary epithelial cells to various essential amino acid administrations
Project description:The bovine mammary gland has a heterogeneous epithelial population which comprises terminally differentiated luminal and myoepithelial cells but also bipotent or lineage restricted progenitors and adult stem cells. The aim of our study was to use a novel surface marker (P-Cadherin) to characterize different mammary subpopulation, to sort adult stem cells with better enrichment and to perform whole RNA-seq among sorted population to identify biological processes or molecular functions associated gene enrichment. We found that only the CD49fhigh/PCadherinneg was enriched for adult mammary stem cells, while in other fractions we could detect luminal progenitors. Analysis of functional enrichments showed that in the stem cell compartment proliferation associated genes were downregulated, while genes relating to adhesion to ECM and to other neighboring cells were upregulated.
Project description:To further investigate the transcriptome profiles of EEF1D knocked-down and control BMECs, total RNA from the knocked-down BMECs treated with EEF1D-1357 or EEF1D-1893 shRNA, and the BMECs treated without shRNA, was extracted and purified, reversely transcribed into cDNA, and synthesized into cRNAs labeled with Cy3 using a Quick Amp Labeling Kit (Agilent). Afterwards, the labeled cRNAs (1.65 μg) were hybridized to a piece of Agilent Bovine Gene Expression 4×44K Chip using a Gene Expression Hybridization Kit (Agilent).
Project description:Purpose: to detect expression profile of differentially expressed mRNAs during bovine mammary epithelial cells (MAC-T) transfected with miR-375 inhibitor or negative control (NC) inhibitor in vitro. Methods: bovine mammary epithelial cells were transfected with miR-375 inhibitor or negative control (NC) inhibitor to assess the expression profiles of mRNAs using RNA-seq. Results: silencing miR-375 down-regulated and upregulated the expression of 48 and 15 mRNAs, respectively, in bovine mammary epithelial cells. Conclusion: miR-375 silencing dysregulated the expression of 63 mRNAs in bMECs. Also, miR-375 silencing increased the expression of NR4A1 and PTPN5 genes, all anti-inflammatory genes, via the MAPK signaling pathway. Given the negative correlation between miR-375 expression and NR4A1 and PTPN5 genes, miR-375 potentially promotes inflammation in the mammary gland through the MAPK signaling pathway. The findings of this study provide a new perspective of treating mastitis in cows.
Project description:Purpose: to detect expression profile of RNA (lncRNA and circRNA) and elucidate differentially expressed RNAs (DElncRNAs and DEcircRNAs) with potential roles during lipopolysaccharide (LPS)-induced inflammation models of bovine mammary epithelial cells MAC-T in vitro. Methods: bovine mammary epithelial cells MAC-T were exposed to LPS for 0, 6 and 12 hours to assess the expression profiles of RNA (lncRNA and circRNA) using RNA-seq. Results: totally 112 DElncRNAs and 71 DEcircRNAs were screened out at different time points. Functional enrichment analysis on target genes of lncRNAs and host genes of circRNAs indicated that these genes were involve in regulating inflammation-related signaling pathways, including Notch, NF-κB, MAPK, PI3K-Akt, mTOR, MAPK and NOD-like receptor signaling pathway. Conclusion: these differentially expressed RNAs (DElncRNAs and DEcircRNAs) may be involved in the regulation of a variety of immune-related processes including inflammatory responses bovine mammary epithelial cells exposed to LPS via some vital signaling pathways. This study lays a foundation for further research on molecular regulation of bovine mastitis, and also provides a reference for breeding strategies based on molecular markers for mastitis resistance in dairy cows.
Project description:Synthesis of milk fat is a complex biochemical process regulated by a series of molecular events. To determine gene expression changes during milk fat synthesis in bovine mammary epithelial cells, we established a cell model with a high capacity for milk fat synthesis using stimulation with acetate and β-hydroxybutyrate. RNA sequencing was used to identify differentially expressed genes (DEGs) between the high-milk-fat and control cells. A total of 625 DEGs (358 upregulated, 267 downregulated) were identified. Among the highly expressed genes, there was enrichment for terms associated with fatty acid synthesis, activation, and triacylglycerol synthesis, consistent with active milk fat synthesis. Kyoto Encyclopedia of Genes and Genomes analysis suggested that DEGs were most enriched in the “lipid metabolism” subcategory of the “metabolism” category, and in the “signal transduction” subcategory of the “environmental information processing” category. Although an in vitro cell model cannot completely simulate in vivo lactation, it eliminates interference from other cell types and from the synthesis of other milk components during transcriptome profile analysis. This work provides a profile of gene expression changes that occur during milk fat synthesis in bovine mammary epithelial cells, which furthers our understanding of the molecular regulation of lipid metabolism.
Project description:<p>Divergence in the targeted amino acid profilings of bovine mammary epithelial cells (BMECs) were systematically captured with regard to 10 individual essential amino acid (EAA) availability. BMECs cultured in 6-well plates were first serum-starved overnight and subsequently assigned to 1 of 12 treatment media (n = 6). DMEM-F12 medium is the positive control treatment (POS), while DMEM-F12 medium devoid of all EAA served as the negative control treatment (NEG). A total of 10 treatments were NEG individually supplemented with arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan or valine. Individual EAA were supplemented to achieve concentrations equal to those of POS. After 6-h treatment, all cell samples were taken at the same time, frozen in liquid nitrogen and stored at -80 °C until required.</p><p><br></p><p><strong>Targeted metabolomics</strong> is reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS3956' rel='noopener noreferrer' target='_blank'><strong>MTBLS3956</strong></a>.</p><p><strong>Untargeted metabolomics</strong> is reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS7789' rel='noopener noreferrer' target='_blank'><strong>MTBLS7789</strong></a>.</p>