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:Staphylococcus aureus small colony variants (SCVs) are a persistence-adapted phenotype associated with chronic and recurrent bovine mastitis. The interleukin-10 receptor ⍺ (IL10RA) is a key regulator of anti-inflammatory signalling in the mammary gland, yet its role in governing transcriptional responses to intracellular staphylococcal challenge remains unknown. Using CRISPR/Cas9-generated IL10RA knockout (KO) MAC-T bovine mammary epithelial cells (MECs) and RNA sequencing, we compared transcriptional responses to infection with S. aureus SCV Heba3231 and its isogenic parental strain (PS). The wild-type (WT) MAC-T cells mounted fundamentally divergent responses to the PS (8 DEGs) and the SCV (461 DEGs), with no transcriptional overlap, indicating strain-specific engagement of distinct molecular programmes. SCV infection of WT cells up-regulated lipid and sterol biosynthetic pathways, and suppressed genes associated with epithelial barrier integrity, including S100A8, S100A9, TGM3 and KRTDAP. IL10RA disruption dramatically amplified transcriptional dysregulation. The IL10RA-KO cells infected with the PS yielded 619 DEGs (77.4-fold increase over WT-PS), while IL10RA-KO cells infected with SCV yielded 1,297 DEGs (2.8-fold increase over WT-SCV). A moderate core of IL10RA-regulated genes (~32–36%) was shared across infection conditions. IL10RA loss derepressed pro-inflammatory cytokine pathways (TNF, IL-17), enhanced pro-apoptotic programmes during SCV infection, and suppressed phagosomal maturation and lipid metabolic gene programmes. Novel candidate genes, including CD79B, WFDC2, AMN and LY6E, were identified as potential biomarkers of IL10RA-dependent mammary immune signalling. These findings establish IL10RA as a broad transcriptional homeostasis regulator in bovine mammary epithelial cells during intracellular staphylococcal infection.