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Both mRNA and miRNA play an important role in the regulation of mammary fatty acid metabolism and milk fat synthesis. Although studies have shown a strong transcriptional control of fatty acid metabolism, less is known about the regulatory mechanisms of milk fat synthesis as a function of miRNA-mRNA interactions. In this study, we carried out transcriptome sequencing using mammary tissues from the early lactation period, peak lactation, mid-lactation and late lactation in dairy cows and identified key genes regulating milk fatty acid metabolism. A total of 32 differentially co-expressed gene were screened out. Large tumor suppressor kinase 2 (LATS2) was chosen for further study using luciferase reporter assays, qRT-PCR and western blotting. The aim was to demonstrate that miR-497 is an upstream regulator of LATS2, i.e. miR-497 and LATS2 are a potential miRNA/mRNA regulatory pair. The results indicated that miR-497 could inhibit the production of triglycerides (TAG) and unsaturated fatty acids in bovine mammary epithelial cells (BMECs). In contrast, LATS2 can promote the production of TAG and unsaturated fatty acids. "Rescue" experiments further verified the miR-497/LATS2 regulatory network. Overall, data underscored that the miR-497/LATS2 pathway exerts control on milk fat metabolism and provides a theoretical approach for improving milk quality via genetic means.

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.

Research on the mechanisms that regulate milk fat synthesis in dairy cows is essential to identify potential molecular targets that in the long term can help develop appropriate molecular breeding programs. Although some studies have revealed that microRNA (miRNA) affect lipid metabolism by targeting specific genes, joint analysis of miRNA and target mRNA data from bovine mammary tissue has revealed few clues regarding the underlying mechanisms controlling milk fat synthesis. The objective of the present study was to use high-throughput sequencing and bioinformatics analysis to identify miRNA and mRNA pairs and explore further their potential roles in regulating milk fat synthesis. A total of 233 pairs of negatively associated miRNA and mRNA pairs were detected. Among those, there were 162 pairs in which the miRNAs were down-regulated and the target mRNAs were up-regulated. Among the identified miRNA, miR-106b can bind the 3'-UTR of the ATP binding cassette subfamily A member 1 ( ABCA1), a gene previously identified as having a positive association with bovine milk fat synthesis. The overexpression of miR-106b in bovine mammary epithelial cells caused a decrease in triglyceride and cholesterol content while the inhibition of miR-106b increased triglyceride and cholesterol content, confirming its role in lipid metabolism. The present study allowed for the construction of a miR-106b- ABCA1 regulatory network map, thus providing a theoretical basis to target this gene in the molecular breeding of dairy cows.

As the main component of the Gram-negative bacterial cell wall, lipopolysaccharide (LPS) is well documented as an inducer of inflammation in bovine mammary cells. Lycium barbarum (goji) polysaccharides (LBP) have been used in nonruminants as prebiotics to improve growth performance, immune ability, and antioxidant capacity. We aimed to investigate the underlying effects of LBPs on proinflammatory responses in LPS-stimulated primary bovine mammary epithelial cells (bMECs). Cells were isolated from mammary tissue of three lactating Holstein cows without clinical disease (30.26 ± 3.1 kg/d of milk yield; 175 ± 6 DIM). For the pre-experimental treatment, bMECs were precultured with serum-free medium for 12 h. Treatments were as follows: pretreatment with culture medium devoid of LPS or LBP for 30 h (CON); CON for 24 h followed by challenge with 2 μg/mL LPS for 6 h (LPS); pretreatment with 100 or 300 μg/mL LBP for 24 h followed by LPS challenge (2 μg/mL) for 6 h (LBP(100)+LPS; LBP(300)+LPS). To further determine if the effect of LBP on immuneregulation is peroxisome proliferator-activated receptor-γ (PPARγ) activation dependent, an inhibitor of PPARγ, GW9662, at a concentration of 1 μM was used. Cells treated with LBP at 100, 300, and 500 μg/mL had upregulated protein abundance of PPARγ, while PGC1α had a higher expression only at 300 μg/mL of LBP treatment. Compared with CON, cells pretreated with LBP at 100 and 300 μg/mL had greater protein abundance of SCD1 and SREBP1. 5-Ethynyl-2′-deoxyuridine (EdU) staining and cell wound healing assays showed that the negative effect of LPS alone on cell proliferation was reversed by pretreatment with LBP at both 100 and 300 μg/mL. Upregulation of gene and protein abundance of proinflammatory factors and cytokines (COX-2, NLRP3, TNF-α, IL-1β, and IL-6) induced by LPS stimulation were alleviated by LBP pretreatment at 300 μg/mL (more than 2-fold decrease). Compared with LPS challenge alone, phosphorylation of proteins involved in NF-κB (IκBα and p65) and MAPK (p38, JNK, and ERK) pathways was downregulated following LBP treatment. Additionally, inhibition of PPARγ by GW9662 weakened the protective effect of LBP on LPS-induced protein abundance of phosphorylated p65, COX-2, IL-1β, and TNF-α. These results indicated that the protective effect of LBP on LPS-induced bMECs inflammatory responses is PPARγ activation-dependent. As such, this knowledge might help design strategies for intervening against the detrimental effects of bovine mastitis. Interpretive summary Current research examined Lycium barbarum polysaccharides (LBP) for combating LPS-induced inflammatory responses in primary bovine mammary epithelial cells. We uncovered a preventive role of LBP in reducing detrimental effects induced by LPS including inhibition of NF-κB and MAPK along with peroxisome proliferator-activated receptor-γ (PPARγ) activation. The decrease in cell proliferation due to LPS was curtailed by pretreatment with LBP. Moreover, the effect of LBP on regulation of inflammatory responses in bovine mammary epithelial cell was PPARγ dependent. Collectively, data suggest that LBP reverses LPS-induced inflammatory response via MAPK/NF-κB signaling in a PPARγ-activation-dependent manner. Thus, the study provides new insights into therapeutic strategies for combating mastitis using LBP and highlighted the link between PPARγ and regulation of mammary cell inflammation. [ABSTRACT FROM AUTHOR]

Background: Lipopolysaccharides (LPS) derived from gram-negative bacterial are often regarded as primary inducer of bovine mammary inflammation. This study evaluated the biological response of metformin activated AMPK signaling on LPS-induced inflammatory responses and metabolic changes in primary bovine mammary epithelial cells (pbMEC). The pbMEC were exposed to either 3 mmol/L Metf. for 12 h as Metf. group (Metf.) or 2 μg/mL LPS for 6 h as LPS group (LPS). Cells pretreated with 3 mmol/L metformin for 12 h followed by washing and 2 μg/mL LPS exposure for 6 h were served as ML group (ML). PBS was added to cells as the control group (Con.). Results: Pre-incubation with Metf. inhibited LPS-induced expression of pro-inflammatory genes (TNF, IL1B, IL6, CXCL8, MYD88 and TLR4) and proteins (IL-1β, TNF-α, NLRP3, Caspase1, ASC) and was accompanied by increased activation of AMPK signaling. Compared with the LPS group, phosphorylation of p65 and IκBα in the ML group were decreased and accumulation of NF-κB in the nucleus was significantly reduced by pretreatment with metformin. Metformin protects the cells from the increase of LPS-induced binding activity of NF-κB on both TNFA and IL1B promoters. Compared with the LPS group, genes (G6PC, PCK2) and proteins (SREBP1, SCD1) related to lipogenesis and carbohydrate metabolism were downregulated while catabolic ones (PPARA, ACSL1, Glut1, HK1) were upregulated in the ML group. Furthermore, increased acetylation of H3K14 by LPS challenge was reversed by pretreatment with metformin. Conclusion: Altogether, our results indicated that pretreatment with metformin dampens LPS-induced inflammatory responses mediated in part by AMPK/NF-κB/NLRP3 signaling and modification of histone H3K14 deacetylation and metabolic changes. [ABSTRACT FROM AUTHOR]

Dairy cow mastitis is a detrimental factor in milk quality and food safety. Mastitis generally refers to inflammation caused by infection by pathogenic microorganisms. Our studies in recent years have revealed the role of miRNA regulation in Staphylococcus aureus‐induced mastitis. In the present study, we overexpressed and suppressed miR‐145 to investigate the function of miR‐145 in Mac‐T cells. Flow cytometry, ELISA and EdU staining were used to detect changes in the secretion of several Mac‐T cytokines and in cell proliferation. We found that overexpression of miR‐145 in Mac‐T cells significantly reduced the secretion of IL‐12 and TNF‐α, but increased the secretion of IFN‐γ; the proliferation of bovine mammary epithelial cells was also inhibited. Using quantitative real‐time PCR (qRT‐PCR), Western blotting and luciferase multiplex verification techniques, we found that miR‐145 targeted and regulated FSCN1. Knock‐down of FSCN1 significantly increased the secretion of IL‐12, while the secretion of TNF‐α was significantly downregulated in Mac‐T cells. Upon S. aureus infection of mammary gland tissue, the body initiated inflammatory responses; Bta‐miR‐145 expression was downregulated, which reduced the inhibitory effect on the FSCN1 gene; and upregulation of FSCN1 expression promoted mammary epithelial cell proliferation to allow the recovery of damaged tissue. The results of the present study will aid in understanding the immune mechanism opposing S. aureus infection in dairy cows and will provide a laboratory research basis for the prevention and treatment of mastitis. [ABSTRACT FROM AUTHOR]

In this study, we identified CTCs using the previously reported CanPatrol CTC enrichment technique from peripheral blood samples of 126 patients with colorectal cancer (CRC) and found that CTCs could be classified into three subpopulations based on expression of epithelial cell adhesion molecule (EpCAM) (E-CTCs), the mesenchymal cell marker vimentin (M-CTCs), or both EpCAM and vimentin (biphenotypic E/M-CTCs). Circulating tumor microemboli (CTMs) were also identified in peripheral blood samples. Meanwhile, E-CTCs, M-CTCs, E/M-CTCs, and CTMs were detected in 76.98%, 42.06%, 56.35%, and 36.51% of the 126 patients, respectively. Interestingly, the presence of CTMs and each CTC subpopulation was significantly associated with blood lymphocyte counts and tumor-node-metastasis stage (P<0.001). Lymphocyte counts and the neutrophil-to-lymphocyte ratio (NLR) in patients lacking CTCs were significantly different from those in patients testing positive for CTMs and each CTC subpopulation (P<0.001). Our results indicate that tumor metastasis is more significantly associated with the presence of CTMs and M-CTCs than with other CTC subpopulations and suggest that EMT may be involved in CTC evasion of lymphocyte-mediated clearance. [ABSTRACT FROM AUTHOR]

Cadmium is a common environmental heavy metal pollutant that can accumulate over long periods of time and cause disease. Thus, analysis of the molecular mechanisms affected by cadmium in the body could be of great significance for the prevention and treatment of cadmium-related diseases. In this study, flow cytometry, immunofluorescence, transmission electron microscopy, H&E (Hematoxylin Eosin) staining and TUNEL (TdT-mediated dUTP Nick-End Labeling) assays were used to verify that cadmium induced apoptosis and immune responses in bovine mammary epithelial cells (BMECs) and in mouse mammary gland. Isolated BMECs cultured with or without cadmium were collected to screen miRNA (microRNA) using high-throughput sequencing. There were 42 differentially-expressed miRNAs among which 27 were upregulated and 15 downregulated including bta-miR-133a, bta-miR-23b-5p, bta-miR-29e, bta-miR-365–5p, bta-miR-615, bta-miR-7, bta-miR-11975, bta-miR-127, and bta-miR-411a. Among those, miR-133a (which can specifically target TGFB2 (Recombinant Transforming Growth Factor Beta 2) was the most significantly downregulated with a fold-change of 5.27 in BMECs cultured with cadmium. Application of the double luciferase reporter system, western blotting, and qRT-PCR (Quantitative Real-time PCR) revealed that circ08409 can directly bind to miR-133a. Experiments demonstrated that circRNA-08409 could adsorb bta-miR-133a. Both circ08409 and TGFB2 significantly increased apoptosis and altered expression level of a series of inflammatory factors in BMECs. In contrast, miR-133a decreased significantly apoptosis and inflammation in the cells. Compared with cultures receiving only cadmium, the miR-133a+cadmium cultures exhibited significant reductions in the occurrence of late apoptosis. Overall, results indicated that circ08409 could relieve the inhibitory effect of miR-133a on TGFB2 expression by combining with miR-133a and subsequently modulating cell proliferation, apoptosis and inflammation. Overall, the data suggested that the circ08409/miR-133a/TGFB2 axis might play a role in mediating the effect of cadmium on BMECs. As such, data provide novel insights into controlling hazards that cadmium could induce in the mammary gland. Cadmium promotes apoptosis and inflammation via the circ08409/miR-133a/TGFB2 [Display omitted] • Cadmium can induce apoptosis and can interfere with the cell cycle at a certain dose. • High-throughput sequencing of miRNA in blank group and Cadmium treatment group. • Cadmium leads to apoptosis and inflammation in BMECs via the circ08409/miR-133a/TGFB2 axis. [ABSTRACT FROM AUTHOR]