Objectives: The correlations between long non‐coding RNAs (lncRNAs) and diverse mammal diseases have been clarified by many researches, but the cognition about bovine mastitis‐related lncRNAs remains limited. This study aimed to investigate the potential role of lncRNA X‐inactive specific transcript (XIST) in the inflammatory response of bovine mammary epithelial cells.Materials and methods: Two inflammatory bovine mammary alveolar cell‐T (MAC‐T) models were established by infecting the cells with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The expressions of pro‐inflammatory cytokines were measured, and the proliferation, viability and apoptosis of the inflammatory cells were evaluated after XIST was knocked down by an siRNA. The relationship among XIST, NF‐κB pathway and NOD‐like receptor protein 3 (NLRP3) inflammasome was investigated using an inhibitor of NF‐κB signal pathway.Results: The expression of XIST was abnormally increased in bovine mastitic tissues and inflammatory MAC‐T cells. Silencing of XIST significantly increased the expression of E. coli or S. aureus‐induced pro‐inflammatory cytokines. Additionally, knockdown of XIST could inhibit cell proliferation, suppress cell viability and promote cell apoptosis under inflammatory conditions. Furthermore, XIST inhibited E. coli or S. aureus‐induced NF‐κB phosphorylation and the production of NLRP3 inflammasome.Conclusions: The expression of XIST was promoted by activated NF‐κB pathway and, in turn, XIST generated a negative feedback loop to regulate NF‐κB/NLRP3 inflammasome pathway for mediating the process of inflammation.

Background/Aims: Transforming growth factor-β1 (TGF-β1) plays a crucial role in chronic inflammation in various tissues, and is related to inflammation-caused organ fibrogenesis associated with the epithelial-mesenchymal transition (EMT) and the deposition of the extracellular matrix (ECM). However, the effect of TGF-β1 on bovine mammary epithelial cells (BMECs) with mastitis, and its mechanism, remain unknown. Methods: We analyzed the level of TGF-β1 in inflamed mammary tissues and cells using western blotting. BMECs were treated with TGF-β1, and EMT-related gene and protein expression changes were evaluated using quantitative real-time polymerase chain reaction (qPCR), western blotting, and immunofluorescence. We also inhibited the TGF/Smad signaling pathway using a receptor inhibitor, and analyzed EMT-related protein expression by western blotting. In addition, we injected TGF-β1 into mice mammary glands to investigate whether it can cause mammary fibrosis in vivo. Results: The TGF-β1 level was up-regulated in mammary tissues with mastitis and in inducible inflammatory BMECs. TGF-β1 treatment activated the TGF/ Smad signaling pathway in BMECs during their transition to the EMT phenotype, as indicated by morphological changes from a cobblestone-like shape to a spindle-like one. TGF-β1 treatment also up-regulated the expression of α-smooth muscle actin, vimentin, and collagen I, albumin, and down-regulated the expression of E-cadherin both in mRNA level and protein level. Furthermore, TGF-β1 enhanced the gene expressions of MMP2, MMP7, and fibronectin in BMECs. TGF-β1 injection induced mice mammary infection and fibrosis. Conclusion: These findings suggested that aberrant up-regulation of TGF-β1 in bovine mastitic mammary glands might play an important role in bovine mammary fibrosis caused by unresolved inflammation.

Increased levels of long noncoding RNA H19 (H19) have been observed in many inflammatory and organ fibrosis diseases including ulcerative colitis, osteoarthritis, liver fibrosis, renal fibrosis and pulmonary fibrosis. However, the role of H19 in bovine mastitis and mastitis-caused fibrosis is still unclear. In our study, H19 was characterized as a novel regulator of EMT induced by transforming growth factor-β1 (TGF-β1) in bovine mammary alveolar cell-T (MAC-T) cell line. We found that H19 was highly expressed in bovine mastitis tissues and inflammatory MAC-T cells induced by virulence factors of pathogens. TGF-β1 was also highly expressed in inflammatory MAC-T cells, and exogenous TGF-β1 could induce EMT, enhance extracellular matrix protein expression, and upregulate H19 expression in epithelial cells. Stable expression of H19 significantly promotes EMT progression and expression of ECM protein induced by TGF-β1 in MAC-T cells. Furthermore, by using a specific inhibitor of the PI3K/AKT pathway, we demonstrated that TGF-β1 upregulated H19 expression through PI3K/AKT pathway. All these observations imply that the lncRNA H19 modulated TGF-β1-induced epithelial to mesenchymal transition in bovine epithelial cells through PI3K/AKT signaling pathway, which suggests that mammary epithelial cells might be one source for myofibroblasts in vivo in the mammary glands under an inflammatory condition, thereby contributing to mammary gland fibrosis.

The long non‐coding RNAs (lncRNAs) are known to transcriptionally regulate a wide spectrum of diseases. Here, we screened for potentially functional lncRNAs in a mammary epithelial cell model of bovine mastitis by RNA‐Seq technology and identified a class of previously undetected mastitis‐related lncRNAs. A novel lncRNA was widely expressed in a variety of bovine tissues with diverse relative abundance and had a relatively low expression in mammary tissue. Given its predicted target gene is TUBA1C, we name it lncRNA‐TUB. We found a higher expression of lncRNA‐TUB in mammary epithelial cells that received a proinflammatory stimulus compared to normal cells. Knockout of lncRNA‐TUB by the CRISPR/Cas9 system revealed that it plays crucial roles in the morphological shape, proliferation, migration and β‐casein secretion of mammary epithelial cells. In addition, lncRNA‐TUB mediates Escherichia coli‐induced inflammatory factor secretion and Staphylococcus aureus adhesion to epithelial cells. Our results suggest that the lncRNAs identified here function in bovine mastitis, and that lncRNA‐TUB affects the basic biological characteristics and functions of bovine mammary epithelial cells in inflammatory conditions, providing valuable insights into the mechanisms of bovine mastitis.

Fibroblasts constitute the majority of the stromal cells within bovine mammary gland, yet the functional contributions of these cells to mastitis and fibrosis and the mechanism are poorly understood. In this study, we demonstrate that inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis had different expression pattern regarding to several extracellular matrix (ECM) proteins, chemokines and cytokines compared to normal fibroblasts (NFs) from dairy cows during lactation. The INFs induced epithelial-mesenchymal transition (EMT) and inflammatory responses of mammary epithelial cells in a vitro co-culture model. These functional contributions of INFs to normal epithelial cells were mediated through their ability to secrete stromal cell-derived factor 1 (SDF-1). SDF-1 was highly secreted/expressed by INFs, lipopolysaccharide (LPS) -treated NFs, lipoteichoic acid (LTA) -treated NFs, as well as mastitic tissue compared to their counterparts. Exogenous SDF-1 promoted EMT on epithelial cells through activating NF-κB pathway, induced inflammation response and inhibited proliferation of epithelial cells. In addition, SDF-1 was able to induce mastitis and slight fibrosis of mouse mammary gland, which was attenuated by a specific inhibitor of the receptor of SDF-1. Our findings indicate that stromal fibroblasts within mammary glands with mastitis contribute to EMT and inflammatory responses of epithelial cells through the secretion of SDF-1, which could result in the inflammation spread and fibrosis within mammary gland.