Role In Mammary Gland Development Research Articles

S100A9 Affects Milk Protein Content by Regulating Amino Acid Transporters and the PI3K-Akt, WNT, and mTOR Signaling Pathways.

Calgranulin B (S100A9) was found to be strongly associated with milk protein percentage in dairy cattle in our previous genome-wide association study. SNPs in S100A9 were identified via pooled sequencing, and genotyping of 1054 cows was performed individually using MassArray with MALDI-TOFMS technology. Association analyses between the S100A9 SNPs and five milk production traits were conducted using SAS 9.2 software. Functional studies of S100A9 were conducted using quantitative PCR, Western blot, CCK-8, and immunofluorescence assays. In the present study, we further verified that two SNPs in S100A9, g.17115387 C>A and g.17115176 C>A, were significantly associated with milk protein percentage. We found that S100A9 could affect the expressions of caseins CSN1S1, CSN2, and CSN3 in MAC-T cells by regulating the expressions of amino acid transporter genes. We investigated the effects of S100A9 on the PI3K-Akt, WNT, and mTOR pathways, which are well known to play important roles in mammary gland development and milk protein synthesis. Our results suggest that S100A9 regulates the expressions of the relevant genes in these pathways, and thus potentially influences the protein synthesis in the mammary gland. This study demonstrates the important role of the S100A9 gene in the milk protein trait of dairy cattle and provides new insights into the molecular mechanism of milk protein content.

Pilot Study on the Effect of Patient Condition and Clinical Parameters on Hypoxia-Induced Factor Expression: HIF1A, EPAS1 and HIF3A in Human Colostrum Cells.

Hypoxia-inducible factor 1 (HIF-1) may play a role in mammary gland development, milk production and secretion in mammals. Due to the limited number of scientific reports on the expression of HIF genes in colostrum cells, it was decided to examine the expression of HIF1A, HIF3A and EPAS1 in the these cells, collected from 35 patients who voluntarily agreed to provide their biological material for research, were informed about the purpose of the study and signed a consent to participate in it. The expression of HIF genes was assessed using qPCR. Additionally, the influence of clinical parameters (method of delivery, occurrence of stillbirths in previous pregnancies, BMI level before pregnancy and at the moment of delivery, presence of hypertension during pregnancy, presence of Escherichia coli in vaginal culture, iron supplement and heparin intake during pregnancy) on the gene expression was assessed, revealing statistically significant correlations. The expression of HIF1A was 3.5-fold higher in the case of patients with the presence of E. coli in vaginal culture (p = 0.041) and 2.5 times higher (p = 0.031) in samples from women who used heparin during pregnancy. Approximately 1.7-fold higher expression of the EPAS1 was observed in women who did not supplement iron during pregnancy (p = 0.046). To our knowledge, these are the first studies showing the relationship between HIF expression in cells from breast milk and the method of delivery and health condition of women giving birth. The assessment of HIF expression requires deeper examination in a larger study group, and the results of further studies will allow to determine whether HIF can become biomarkers in pregnancy pathology states.

Stem Cell Division and Its Critical Role in Mammary Gland Development and Tumorigenesis: Current Progress and Remaining Challenges.

The origin of breast cancer (BC) has traditionally been a focus of medical research. It is widely acknowledged that BC originates from immortal mammary stem cells and that these stem cells participate in two division modes: symmetric cell division (SCD) and asymmetrical cell division (ACD). Although both of these modes are key to the process of breast development and their imbalance is closely associated with the onset of BC, the molecular mechanisms underlying these phenomena deserve in-depth exploration. In this review, we first outline the molecular mechanisms governing ACD/SCD and analyze the role of ACD/SCD in various stages of breast development. We describe that the changes in telomerase activity, the role of polar proteins, and the stimulation of ovarian hormones subsequently lead to two distinct consequences: breast development or carcinogenesis. Finally, gene mutations, abnormalities in polar proteins, modulation of signal-transduction pathways, and alterations in the microenvironment disrupt the balance of BC stem cell division modes and cause BC. Important regulatory factors such as mammalian Inscuteable mInsc, Numb, Eya1, PKCα, PKCθ, p53, and IL-6 also play significant roles in regulating pathways of ACD/SCD and may constitute key targets for future research on stem cell division, breast development, and tumor therapy.

C/EBPβ deletion in macrophages impairs mammary gland alveolar budding during the estrous cycle.

Macrophages have important roles in mammary gland development and tissue homeostasis, but the specific mechanisms that regulate macrophage function need further elucidation. We have identified C/EBPβ as an important transcription factor expressed by multiple macrophage populations in the normal mammary gland. Mammary glands from mice with C/EBPβ-deficient macrophages (Cebpb ΔM) show a significant decrease in alveolar budding during the diestrus stage of the reproductive cycle, whereas branching morphogenesis remains unchanged. Defects in alveolar budding were found to be the result of both systemic hormones and local macrophage-directed signals. RNA sequencing shows significant changes in PR-responsive genes and alterations in the Wnt landscape of mammary epithelial cells of Cebpb ΔM mice, which regulate stem cell expansion during diestrus. Cebpb ΔM macrophages demonstrate a shift from a pro-inflammatory to a tissue-reparative phenotype, and exhibit increased phagocytic capacity as compared to WT. Finally, Cebpb ΔM macrophages down-regulate Notch2 and Notch3, which normally promote stem cell expansion during alveolar budding. These results suggest that C/EBPβ is an important macrophage factor that facilitates macrophage-epithelial crosstalk during a key stage of mammary gland tissue homeostasis.

Abstract PO4-24-06: The function of GATA3-PARP1 complex in breast cancer during GATA3-mediated cellular reprogramming

Abstract In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins. Chromatin acts as a physical barrier for many transcription factors, preventing them from efficiently binding to their recognition sequences within the nucleosome. Pioneer factors are a class of transcription factors that bind to nucleosomes and activate silent chromatin. Pioneer factors are frequently involved in various immune cell pathways, tissue development, and homeostasis, thereby, underscoring their important roles in development and disease. Previous research found that pioneer factors have at least three fundamental properties: However, the molecular mechanisms of pioneer factor-mediated cellular reprogramming (mesenchymal-to-epithelial transition (MET)) are largely unknown. For this study, I will use GATA3-mediated MET to study the pioneer factor-mediated cellular reprogramming. GATA3 is a transcription factor that plays a pivotal role in mammary gland development, luminal epithelial differentiation, and cellular reprogramming. Additionally, it has been implicated in various cellular processes such as proliferation, migration, and invasion and is a critical regulator in breast cancer, including triple-negative breast cancer (TNBC). In TNBC, GATA3 expression is often downregulated, correlating with poor prognosis. Our lab and others have shown that GATA3 functions as a pioneer factor that actively changes the chromatin state from closed to open. In luminal breast cancer cells, ER-alpha and FOXA1 are well-known GATA3 co-factors. In mesenchymal breast cancer cells, GATA3 can suppress tumor metastasis by inducing MET in the absence of well-known GATA3 co-factors FOXA1 and ER-alpha1. These findings suggest that GATA3 works with additional, unknown co-factors during MET. We have been studying how GATA3 activates silent chromatin. More than 7 million GATA3 motifs exist in the human genome, yet the experimental data from GATA3 ChIP-seq analysis indicates less than 1% of the motifs are occupied by the pioneer factor, GATA3. In addition to binding selectivity, we have shown that the pioneer factor action is site-specific (context-dependent) and only induces chromatin opening and enhancer formation at a subset of binding sites. To identify a novel co-factor that is involved in GATA3-mediated MET, we performed a rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) assay, which identified the chromatin-modification enzyme, Poly ADP-ribose polymerase 1 (PARP1), as a potential GATA3 co-factor. PARP1 is essential for gene expression regulation and initiating DNA repair. Previous studies have shown PARP1 as a co-factor to the pioneer factor, SOX2. Our genomics data strongly suggest that PARP1 is involved in such context-dependent action of GATA3. Our overall goal is to investigate GATA3-PARP1 interaction during MET to understand the role of pioneer factors in cellular reprogramming. To do so, we are dissecting the GATA2-PARP1 function in two distinct steps: Citation Format: Mikhala Cooper, Motoki Takaku. The function of GATA3-PARP1 complex in breast cancer during GATA3-mediated cellular reprogramming [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-06.

SNAT2-mediated regulation of estrogen and progesterone in the proliferation of goat mammary epithelial cells

The development of the goat mammary gland is mainly under the control of ovarian hormones particularly estrogen and progesterone (P4). Amino acids play an essential role in mammary gland development and milk production, and sodium-coupled neutral amino acid transporter 2 (SNAT2) was reported to be expressed in the mammary gland of rats and bovine mammary epithelial cells, which may affect the synthesis of milk proteins or mammary cell proliferation by mediating prolactin, 17β-estradiol (E2) or methionine function. However, whether SNAT2 mediates the regulatory effects of E2 and P4 on the development of the ruminant mammary gland is still unclear. In this study, we show that E2 and P4 could increase the proliferation of goat mammary epithelial cells (GMECs) and regulate SNAT2 mRNA and protein expression in a dose-dependent manner. Further investigation revealed that SNAT2 is abundantly expressed in the mammary gland during late pregnancy and early lactation, while knockdown and overexpression of SNAT2 in GMECs could inhibit or enhance E2- and P4-induced cell proliferation as well as mammalian target of rapamycin (mTOR) signaling. We also found that the accelerated proliferation induced by SNAT2 overexpression in GMECs was suppressed by the mTOR signaling pathway inhibitor rapamycin. This indicates that the regulation of GMECs proliferation mediated by SNAT2 in response to E2 and P4 is dependent on the mTOR signaling pathway. Finally, we found that the total content of the amino acids in GMECs changed after knocking-down and overexpressing SNAT2. In summary, the results demonstrate that the regulatory effects of E2 and P4 on GMECs proliferation may be mediated by the SNAT2-transported amino acid pathway. These results may offer a novel nutritional target for improving the development of the ruminant mammary gland and milk production.

Mcam inhibits macrophage-mediated development of mammary gland through non-canonical Wnt signaling

While canonical Wnt signaling is well recognized for its crucial regulatory functions in cell fate decisions, the role of non-canonical Wnt signaling in adult stem cells remains elusive and contradictory. Here, we identified Mcam, a potential member of the non-canonical Wnt signaling, as an important negative regulator of mammary gland epithelial cells (MECs) by genome-scale CRISPR-Cas9 knockout (GeCKO) library screening. Loss of Mcam increases the clonogenicity and regenerative capacity of MECs, and promotes the proliferation, differentiation, and ductal morphogenesis of mammary epithelial in knockout mice. Mechanically, Mcam knockout recruits and polarizes macrophages through the Il4-Stat6 axis, thereby promoting secretion of the non-canonical Wnt ligand Wnt5a and its binding to the non-canonical Wnt signaling receptor Ryk to induce the above phenotypes. These findings reveal Mcam roles in mammary gland development by orchestrating communications between MECs and macrophages via a Wnt5a/Ryk axis, providing evidences for non-canonical Wnt signaling in mammary development.

The role of endoplasmic reticulum stress in metabolic diseases and mammary epithelial cell homeostasis in dairy cows.

High-yielding dairy cows undergo various physiological stresses during the transitional phase of the calving cycle. In this period, they experience negative energy balance, subjecting the liver to significant metabolic stress from an influx of nonesterified fatty acids. This metabolic stress not only impairs liver function but also diminishes milk production. Early lactation dairy cows may develop endoplasmic reticulum (ER) stress in the liver, potentially leading to liver-related diseases and contributing to ER stress in mammary epithelial cells, resulting in decreased milk production. Natural products that alleviate ER stress have been identified, and if further in vivo studies confirm their efficacy, they have potential as feed additives to prevent disease and reduce milk yield. Conversely, physiological levels of ER stress play a role in mammary gland development and positively influence protein synthesis in milk. Understanding the threshold level of ER stress in mammary tissue and its detailed mechanisms will be crucial in dairy farming.

Kindlin-2 in myoepithelium controls luminal progenitor commitment to alveoli in mouse mammary gland

Myoepithelium plays an important role in mammary gland development, but less is known about the molecular mechanism underlying how myoepithelium controls acinus differentiation during gestation. Herein, we found that loss of Kindlin-2 in myoepithelial cells impaired mammary morphogenesis, alveologenesis, and lactation. Using five genetically modified mouse lines combined with single-cell RNA sequencing, we found a Kindlin-2–Stat3–Dll1 signaling cascade in myoepithelial cells that inactivates Notch signaling in luminal cells and consequently drives luminal progenitor commitment to alveolar cells identity. Single-cell profiling revealed that Kindlin-2 loss significantly reduces the proportion of matured alveolar cells. Mechanistically, Kindlin-2 depletion in myoepithelial cells promotes Stat3 activation and upregulates Dll1, which activates the Notch pathway in luminal cells and inhibits luminal progenitor differentiation and maturation during gestation. Inhibition of Notch1 with tangeretin allowed luminal progenitors to regain commitment ability in the pregnant mice with Kindlin-2 depletion in myoepithelium. Taken together, we demonstrated that Kindlin-2 is essential to myoepithelium-controlled luminal progenitors to alveoli transition during gestation.

Productive and physiological implications of top-dress addition of branched-chain amino acids and arginine on lactating sows and offspring

BackgroundBranched-chain amino acids (BCAAs), including L-leucine (L-Leu), L-isoleucine (L-Ile), L-valine (L-Val), and L-arginine (L-Arg), play a crucial role in mammary gland development, secretion of milk and regulation of the catabolic state and immune response of lactating sows. Furthermore, it has recently been suggested that free amino acids (AAs) can also act as microbial modulators. This study aimed at evaluating whether the supplementation of lactating sows with BCAAs (9, 4.5 and 9 g/d/sow of L-Val, L-Ile and L-Leu, respectively) and/or L-Arg (22.5 g/d/sow), above the estimated nutritional requirement, could influence the physiological and immunological parameters, microbial profile, colostrum and milk composition and performance of sows and their offspring.ResultsAt d 41, piglets born from the sows supplemented with the AAs were heavier (P = 0.03). The BCAAs increased glucose and prolactin (P < 0.05) in the sows’ serum at d 27, tended to increase immunoglobulin A (IgA) and IgM in the colostrum (P = 0.06), increased the IgA (P = 0.004) in the milk at d 20 and tended to increase lymphocyte% in the sows’ blood at d 27 (P = 0.07). Furthermore, the BCAAs tended to reduce the Chao1 and Shannon microbial indices (P < 0.10) in the sows’ faeces. The BCAA group was discriminated by Prevotellaceae_UCG-004, Erysipelatoclostridiaceae UCG-004, the Rikenellaceae_RC9_gut_group and Treponemaberlinense. Arginine reduced piglet mortality pre- (d 7, d 14) and post-weaning (d 41) (P < 0.05). Furthermore, Arg increased the IgM in the sow serum at d 10 (P = 0.05), glucose and prolactin (P < 0.05) in the sow serum at d 27 and the monocyte percentage in the piglet blood at d 27 (P = 0.025) and their jejunal expression of NFKB2 (P = 0.035) while it reduced the expression of GPX-2 (P = 0.024). The faecal microbiota of the sows in Arg group was discriminated by Bacteroidales. The combination of BCAAs and Arg tended to increase spermine at d 27 (P = 0.099), tended to increase the Igs (IgA and IgG, P < 0.10) at d 20 in the milk, favoured the faecal colonisation of Oscillospiraceae UCG-005 and improved piglet growth.ConclusionFeeding Arg and BCAAs above the estimated requirements for milk production may be a strategy to improve sow productive performance in terms of piglet average daily gain (ADG), immune competence and survivability via modulation of the metabolism, colostrum and milk compositions and intestinal microbiota of the sows. The synergistic effect between these AAs, noticeable by the increase of Igs and spermine in the milk and in the improvement of the performance of the piglets, deserves additional investigation.

Abstract P5-15-01: Rediscovering IRF5: A prognostic indicator with novel roles in mammary gland development, ribosome biogenesis, tumor initiation, and metastasis

Abstract Breast cancer is the second leading cause of death for women in the United States. However, only 10% of breast cancers have been linked to inherited genomic mutations. Thus, identifying biomarkers to predict cancer progression and metastasis remains a clear need in the research and medical world. Here, we report a novel role for Interferon Regulatory Factor 5 (IRF5) in mammary gland development, tumorigenesis, and metastasis. Historically, IRF5 has been studied as a transcription factor in the context of genetic risk for autoimmune diseases. However, mining of The Cancer Genome Atlas revealed that loss of IRF5 expression in human breast cancer is significantly linked with progression to high grade carcinoma, increased metastasis, and decreased overall and recurrence-free survival. Supporting these analyses, we demonstrated that female Irf5-/- BALB/c mice have higher incidence of spontaneous atypical ductal hyperplasia (ADH), increased progression to DCIS, and ultimately, increased incidence of IDC. Using qPCR, FISH and IHC, we confirmed that IRF5 is expressed in both luminal and basal myoepithelial cells, but expression is higher in basal cells. Histologic analysis of whole mount preparations of Irf5-/- mammary glands revealed aberrant ductal morphogenesis, characterized by expansion of luminal and basal myoepithelial cells with a loss of organized glandular structure. RNAseq of primary mammary epithelial cells from wild type and Irf5-/- littermate mice showed Irf5-/- mammary epithelial cells to be enriched in ribosome biogenesis pathways, the physiologic consequences of which were demonstrated through increased rates of protein synthesis. Transferring our studies in vivo, we demonstrated that loss of tumor IRF5 expression resulted in decreased tumor-infiltrating lymphocytes and increased pulmonary metastasis in the murine orthotopic 4T1 implantation model. Mechanistically, we found that—as in our studies utilizing primary mammary epithelial cells—IRF5 expression in tumors regulated protein translation and ribosome biogenesis. In light of these findings, we propose IRF5 as a novel prognostic biomarker, loss of which alters mammary gland development, drives tumor initiation and metastasis, and dysregulates mammary epithelial cell protein synthesis. Citation Format: Betsy Barnes, Zarina Brune, Matthew Rice, Carter Somerville. Rediscovering IRF5: A prognostic indicator with novel roles in mammary gland development, ribosome biogenesis, tumor initiation, and metastasis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-15-01.

Emerging functions of C/EBPβ in breast cancer.

Breast tumorigenesis relies on complex interactions between tumor cells and their surrounding microenvironment, orchestrated by tightly regulated transcriptional networks. C/EBPβ is a key transcription factor that regulates the proliferation and differentiation of multiple cell types and modulates a variety of biological processes such as tissue homeostasis and the immune response. In addition, C/EBPβ has well-established roles in mammary gland development, is overexpressed in breast cancer, and has tumor-promoting functions. In this review, we discuss context-specific roles of C/EBPβ during breast tumorigenesis, isoform-specific gene regulation, and regulation of the tumor immune response. We present challenges in C/EBPβ biology and discuss the importance of C/EBPβ isoform-specific gene regulation in devising new therapeutic strategies.

GATA-3 expression and its correlation with prognostic factors and survival in canine mammary tumors.

The transcription factor GATA-3 plays a significant role in mammary gland development and differentiation. Recent studies on human oncology have demonstrated its association with favorable pathologic factors in breast cancer. Canine mammary tumours, proposed as comparative and translational study models, have epidemiological, clinical, biological, and genetic characteristics similar to those of human breast cancers. Here, we evaluated the frequency of GATA-3 expression in mammary tumors of dogs and its relationship with prognostic factors and survival. Tumor samples were obtained from 40 female dogs and grouped according to histological type into benign tumors (n = 10), carcinoma in mixed tumors (CMTs) (n = 20), and aggressive tumors (n = 10). CMTs were further separated according to histological grade, and data on clinical staging and diagnosis, histopathological grading, and survival rate were collected. GATA-3 and estrogen receptor (ER) expression were higher in benign and well-differentiated carcinomas than in aggressive tumors, which showed greater Ki-67 expression. The expression rate of ER in the studied groups was equivalent to that of GATA-3. We identified a strong positive correlation between GATA-3 and ER expression frequencies and a negative correlation between those of GATA-3 and Ki-67. There were associations between GATA-3 (p < 0.001), Ki-67 (p = 0.003), tumor size (p < 0.001), clinical stage (p = 0.002), lymph node metastasis (p < 0.001), and histological grade (p < 0.001) by univariate survival analysis. The parameters ER (p = 0.015) and GATA-3 (p = 0.005) also influenced survival in a multifactorial manner. Kaplan-Meier analysis of survival curves validated our previous findings that dogs with GATA-3 expression in ≥79.4% of cells had significantly higher survival rates (p < 0.001). The performance analysis showed that the expression of GATA-3 in ≥79.4% of cells effectively predicted survival or death in dogs with mammary tumors. Collectively, these results suggest that GATA-3 can be a relevant marker in the study of mammary tumor progression and has potential as a prognosis marker for predicting outcomes in canine mammary tumors.

MicroRNAs: A Link between Mammary Gland Development and Breast Cancer

Breast cancer is among the most common cancers in women, second to skin cancer. Mammary gland development can influence breast cancer development in later life. Processes such as proliferation, invasion, and migration during mammary gland development can often mirror processes found in breast cancer. MicroRNAs (miRNAs), small, non-coding RNAs, can repress post-transcriptional RNA expression and can regulate up to 80% of all genes. Expression of miRNAs play a key role in mammary gland development, and aberrant expression can initiate or promote breast cancer. Here, we review the role of miRNAs in mammary development and breast cancer, and potential parallel roles. A total of 32 miRNAs were found to be expressed in both mammary gland development and breast cancer. These miRNAs are involved in proliferation, metastasis, invasion, and apoptosis in both processes. Some miRNAs were found to have contradictory roles, possibly due to their ability to target many genes at once. Investigation of miRNAs and their role in mammary gland development may inform about their role in breast cancer. In particular, by studying miRNA in development, mechanisms and potential targets for breast cancer treatment may be elucidated.

Discoidin domain receptor 1 regulates ErbB2/ErbB3 signaling in mammary epithelial cells.

The ErbB2 receptor tyrosine kinase plays a key role in mammary gland development. It forms large clusters which serve as signaling platforms for integration of extracellular information. The discoidin domain receptor (DDR) family are collagen receptor tyrosine kinases which, together with ErbB2, are involved in many physiological and pathological processes. Here, we investigated the interaction of ErbB2 and DDR1 receptors in breast cancer cells. In contrast to beta1-integrin, DDR1 colocalizes with ErbB2 in membrane clusters regardless of their expression levels. We demonstrated that this spatial coexistence is a consequence of the physical interaction between these receptors. In addition, these receptors are coexpressed in the normal mammary gland but not in breast tumor samples. Together, these results present DDR1 as a novel modulator of the ErbB2/ErbB3 signaling pathway.

LncRNA MPFAST Promotes Proliferation and Fatty Acid Synthesis of Bovine Mammary Epithelial Cell by Sponging miR-103 Regulating PI3K-AKT Pathway.

Long noncoding RNAs (lncRNAs) have an essential role in mammary gland development and lactation. Our earlier study showed that the lncRNA mammary proliferation and fatty acid synthesis-associated transcript (MPFAST) is highly expressed in the Holstein cow mammary gland during the middle lactation period compared to the dry period, which indicates its potential role in lactation. Therefore, gain- and loss-of-function experiments were performed on bovine mammary epithelial cells (BMECs) by cell counting kit 8 (CCK8), 5-ethynyl-2'-deoxyuridine (EdU), real-time quantitative polymerase chain reaction (RT-qPCR), and western blot. The results indicated that MPFAST promoted the viability and proliferation of BMECs. The oil red O staining and cellular triglyceride assay further showed that MPFAST promoted the number of lipid droplets and cellular triglyceride synthesis in BMECs. Bioinformatics analysis showed that MPFAST could act as a molecular sponge for miR-103, and PIK3R1 was a potential target of miR-103, which was further confirmed by the dual-luciferase reporter assay, RT-qPCR, and western blot. The overexpression of MPFAST promoted the expression of PIK3R1 at mRNA and protein levels. It also significantly increased the mRNA relative expression levels of AKT, mTOR, and SREBP1, and the protein relative expression levels of AKT and p-AKT in the PI3K-AKT signaling pathway. In contrast, the inhibition of MPFAST resulted in the downregulation of the PI3K-AKT signaling pathway genes. These results indicated that MPFAST regulates the expression of the genes in the PI3K-AKT signaling pathway through sponging miR-103 and promotes the proliferation and synthesis of fatty acids of BMECs. Our results would provide a new direction for further exploring the regulatory mechanism of lncRNA in the mammary gland.

Evaluation of biological mechanisms of artemisinin on bovine mammary epithelial cells by integration of network pharmacology and TMT-based quantitative proteomics.

The sesquiterpene lactone, artemisinin, is a primary component of the medicinal plant Artemisia annua L., which has anti-inflammatory, antibacterial and antioxidant activities. However, the potential effects of artemisinin on the mammary gland of dairy cows and the underlying molecular mechanisms remain unclear. Here, we utilized systematic network pharmacology and proteomics to elucidate the mechanism by which artemisinin affects milk production and the proliferation of bovine mammary epithelial cells (BMECs). Nineteen bioactive compounds and 56 key targets were identified through database mining. To delineate the mechanism of artemisia’s activity, a protein-protein interaction network and integrated visual display were generated from bioinformatics assays to explore the relationships and interactions among the bioactive molecules and their targets. The gene ontology (GO) terms and kyoto encyclopedia of genes and genomes annotation suggested that the apoptotic process, cell division, p53 pathway, prolactin and PI3K-Akt pathways played vital roles in mammary gland development. Using proteomics analysis, we identified 122 up-regulated and 96 down-regulated differentially significant expressed proteins (DSEPs). The differentially significant expressed proteins had multiple biological functions associated with cell division, apoptosis, differentiation, and migration. Gene ontology enrichment analysis suggested that differentially significant expressed proteins may promote cell proliferation and regulate apoptosis in bovine mammary epithelial cells. Kyoto encyclopedia of genes and genomes pathway analysis indicated that several biological pathways, such as those involved in antigen processing and presentation, cell adhesion molecules and ribosomes, played significant roles in the effects of artemisinin on bovine mammary epithelial cells. These findings contribute to a comprehensive understanding of the mechanism by which artemisinin affects bovine mammary epithelial cells to improve mammary gland turnover by inducing cell proliferation and mammary gland development.

Identification and profiling of microRNAs involved in the regenerative involution of mammary gland

Regenerative involution is important for the subsequent lactation, but molecular mechanism has not been revealed. The crucial miRNA in tissue development indicates that miRNAs might participate in regenerative involution. In the present study, the mammary tissues of the dairy goats (n = 3) were collected via biopsy at wk-8 (time to dry off), –6, –4, –1, and + 1 relative to lambing for the Hematoxylin and Eosin staining and miRNA sequencing. Alveolar structures collapsed during regenerative involution, but the structures remained intact and distended. Among the 50 miRNA expression trajectories categorized by short time-series expression miner, two significant patterns were clustered. The differentially expressed miRNAs in the two patterns were mainly related to the self-renewal of tissue and enriched in pathways containing vesical-mediated transport, tissue development, tube development, vasculature development and epithelial development. The identification of the miRNAs will help in elucidating their regulatory roles in mammary gland development.

Regulation of Tight Junctions by Sex Hormones in Goat Mammary Epithelial Cells.

Simple SummaryHow ovarian hormones affect goat lactation by regulating cell–cell junctions is still unclear. Through the in vivo and in vitro assays, we found that ovarian hormones could elevate cell–cell junction protein expression, which may affect the intercellular space and molecule transportation between the goat mammary epithelial cells. Our assessment suggests that ovarian hormones may affect goat milk production by regulating the cell–cell junction protein expression between mammary epithelial cells.The sex hormones of estrogen and progesterone (P4) play a vital role in mammary gland development and milk lactation in ruminants. The tight junction (TJ) between adjacent secretory epithelial cells is instrumental in establishing the mammary blood–milk barrier. However, whether estrogen and P4 exert their effect on mammary function via regulating TJ remain unclear. Here, to clarify the role of 17-β estradiol (E2) and P4 in the regulation of TJ in goat mammary gland, we first explored the relationships between the concentrations of E2, P4, and the protein expression of claudin-1, claudin-3, occludin, and ZO-1 during the mammary gland development in goat. Then, we further explored the mRNA and protein expression of claudin-1, claudin-3, occludin, and ZO-1 in the goat mammary epithelial cells (GMECs) in vitro under different concentrations of E2 and P4. The results demonstrated that the protein expression of claudin-1 decreased, but occludin and ZO-1 increased with the decline in E2 and P4 during the transition from pregnancy to lactation. In the in vitro studies, E2 exerted a positive effect on the mRNA expression of claudin-1, and accelerated the proteins’ expression of claudin-1 and ZO-1 in GMECs; P4 upregulated the mRNA expression of claudin-1, claudin-3, occludin, and ZO-1, and also improved the protein expression of claudin-1, claudin-3, and ZO-1 in the GMECs. The results demonstrated that E2 and P4 play an important role in regulating the expression of the mammary TJ components, which may ultimately affect the mammary gland development and milk lactation.

156 Genome-wide DNA Methylation Profile of Mammary Gland Tissues from Holstein Cattle Producing Various Milk Protein Yields

Abstract Milk protein content is attracting considerable attention due to increasing demand for high-quality products. Epigenetic marks including DNA methylation have demonstrated considerable potential to contribute to the achievement of increased gain in bovine milk traits. To investigate potential roles of DNA methylation in regulating milk protein productivity, whole genome bisulfite sequencing was used to profile the global DNA methylation of mammary gland (MG) tissues from Canadian Holstein cows producing milk with high protein (HMP) (&amp;gt;4.0%, n = 5) or low protein (LMP) (&amp;lt; 3.0%, n = 7) contents. Bioinformatics processing using standard tools and construction of the methylation landscape of MG was based on methylation sites with ≥10× coverage. A total of 2420 differentially methylated CpG (DMC) sites, including 1842 with higher and 578 with lower methylation levels (FDR&amp;lt; 0.1) were identified when HMP group was compared with LMP group. DMCs were most abundant on chromosomes 2 (n=347) and 27 (N=535), respectively. The most DMCs were annotated to mRNAs, followed by lncRNAs, pseudogenes and rRNAs. Eight genes harbored five or more DMCs each, including LOC112443250 (5S ribosomal RNA) and LOC112443149 (liprin-alpha-1-like, LIP1) (harbored 20 hypermethylated and 1 hypomethylated sites, each), followed by miR-2287-1 (12 hypomethylated sites), etc. LIP1, a liprin family member interacts with LAR family members to play important roles in MG development. MiR-2287-1 and other family members are frequently identified in MG tissues and milk and are associated with many livestock diseases. Interestingly, among the DMCs that overlap with QTLs related to milk production were clusters on chromosomes 27 (N=534) and 14 (N=91) that associated with milk protein yield. In conclusion, DNA methylation showed potential involvement in the genetic variation underlying milk protein content and MG biological processes. More analysis with a higher sample size is however necessary to adequately quantify the involvement of DNA methylation in the regulation of milk protein content and mammary gland development.