Porcine reproductive and respiratory syndrome virus (PRRSV), the most economically important infectious disease of pigs, elicits poor innate and adaptive immune responses. Soluble CD83 (sCD83), a ...secretion from various immune cell populations, especially MoDCs, is involved in negatively regulating the immune response. We speculate sCD83 may be a critical factor in the process of PRRSV-coordinated macrophage polarization. In this study, we found that PAMs co-cultured with PRRSV-infected MoDCs inhibited the M1 macrophage while enhancing the M2 macrophage. This was accompanied by a decrease in the pro-inflammatory cytokine TNF-α and iNOS and an increase in the anti-inflammatory cytokine IL-10 and Arg1. Meanwhile, sCD83 incubation causes the same specific effects lead to a switch in macrophage from M1 to M2. Neutralization of sCD83 removes the inhibitory effects of PRRSV on PAMs. Using reverse genetics, we generated recombinant PRRSVs with mutations in N protein, nsp1α, and nsp10 (knockout sCD83-concerned key amino acid site). Four mutant viruses lost the suppression of M1 macrophage markers, in contrast to the restriction of the upregulation of M2 macrophage markers. These findings suggest that PRRSV modulates the switch of macrophage polarization from M1 to M2 by upregulating the MoDC-induced secretion of CD83, providing new insights into the mechanism by which PRRSV regulates host immunity.
MicroRNAs (miRNAs) are small noncoding RNA molecules that serve as important post-transcriptional gene expression regulators by targeting messenger RNAs for post-transcriptional endonucleolytic ...cleavage or translational inhibition. miRNAs play important roles in many biological processes. Extensive high-throughput sequencing studies of miRNAs have been performed in several animal models. However, little is known about the diversity of these regulatory RNAs in goat (Capra hircus), which is one of the most important agricultural animals and the oldest domesticated species raised worldwide. Goats have long been used for their milk, meat, hair (including cashmere), and skins throughout much of the world.
In this study, two small RNA libraries were constructed based on dry period and peak lactation dairy goat mammary gland tissues and sequenced using the Illumina-Solexa high-throughput sequencing technology. A total of 346 conserved and 95 novel miRNAs were identified in the dairy goat. miRNAs expression was confirmed by qRT-PCR in nine tissues and in the mammary gland during different stages of lactation. In addition, several candidate miRNAs that may be involved in mammary gland development and lactation were found by comparing the miRNA expression profiles in different tissues and developmental stages of the mammary gland.
This study reveals the first miRNAs profile related to the biology of the mammary gland in the dairy goat. The characterization of these miRNAs could contribute to a better understanding of the molecular mechanisms of lactation physiology and mammary gland development in the dairy goat.
Scope
Understanding the regulatory mechanism of milk protein synthesis is important to develop strategies to improve milk protein and enhance lactation performance. The mammalian target of rapamycin ...(mTOR) pathway is a crucial modulator of protein synthesis. In this study, we want to investigate if T1R1/T1R3 can regulate milk protein synthesis and mediate the mTOR pathway in the mice mammary gland in vivo.
Methods and results
T1R1 knockout mice, WT mice, and mammary explants were used. The weigh‐suckle‐weigh method was used to quantify the milk yield. The expression level of β‐casein and AA transporter mRNA were analyzed by qPCR. Western blot was used to analyze protein abundance of members of the mTOR pathway. As expected, the knockout of T1R1 not only reduced the total milk yield in the mice mammary glands, but also repressed β‐casein synthesis. Additionally, the phosphorylation of 4EBP1 and S6K was significantly decreased in T1R1 knockout mice. The T1R1 knockout also increased the protein abundance of the AA transporter SLC3A2 and mRNA expression of SLC7A5/SLC3A2 and SLC1A5. Activation of the mTOR pathway was repressed by inhibition of T1R3 or T1R1 knockout in mammary gland explants.
Conclusion
T1R1/T1R3 modulates the mTOR pathway to regulate milk protein synthesis in the mouse mammary gland in vivo.
T1R1/T1R3 acts as a sensor of extracellular amino acids and is involved in milk protein synthesis in mouse mammary epithelial cells. This study investigates whether T1R1/T1R3 regulates milk protein synthesis in the mouse mammary gland. The use of T1R1 knockout mice reveals that T1R1/T1R3 mediates the mTOR pathway to regulate milk protein synthesis in the mouse mammary gland in vivo.
microRNAs are a kind of endogenous, non-coding, single-strand small RNA. They have been reported as an important regulatory factor in skeletal myogenesis. In this study, miR-452 was selected from RNA ...high-throughput sequencing data to explore its regulatory role in myogenesis. Functionally, miR-452 overexpression could promote C2C12 myoblast proliferation while inhibiting myogenic differentiation. On the contrary, inhibition of miR-452 could suppress C2C12 myoblast proliferation but accelerate myogenic differentiation. Bioinformatics analysis and dual luciferase report assays showed that
(
),
, and
were the potential target genes of miR-452. To further confirm the target relationship between
,
, and
with miR-452, the mRNA level and protein level of these genes were detected by using RT-qPCR and Western blot, respectively. Result analysis indicated that
was a target gene of miR-452. In addition, knockdown of
could obviously promote C2C12 myoblast proliferation but block their differentiation. In summary, these results demonstrated that miR-452 promoted C2C12 myoblast proliferation and inhibited their differentiation
targeting
.
Purpose
The mechanism of dietary amino acids in regulating milk protein synthesis at the translational level is not well understood. Numerous studies have shown that the amino acid signal is ...transferred through the mammalian target of rapamycin (mTOR) pathway; however, the extracellular amino acid-sensing mechanism that activates mTOR complex 1 is unknown. We tested the hypotheses that the T1R1/T1R3 heterodimer functions as a direct sensor of the fed state and amino acid availability preceding the mTOR pathway and affects milk protein synthesis in mammary epithelial cells.
Methods
The expression of T1R1 was repressed by T1R1 siRNA in mouse mammary epithelial cells model (HC11). Western blot was used to analyze activity of the mTOR pathway and β-casein expression, and quantitative real-time RT-PCR was used to analyze the change in mRNA abundance of amino acid transporters.
Results
The transcripts and proteins of T1R1 and T1R3 were detected in HC11 cells and mouse mammary gland tissue. siRNA silencing of T1R1 repressed β-casein synthesis in HC11 cells both with and without essential amino acids present in the culture medium. The phosphorylation of mTOR, S6K, and 4EBP1 in T1R1 knockdown HC11 cells declined to 25, 50, and 30 %, indicating T1R1 knockdown repressed the activity of the mTOR pathway. T1R1 knockdown increased the mRNAs coding three important amino acid transporters (SLC1A5 and SLC3A2/SLC7A5). Activation of the mTOR pathway was partially repressed by T1R1 siRNA or SLC7A5/SLC3A2 inhibitor (BCH, 10 mM), and the combination of these two treatments further repressed the activity of this pathway.
Conclusion
T1R1/T1R3 serves as sensor of extracellular amino acids in mouse mammary epithelial cells and involved in milk protein synthesis regulation.
Though miRNAs have been reported to regulate bovine myoblast proliferation, but many miRNAs still need to be further explored. Specifically, miR-152 is a highly expressed miRNA in cattle skeletal ...muscle tissues, but its function in skeletal muscle development is unknown. Herein, we aimed to investigate the role of miR-152 in regulating bovine myoblast proliferation. Functionally, RT-qPCR, Western blotting, EdU assay, and flow cytometry detection results showed that miR-152 inhibited bovine myoblast proliferation. Mechanistically, we demonstrated transcription factor KLF6 was a target gene of miR-152 by means of bioinformatics software prediction and dual-luciferase report analysis, which had been demonstrated to be favorable for myoblast proliferation. Collectively, our research suggested that miR-152 inhibits bovine myoblast proliferation via targeting KLF6.
Goat is an important agricultural animal for meat production. Functional studies have demonstrated that microRNAs (miRNAs) regulate gene expression at the post-transcriptional level and play an ...important role in various biological processes. Although studies on miRNAs expression profiles have been performed in various animals, relatively limited information about goat muscle miRNAs has been reported. To investigate the miRNAs involved in regulating different periods of skeletal muscle development, we herein performed a comprehensive research for expression profiles of caprine miRNAs during two developmental stages of skeletal muscles: fetal stage and six month-old stage. As a result, 15,627,457 and 15,593,721 clean reads were obtained from the fetal goat library (FC) and the six month old goat library (SMC), respectively. 464 known miRNAs and 83 novel miRNA candidates were identified. Furthermore, by comparing the miRNA profile, 336 differentially expressed miRNAs were identified and then the potential targets of the differentially expressed miRNAs were predicted. To understand the regulatory network of miRNAs during muscle development, the mRNA expression profiles for the two development stages were characterized and 7322 differentially expressed genes (DEGs) were identified. Then the potential targets of miRNAs were compared to the DEGs, the intersection of the two gene sets were screened out and called differentially expressed targets (DE-targets), which were involved in 231 pathways. Ten of the 231 pathways that have smallest P-value were shown as network figures. Based on the analysis of pathways and networks, we found that miR-424-5p and miR-29a might have important regulatory effect on muscle development, which needed to be further studied. This study provided the first global view of the miRNAs in caprine muscle tissues. Our results help elucidation of complex regulatory networks between miRNAs and mRNAs and for the study of muscle development.
The highly expressed circHIPK3 is a circular RNA that has been previously reported to regulate the growth of human cells. In this study, we found an increased expression of circHIPK3 in bovine ...mammary epithelial cells treated with prolactin (PRL) in high-throughput sequencing data. Thus, we further investigated the effect of circHIPK3 on the proliferation and differentiation of mammary epithelial cells. We used qRT-PCR/Cell Counting Kit-8 (CCK-8) and a Western blotting analysis to evaluate the effects on cell proliferation. We found that circHIPK3 promotes the proliferation of mammary epithelial cells. The STAT5 signaling pathway was previously associated with the prolactin response and when the STAT5 was suppressed, the expression of circHIPK3 decreased. The results suggest that the response to prolactin and the associated STAT5 signaling pathway affect the expression of circHIPK3, which subsequently affects the proliferation of mammary epithelial cells in dairy cows.
Sweet potato vine tips are abundant in chlorogenic acid (CGA). In this study, CGA was extracted from vegetable and conventional sweet potato vine tips using ethanol, followed by subsequent ...purification of the extract through a series of sequential steps. Over 4 g of the purified product was obtained from 100 g of sweet potato vine tip powder, producing more than 85% of purified CGA. The LC-MS analysis of all samples indicated that 4-CQA was the predominant isomer in both sweet potato cultivars. Significant variations of p-coumaroyl quinic acids, feruloyl quinic acids, dicaffeoyl quinic acids, and tricaffeoyl quinic acid were identified, whereas the mono-caffeoyl quinic acids did not vary when the two sweet potato varieties were compared. Compared to conventional sweet potatoes, vegetable sweet potatoes exhibit a high negative correlation between 4-CQA and 5-pCoQA, while showing a high positive correlation between 3,5-CQA and 3-pCoQA. A series of principal component analyses (PCA) using CGA isomers enables a clear differentiation between vine tips derived from vegetable and conventional sweet potatoes. The model of linear discriminant analysis, based on the characteristic CGA, achieved a 100% accuracy rate in distinguishing between vegetable and conventional sweet potatoes. The high purity of sweet potato CGA (SCGA) exhibited potent anti-breast cancer activity. The results demonstrated that SCGA significantly suppressed the clonogenicity of MB231 and MCF7 cells, and impeded the migratory, invasive, and lung metastatic potential of MB231 cells.
Circular RNAs (circRNAs), a novel class of RNAs, perform important functions in biological processes. However, the role of circRNAs in the mammary gland remains unknown. The present study is aimed at ...identifying and characterizing the circRNAs expressed in the mammary gland of lactating rats.
Deep sequencing of RNase R-enriched rat lactating mammary gland samples was performed and circRNAs were predicted using a previously reported computational pipeline. Gene ontology terms of circRNA-producing genes were also analyzed.
A total of 6,824 and 4,523 circRNAs were identified from rat mammary glands at two different lactation stages. Numerous circRNAs were specifically expressed at different lactation stages, and only 1,314 circRNAs were detected at both lactation stages. The majority of the candidate circRNAs map to noncoding intronic and intergenic regions. The results demonstrate a circular preference or specificity of some genes. DAVID analysis revealed an enrichment of protein kinases and related proteins among the set of genes encoding circRNAs. Interestingly, four protein-coding genes (Rev3l, IGSF11, MAML2, and LPP) that also transcribe high levels of circRNAs have been reported to be involved in cancer.
Our findings provide the basis for comparison between breast cancer profiles and for selecting representative circRNA candidates for future functional characterization in breast development and breast cancer.
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