Metastasis is the leading cause of death in breast cancer patients. CD73, also known as ecto-5'-nucleotidase, plays a critical role in cancer development including metastasis. The existing researches indicate that overexpression of CD73 promotes growth and metastasis of breast cancer. Therefore, CD73 inhibitor can offer a promising treatment for breast cancer. Here, we determined whether tiamulin, which was found to inhibit CD73, was able to suppress breast cancer development and explored the related mechanisms.We firstly measured the effect of tiamulin hydrogen fumarate (THF) on CD73 using high performance liquid chromatography (HPLC). Then, we investigated cell proliferation, migration and invasion in MDA-MB-231 human breast cancer cell line and 4 T1 mouse breast cancer cell line treated with THF by migration assay, invasion assay and activity assay. Besides, we examined the effect of THF on syngeneic mammary tumors of mice by immunohistochemistry.Our data demonstrated that THF inhibited CD73 by decreasing the activity instead of the expression of CD73. In vitro, THF inhibited the proliferation, migration and invasion of MDA-MB-231 and 4 T1 cells by suppressing CD73 activity. In vivo, animal experiments showed that THF treatment resulted in significant reduction in syngeneic tumor growth, microvascular density and lung metastasis rate.Our results indicate that THF inhibits growth and metastasis of breast cancer by blocking the activity of CD73, which may offer a promising treatment for breast cancer therapy.
Upon fertilization, extensive chromatin reprogramming occurs during preimplantation development. Growing evidence reveals species-dependent regulations of this process in mammals. ATP-dependent chromatin remodeling factor SMARCA5 (also known as SNF2H) is required for peri-implantation development in mice. However, the specific functional role of SMARCA5 in preimplantation development and if it is conserved among species remain unclear. Herein, comparative analysis of public RNA-seq datasets reveals that SMARCA5 is universally expressed during oocyte maturation and preimplantation development in mice, cattle, humans, and pigs with species-specific patterns. Immunostaining analysis further describes the temporal and spatial changes of SMARCA5 in both mouse and bovine models. siRNA-mediated SMARCA5 depletion reduces the developmental capability and compromises the specification and differentiation of inner cell mass in mouse preimplantation embryos. Indeed, OCT4 is not restricted into the inner cell mass and the formation of epiblast and primitive endoderm disturbed with reduced NANOG and SOX17 in SMARCA5-deficient blastocysts. RNA-seq analysis shows SMARCA5 depletion causes limited effects on the transcriptomics at the morula stage, however, dysregulates 402 genes, including genes involved in transcription regulation and cell proliferation at the blastocyst stage in mice. By comparison, SMARCA5 depletion does not affect the development through the blastocyst stage but significantly compromises the blastocyst quality in cattle. Primitive endoderm formation is greatly disrupted with reduced GATA6 in bovine blastocysts. Overall, our studies demonstrate the importance of SMARCA5 in fostering the preimplantation development in mice and cattle while there are species-specific effects.
The maternal nucleolus plays an indispensable role in zygotic genome activation (ZGA) and early embryonic development in mice. During oocyte-to-embryo transition, the nucleolus is subject to substantial transformation. Despite the primary role of the nucleolus is ribosome biogenesis, accumulating evidence has uncovered its functions in various other cell processes. However, the regulation of nucleolar maturation and ribosome biogenesis and the molecules involved remain unclear during early embryonic development. In this study, we observed that nucleolar protein 2 (NOP2) is restrictedly localized within the nucleolus, first detected in the late two-cell embryos, and increases to a peak level at the eight-cell stage in mice. RNAi-mediated NOP2 depletion leads to a developmental arrest during the morula-to-blastocyst transition. RNA-seq analyses reveal that 208 genes are differentially expressed, including multiple lineage-specific genes and several genes encoding ribosome proteins. Indeed, we observe a failure of the first lineage specification with reduced TEA domain transcription factor 4(TEAD4) (trophectoderm-specific), tir na nog (NANOG), and kruppel-like factor 4 (KLF4) (inner cell mass-specific). Importantly, by Transmission Electron Microscopy (TEM), we noted a decrease in the ratio of the nucleolus size and an increase in the ratio of the size of the nucleolus precursor body, suggesting the nucleolar maturation is disrupted. Moreover, both qPCR and Fluorescence In Situ Hybridization (FISH) data showcase a significant decrease in the abundance of ribosome RNAs. Similarly, NOP2 depletion causes reduced developmental potential and decreased rRNA level in bovine early embryos, suggesting a functional conservation of NOP2 in mammals. Taken together, these results suggest that NOP2 is required for mammalian preimplantation development, presumably by regulating nucleolar maturation and ribosome biogenesis.
ABSTRACT The genome is transcriptionally inert at fertilization and must be activated through a remarkable developmental process called zygotic genome activation (ZGA). Epigenetic reprogramming contributes significantly to the dynamic gene expression during ZGA; however, the mechanism has yet to be resolved. Here, we find histone deacetylases 1 and 2 (HDAC1/2) can regulate ZGA through lysine deacetylase activity. Notably, in mouse embryos, overexpression of a HDAC1/2 dominant-negative mutant leads to developmental arrest at the two-cell stage. RNA-seq reveals that 64% of downregulated genes are ZGA genes and 49% of upregulated genes are developmental genes. Inhibition of the deacetylase activity of HDAC1/2 causes a failure of histone deacetylation at multiple sites, including H4K5, H4K16, H3K14, H3K18 and H3K27. ChIP-seq analysis exhibits an increase and decrease of H3K27ac enrichment at promoters of up- and downregulated genes, respectively. Moreover, HDAC1 mutants prohibit the removal of H3K4me3 by impeding expression of Kdm5 genes. Importantly, the developmental block can be greatly rescued by Kdm5b injection and by partially correcting the expression of the majority of dysregulated genes. Similar functional significance of HDAC1/2 is conserved in bovine embryos. Overall, we propose that HDAC1/2 are indispensable for ZGA by creating correct transcriptional repressive and active states in mouse and bovine embryos.
Consistent with the gene dosage effect hypothesis, renal cysts can arise in transgenic murine models overexpressing either PKD1 or PKD2, which are causal genes for autosomal dominant polycystic kidney disease (ADPKD). To determine whether PKD gene overexpression is a universal mechanism driving cystogenesis or is merely restricted to rodents, other animal models are required. Previously, we failed to observe any renal cysts in a transgenic porcine model of PKD2 overexpression partially due to epigenetic silencing of the transgene. Thus, to explore the feasibility of porcine models and identify potential genes/pathways affected in ADPKD, LLC-PK1 cells with high PKD2 expression were generated. mRNA sequencing (RNA-seq) was performed, and MYC, IER3, and ADM were found to be upregulated genes common to the different PKD2 overexpression cell models. MYC is a well-characterized factor contributing to cystogenesis, and ADM is a biomarker for chronic kidney disease. Thus, these genes might be indicators of disease progression. Additionally, some ADPKD-associated pathways, e.g., the mitogen-activated protein kinase (MAPK) pathway, were enriched in the cells. Moreover, gene ontology (GO) analysis demonstrated that proliferation, apoptosis, and cell cycle regulation, which are hallmarks of ADPKD, were altered. Therefore, our experiment identified some biomarkers or indicators of ADPKD, indicating that high PKD2 expression would likely drive cystogenesis in future porcine models.
Abstract The NOTCH signaling pathway plays an important role in regulating various biological processes, including lineage specification and apoptosis. Multiple components of the NOTCH pathway have been identified in mammalian preimplantation embryos. However, the precise role of the NOTCH pathway in early embryonic development is poorly understood, especially in large animals. Here, we show that the expression of genes encoding key transcripts of the NOTCH pathway is dynamic throughout early embryonic development. We also confirm the presence of active NOTCH1 and RBPJ. By using pharmacological and RNA interference tools, we demonstrate that the NOTCH pathway is required for the proper development of bovine early embryos. This functional consequence could be partly attributed to the major transcriptional mediator, Recombination Signal Binding Protein For Immunoglobulin Kappa J Region (RBPJ), whose deficiency also compromised the embryo quality. Indeed, both NOTCH1 and RBPJ knockdown cause a significant increase of histone H3 serine 10 phosphorylation (pH3S10, a mitosis marker) positive blastomeres, suggesting a cell cycle arrest at mitosis. Importantly, RNA sequencing analyses reveal that either NOTCH1 or RBPJ depletion triggers a reduction in H1FOO that encodes the oocyte-specific linker histone H1 variant. Interestingly, depleting H1FOO results in detrimental effects on the developmental competence of early embryos, similar with NOTCH1 inhibition. Overall, our results reveal a crucial role for NOTCH pathway in regulating bovine preimplantation development, likely by controlling cell proliferation and maintaining H1FOO expression.
Using Luminex xMAP (x = analyte, MAP = multi-analyte profiling) technology, a serological method for the simultaneous detection of antibodies to Newcastle disease virus (NDV) and avian influenza virus (AIV) was established. Nano-magnetic beads coated with purified NDV protein and AIV nucleoprotein were incubated with serum samples. Using biotinylated rabbit anti-chicken IgY and streptavidin-R-phycoerythrin, the optical signals measured by a Luminex 200 detection system indicated the quantification of NDV or AIV antibodies in the serum. Specific pathogen-free (SPF) chicken serum was used as a negative control. The Luminex xMAP assay developed in this study demonstrated high specificity as there was no cross-reaction with antibodies to infectious laryngotracheitis virus, infectious bronchitis virus, infectious bursal disease virus, avian leukosis virus, and Marek's disease virus. The results from reproducibility experiments showed that intra-coefficients of variation were 3.36 and 9.23% and inter-coefficients of variation were 6.50 and 7.66% for NDV and AIV, respectively. The results also indicated that the Luminex xMAP assay was 16 times more sensitive for NDV antibody detection and 1,024 times more sensitive for AIV antibody detection compared to the enzyme-linked immunosorbent assay (ELISA). A total of 300 chicken serum samples were subjected to both Luminex xMAP assay and ELISA, showing the coincidence rates of 98.67 and 98% for NDV and AIV antibody detection, respectively. This study provides a new method for the simultaneous detection NDV and AIV antibodies in the serum with high specificity and sensitivity.
Abstract Background: Ectromelia virus (ECTV) is the pathogen of mousepox, which has a limited host range and a high mortality rate. In the past century, ECTV has affected laboratory mouse colonies worldwide. Recombinant polymerase amplification (RPA), which is widely used in virus detection, is an isothermal amplification method. Results: In this study, a probe-based RPA detection method was established for rapid and sensitive detection of ECTV.Primers were designed for the highly conserved region of crmD gene, the main core protein of recessive poxvirus, and standard plasmids were constructed.The sensitivity of the RPA assay is 100 copies of the genome per reaction. In addition, the method showed high specificity and did not cross-react with other common mouse viruses.Therefore, the practicability of the RPA method in the field was confirmed by the detection of 52 clinical samples. The real-time RPA assay was very similar to the ECTV real-time PCR assay, with 100% agreement. Conclusions: In conclusion, this RPA assay, especially in low-resource settings, provides a novel alternative for sensitive, simple, and specific identification of ECTV.
Currently, antibiotics are the most common treatment for bacterial infections in clinical practice. However, with the abuse of antibiotics and the emergence of drug-resistant bacteria, the use of antibiotics has faced an unprecedented challenge. It is imminent to develop nonantibiotic antimicrobial agents. Based on the cation-π structure of barnacle cement protein, a polyphosphazene-based polymer poly[(N,N-dimethylethylenediamine)-g-(N,N,N,N-dimethylaminoethyl p-ammonium bromide (ammonium bromide)-g-(N,N,N,N-dimethylaminoethyl acetate ethylammonium bromide)] (PZBA) with potential adhesion and inherent antibacterial properties was synthesized, and a series of injectable antibacterial adhesive hydrogels (PZBA-PVA) were prepared by cross-linking with poly(vinyl alcohol) (PVA). PZBA-PVA hydrogels showed good biocompatibility, and the antibacterial rate of the best-performed hydrogel reached 99.81 ± 0.04% and 98.80 ± 2.16% against Staphylococcus aureus and Escherichia coli within 0.5 h in vitro, respectively. In the infected wound model, the healing rate of the PZBA-PVA-treated group was significantly higher than that of the Tegaderm film group due to the fact that the hydrogel suppressed inflammatory responses and modulated the infiltration of immune cells. Moreover, the wound healing mechanism of the PZBA-PVA hydrogel was further evaluated by real-time polymerase chain reaction and total RNA sequencing. The results indicated that the process of hemostasis and tissue development was prompted and the inflammatory and immune responses were suppressed to accelerate wound healing. Overall, the PZBA-PVA hydrogel is shown to have the potential for infected wound healing application.
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