ABSTRACT
Skeletal muscle is not only the largest organ in the body that is responsible for locomotion and exercise but also crucial for maintaining the body's energy metabolism and endocrine secretion. The trimethylation of histone H3 lysine 27 (H3K27me3) is one of the most important histone modifications that participates in muscle development regulation by repressing the transcription of genes. Previous studies indicate that the RASGRP1 gene is regulated by H3K27me3 in embryonic muscle development in pigs, but its function and regulatory role in myogenesis are still unclear. In this study, we verify the crucial role of H3K27me3 in RASGRP1 regulation. The gain/loss function of RASGRP1 in myogenesis regulation is performed using mouse myoblast C2C12 cells and primarily isolated porcine skeletal muscle satellite cells (PSCs). The results of qPCR, western blot analysis, EdU staining, CCK-8 assay and immunofluorescence staining show that overexpression of RASGRP1 promotes cell proliferation and differentiation in both skeletal muscle cell models, while knockdown of RASGRP1 leads to the opposite results. These findings indicate that RASGRP1 plays an important regulatory role in myogenesis in both mice and pigs.
Subject(s)
Histones , Myoblasts , Animals , Mice , Swine , Histones/metabolism , Cell Differentiation/genetics , Cell Proliferation/genetics , Myoblasts/metabolism , Muscle, Skeletal/metabolism , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolismABSTRACT
The formation of embryonic muscle fibers determines the amount of postnatal muscles and is regulated by a variety of signaling pathways and transcription factors. Previously, by using chromatin immunoprecipitation-sequencing and RNA-Seq techniques, we identified a large number of genes that are regulated by H3K27me3 in porcine embryonic skeletal muscles. Among these genes, we found that ITGB6 is regulated by H3K27me3. However, its function in muscle development is unknown. In this study, we first verified that ITGB6 was differentially regulated by H3K27me3 and that its expression levels were upregulated in porcine skeletal muscles at embryonic Days 33, 65, and 90. Then, we performed gain- or loss-of-function studies on porcine skeletal muscle satellite cells to study the role of ITGB6 in porcine skeletal muscle development. The proliferation of porcine skeletal muscle satellite cells was studied through real-time polymerase chain reaction, Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine staining, Western blot, and flow cytometry analyses. We found that the ITGB6 gene was regulated by H3K27me3 during muscle development and had an inhibitory effect on the proliferation of porcine skeletal muscle satellite cells.
Subject(s)
Cell Proliferation , Integrin beta Chains/metabolism , Muscle Development , Satellite Cells, Skeletal Muscle/metabolism , Animals , Cells, Cultured , Gene Expression Regulation, Developmental , Histones/metabolism , Integrin beta Chains/genetics , Methylation , Protein Processing, Post-Translational , Signal Transduction , Sus scrofa , Time FactorsABSTRACT
The two-component systems, which could sense and respond to environmental changes, widely exist in bacteria as a signal transduction pathway. The bacterial CckA/CtrA, ArcA/ArcB and PhoP/PhoQ two-component systems are associated with initiation of DNA replication and cell division, however, the effects of the TorS/TorR system on cell cycle and DNA replication remains unknown. The TorS/TorR system in Escherichia coli can sense changes in trimethylamine oxide (TMAO) concentration around the cells. However, it is unknown if it also affects initiation of DNA replication. We detected DNA replication patterns in ΔtorS and ΔtorR mutant strains by flow cytometry. We found that the average number of replication origins (oriCs) per cell and doubling time in ΔtorS mutants were the same while the average number of oriCs in ΔtorR mutants was increased compared with that in wild-type cells. These results indicated that absence of TorR led to an earlier initiation of DNA replication than that in wild-type cells. Strangely, neither overexpression of TorR nor co-expression of TorR and TorS could restore ΔtorR mutant phenotype to the wild type. However, overexpression of SufD in both wild type and ΔtorR mutants promoted initiation of DNA replication, while mutation of SufD delayed it in ΔtorR mutants. Thus, TorR may affect initiation of DNA replication indirectly through regulating gene expression of sufD.
Subject(s)
DNA Replication , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Phosphotransferases/metabolism , Transcription Factors/metabolism , Cell Division , Escherichia coli/cytology , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Phosphotransferases/genetics , Replication Origin , Transcription Factors/geneticsABSTRACT
Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors in the contemporary era, representing a significant global health concern. Early HCC patients have mild symptoms or are asymptomatic, which promotes the onset and progression of the disease. Moreover, advanced HCC is insensitive to chemotherapy, making traditional clinical treatment unable to block cancer development. Sorafenib (SFB) is a first-line targeted drug for advanced HCC patients with anti-angiogenesis and anti-tumor cell proliferation effects. However, the efficacy of SFB is constrained by its off-target distribution, rapid metabolism, and multi-drug resistance. In recent years, nanoparticles based on a variety of materials have been demonstrated to enhance the targeting and therapeutic efficacy of SFB against HCC. Concurrently, the advent of joint drug delivery systems has furnished crucial empirical evidence for reversing SFB resistance. This review will summarize the application of nanotechnology in the field of HCC treatment over the past five years. It will focus on the research progress of SFB delivery systems combined with multiple therapeutic modalities in HCC treatment.
Subject(s)
Antineoplastic Agents , Carcinoma, Hepatocellular , Drug Delivery Systems , Liver Neoplasms , Sorafenib , Humans , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/pathology , Sorafenib/administration & dosage , Sorafenib/therapeutic use , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , Drug Delivery Systems/methods , Animals , Nanoparticles , Drug Resistance, NeoplasmABSTRACT
Regulation of the cell cycle process is an effective measure to ensure the stability and fidelity of genetic material during the reproduction of bacteria under different stresses. The small RNA DsrA helps bacteria adapt to environments by binding to multiple targets, but its association with the cell cycle remains unclear. Detection by flow cytometry, we first found that the knockout of dsrA promoted replication initiation, and corresponding overexpression of DsrA inhibited replication initiation in Escherichia coli. The absence of the chaperone protein Hfq, the DNA replication negative regulator protein Dps, or the transcription factor OxyR, was found to cause DsrA to no longer inhibit replication initiation. Excess DsrA promotes expression of the oxyR and dps gene, whereas ß-galactosidase activity assay showed that deleting oxyR limited the enhancement of dps promoter transcriptional activity by DsrA. OxyR is a known positive regulator of Dps. Our data suggests that the effect of DsrA on replication initiation requires Hfq and that the upregulation of Dps expression by OxyR in response to DsrA levels may be a potential regulatory pathway for the negative regulation of DNA replication initiation.
Subject(s)
Escherichia coli Proteins , RNA, Small Untranslated , Escherichia coli/genetics , Escherichia coli/metabolism , RNA/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA Replication/genetics , Gene Expression Regulation, Bacterial , RNA, Small Untranslated/genetics , RNA, Small Untranslated/metabolismABSTRACT
Trimethylamine N-oxide (TMAO), an important intestinal flora-derived metabolite, plays a role in the development of cardiovascular disease and tumor immunity. Here, we determined the minimum inhibitory concentration (MIC) of antibiotics against Escherichia coli under gradient concentrations of TMAO and performed a bacterial killing analysis. Overall, TMAO (in the range of 10 ~ 100 mM) increased the MIC of quinolones, aminoglycosides, and ß-lactams in a concentration-dependent manner, and increased the lethal dose of antibiotics against E. coli. It implies that TMAO is a potential risk for failure of anti-infective therapy, and presents a case for the relationship between intestinal flora-derived metabolites and antibiotic resistance. Further data demonstrated that the inhibition of antibiotic efficacy by TMAO is independent of the downstream metabolic processes of TMAO and the typical bacterial resistance mechanisms (mar motif and efflux pump). Interestingly, TMAO protects E. coli from high-protein denaturant (urea) stress and improves the viability of bacteria following treatment with two disinfectants (ethanol and hydrogen peroxide) that mediate protein denaturation by chemical action or oxidation. Since antibiotics can induce protein inactivation directly or indirectly, our work suggests that disruption of protein homeostasis may be a common pathway for different stress-mediated bacterial growth inhibition/cell death. In addition, we further discuss this possibility, which provides a different perspective to address the global public health problem of antibiotic resistance.
ABSTRACT
Imprinted genes play important regulatory roles in the growth and development of placentas and foetuses during pregnancy. In a previous study, we found that the imprinted gene Neuronatin (NNAT) is involved in foetal development; NNAT expression was significantly lower in the placentas of piglets that died neonatally compared to the placentas of surviving piglets. However, the function and mechanism of NNAT in regulating porcine placental development is still unknown. In this study, we collected the placentas of high- and low-weight foetuses at gestational day (GD 65, 90), (n = 4-5 litters/GD) to investigate the role of NNAT in regulating foetal growth and development. We found that the mRNA and protein levels of NNAT were significantly higher in the placentas of high-weight than low-weight foetuses. We then overexpressed NNAT in porcine placental trophoblast cell lines (pTr2) and demonstrated that NNAT activated the PI3K-AKT pathway, and further promoted the expression of glucose transporter 1 (GLUT1) and increased cellular calcium ion levels, which improved glucose transport in placental trophoblast cells in vitro. To conclude, our study suggests that NNAT expression impacts porcine foetal development by regulating placental glucose transport.
Subject(s)
Gene Expression Regulation, Developmental , Glucose/metabolism , Nerve Tissue Proteins/genetics , Animals , Calcium Signaling/genetics , Cell Line , Female , Fetal Weight/genetics , Glucose/genetics , Glucose Transporter Type 1/genetics , Glucose Transporter Type 1/metabolism , Glucose Transporter Type 3/genetics , Glucose Transporter Type 3/metabolism , Nerve Tissue Proteins/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Placenta , Pregnancy , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Swine , Trophoblasts/metabolism , Trophoblasts/physiologyABSTRACT
The proper methylation status of histones is essential for appropriate cell lineage and organogenesis. EZH2, a methyltransferase catalyzing H3K27me3, has been abundantly studied in human and mouse embryonic development. The pig is an increasing important animal model for molecular study and pharmaceutical research. However, the transcript variant and temporal expression pattern of EZH2 in the middle and late porcine fetus are still unknown. Here, we identified the coding sequence of the EZH2 gene and characterized its expression pattern in fetal tissues of Duroc pigs at 65- and 90-day postcoitus (dpc). Our results showed that the coding sequence of EZH2 was 2241 bp, encoding 746 amino acids. There were 9 amino acid insertions and an amino acid substitution in this transcript compared with the validated reference sequence in NCBI. EZH2 was ubiquitously expressed in the fetal tissues of two time points with different expression levels. These results validated a different transcript in pigs and characterized its expression profile in fetal tissues of different gestation stages, which indicated that EZH2 played important roles during porcine embryonic development.