Extracellular MicroRNAs in Urologic Malignancies: Chances and Challenges

Small noncoding RNAs that are 19–23 nucleotides long, known as microRNAs (miRNAs), are involved in almost all biological mechanisms during carcinogenesis. Recent studies show that miRNAs released from live cells are detectable in body fluids and may be taken up by other cells to confer cell-cell communication. These released miRNAs (here referred to as extracellular miRNAs) are often protected by RNA-binding proteins or embedded inside circulating microvesicles. Due to their relative stability, extracellular miRNAs are believed to be promising candidates as biomarkers for diagnosis and prognosis of disease, or even as therapeutic agents for targeted treatment. In this review, we first describe biogenesis and characteristics of these miRNAs. We then summarize recent publications involving extracellular miRNA profiling studies in three representative urologic cancers, including: prostate cancer, bladder cancer, and renal cell carcinoma. We focus on the diagnostic, prognostic, and therapeutic potential of these miRNAs in biological fluids, such as serum, plasma, and urine. Finally, we discuss advantages and challenges of these miRNAs in clinical applications.     

Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy

Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4–10), osmolarity (50–1000 mOsm/L), temperature (15–60 °C), and surfactant Triton X-100 (10–500 μM)). Their stability was evaluated by LS by considering the hydrodynamic radius (R_h), intensity of scattered light (I), and the shape parameter (ρ). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS–citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50–1000 mOsm/L, pH 4–10) had no significant effect on the R_h (=100–130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in I of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material.     

Extracellular Vesicles Mediate Communication between Endothelial and Vascular Smooth Muscle Cells

The recruitment of pericytes and vascular smooth muscle cells (SMCs) that enwrap endothelial cells (ECs) is a crucial process for vascular maturation and stabilization. Communication between these two cell types is crucial during vascular development and in maintaining vessel homeostasis. Extracellular vesicles (EVs) have emerged as a new communication tool involving the exchange of microRNAs between cells. In the present study, we searched for microRNAs that could be transferred via EVs from ECs to SMCs and vice versa. Thanks to a microRNA profiling experiment, we found that two microRNAs are more exported in each cell type in coculture experiments: while miR-539 is more secreted by ECs, miR-582 is more present in EVs from SMCs. Functional assays revealed that both microRNAs can modulate both cell-type phenotypes. We further identified miR-539 and miR-582 targets, in agreement with their respective cell functions. The results obtained in vivo in the neovascularization model suggest that miR-539 and miR-582 might cooperate to trigger the process of blood vessel coverage by smooth muscle cells in a mature plexus. Taken together, these results are the first to highlight the role of miR-539 and miR-582 in angiogenesis and communication between ECs and SMCs.     

Plant Extracellular Vesicles and Nanovesicles: Focus on Secondary Metabolites,Proteins and Lipids with Perspectives on Their Potential and Sources

While human extracellular vesicles (EVs) have attracted a big deal of interest and have been extensively characterized over the last years, plant-derived EVs and nanovesicles have earned less attention and have remained poorly investigated. Although a series of investigations already revealed promising beneficial health effects and drug delivery properties, adequate (pre)clinical studies are rare. This fact might be caused by a lack of sources with appropriate qualities. Our study introduces plant cell suspension culture as a new and well controllable source for plant EVs. Plant cells, cultured in vitro, release EVs into the growth medium which could be harvested for pharmaceutical applications. In this investigation we characterized EVs and nanovesicles from distinct sources. Our findings regarding secondary metabolites indicate that these might not be packaged into EVs in an active manner but enriched in the membrane when lipophilic enough, since apparently lipophilic compounds were associated with nanovesicles while more hydrophilic structures were not consistently found. In addition, protein identification revealed a possible explanation for the mechanism of EV cell wall passage in plants, since cell wall hydrolases like 1,3-β-glucosidases, pectinesterases, polygalacturonases, β-galactosidases and β-xylosidase/α-L-arabinofuranosidase 2-like are present in plant EVs and nanovesicles which might facilitate cell wall transition. Further on, the identified proteins indicate that plant cells secrete EVs using similar mechanisms as animal cells to release exosomes and microvesicles.     

Therapeutic Potential of Mesenchymal Stem Cells (MSCs) and MSC-Derived Extracellular Vesicles for the Treatment of Spinal Cord Injury

Spinal cord injury (SCI) is a life-threatening condition that leads to permanent disability with partial or complete loss of motor, sensory, and autonomic functions. SCI is usually caused by initial mechanical insult, followed by a cascade of several neuroinflammation and structural changes. For ameliorating the neuroinflammatory cascades, MSC has been regarded as a therapeutic agent. The animal SCI research has demonstrated that MSC can be a valuable therapeutic agent with several growth factors and cytokines that may induce anti-inflammatory and regenerative effects. However, the therapeutic efficacy of MSCs in animal SCI models is inconsistent, and the optimal method of MSCs remains debatable. Moreover, there are several limitations to developing these therapeutic agents for humans. Therefore, identifying novel agents for regenerative medicine is necessary. Extracellular vesicles are a novel source for regenerative medicine; they possess nucleic acids, functional proteins, and bioactive lipids and perform various functions, including damaged tissue repair, immune response regulation, and reduction of inflammation. MSC-derived exosomes have advantages over MSCs, including small dimensions, low immunogenicity, and no need for additional procedures for culture expansion or delivery. Certain studies have demonstrated that MSC-derived extracellular vesicles (EVs), including exosomes, exhibit outstanding chondroprotective and anti-inflammatory effects. Therefore, we reviewed the principles and patho-mechanisms and summarized the research outcomes of MSCs and MSC-derived EVs for SCI, reported to date.     

Expression Profiling of Exosomal miRNAs Derived from Human Esophageal Cancer Cells by Solexa High-Throughput Sequencing

Cellular genetic materials, such as microRNAs (miRNAs), mRNAs and proteins, are packaged inside exosomes, small membrane vesicles of endocytic origin that are released into the extracellular environment. These cellular genetic materials can be delivered into recipient cells, where they exert their respective biological effects. However, the miRNA profiles and biological functions of exosomes secreted by cancer cells remain unknown. The present study explored the miRNA expression profile and distribution characteristics of exosomes derived from human esophageal cancer cells through Solexa high-throughput sequencing. Results showed that 56,421 (2.94%) unique sequences in cells and 7727 (0.63%) in exosomes matched known miRNAs. A total of 342 and 48 known miRNAs were identified in cells and exosomes, respectively. Moreover, 64 and 32 novel miRNAs were predicted in cells and exosomes, respectively. Significant differences in miRNA expression profiles were found between human esophageal cancer cells and exosomes. These findings provided new insights into the characteristics of miRNAs in exosomes derived from human esophageal cancer cells and the specific roles of miRNAs in intercellular communication mediated by exosomes in esophageal cancer.     

植物生物反应器生产转基因植物可食疫苗的研究进展

利用转基因植物作为生物反应器规模化生产可食疫苗是一个新兴的研究领域。随着生物技术的不断发展,植物生物反应器将会成为疫苗生产的有效途径。本文就转基因植物可食疫苗的优点、免疫原理、研究现状及存在的问题作一综述。     

Extracellular Vesicles from Airway Secretions: New Insights in Lung Diseases

Lung diseases (LD) are one of the most common causes of death worldwide. Although it is known that chronic airway inflammation and excessive tissue repair are processes associated with LD such as asthma, chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF), their specific pathways remain unclear. Extracellular vesicles (EVs) are heterogeneous nanoscale membrane vesicles with an important role in cell-to-cell communication. EVs are present in general biofluids as plasma or urine but also in secretions of the airway as bronchoalveolar lavage fluid (BALF), induced sputum (IS), nasal lavage (NL) or pharyngeal lavage. Alterations of airway EV cargo could be crucial for understanding LD. Airway EVs have shown a role in the pathogenesis of some LD such as eosinophil increase in asthma, the promotion of lung cancer in vitro models in COPD and as biomarkers to distinguishing IPF in patients with diffuse lung diseases. In addition, they also have a promising future as therapeutics for LD. In this review, we focus on the importance of airway secretions in LD, the pivotal role of EVs from those secretions on their pathophysiology and their potential for biomarker discovery.     

高效液相色谱法测定食用植物油中的游离棉酚

研究采用高效液相色谱法测定食用植物油中游离棉酚的方法,最佳色谱条件:色谱柱C18柱,流动相为甲醇-磷酸溶液(1%)=84+16,波长240 nm,流速为1.0 mL/min,柱温30℃。曲线方程y=3.1033×10-5x,相关系数r=0.999871,线性关系良好。检出限为2.5 mg/kg,回收率为98.0%～100.2%。该法简便,快速,准确性高,重现性好。     

Differential Therapeutic Effect of Extracellular Vesicles Derived by Bone Marrow and Adipose Mesenchymal Stem Cells on Wound Healing of Diabetic Ulcers and Correlation to Their Cargoes

Extracellular vesicles (EVs) derived from mesenchymal stem cells isolated from both bone marrow (BMSCs) and adipose tissue (ADSCs) show potential therapeutic effects. These vesicles often show a similar beneficial effect on tissue regeneration, but in some contexts, they exert different biological properties. To date, a comparison of their molecular cargo that could explain the different biological effect is not available. Here, we demonstrated that ADSC-EVs, and not BMSC-EVs, promote wound healing on a murine model of diabetic wounds. Besides a general similarity, the bioinformatic analysis of their protein and miRNA cargo highlighted important differences between these two types of EVs. Molecules present exclusively in ADSC-EVs were highly correlated to angiogenesis, whereas those expressed in BMSC-EVs were preferentially involved in cellular proliferation. Finally, in vitro analysis confirmed that both ADSC and BMSC-EVs exploited beneficial effect on cells involved in skin wound healing such as fibroblasts, keratinocytes and endothelial cells, but through different cellular processes. Consistent with the bioinformatic analyses, BMSC-EVs were shown to mainly promote proliferation, whereas ADSC-EVs demonstrated a major effect on angiogenesis. Taken together, these results provide deeper comparative information on the cargo of ADSC-EVs and BMSC-EVs and the impact on regenerative processes essential for diabetic wound healing.     

The Blocking of Integrin-Mediated Interactions with Maternal Endothelial Cells Reversed the Endothelial Cell Dysfunction Induced by EVs,Derived from Preeclamptic Placentae

Placental extracellular vesicles (EVs) have increasingly been recognized as a major mediator of feto-maternal communication. However, the cellular and molecular mechanisms of the uptake of placental EVs by recipient cells are still not well-understood. We previously reported that placental EVs target a limited number of organs in vivo. In the current study, we investigated the mechanisms underlying the uptake of placental EVs into target cells. Placental EVs were derived from explant cultures of normal or preeclamptic placentae. The mechanisms underlying the uptake of placental EVs were elucidated, using the phagocytosis or endocytosis inhibitor, trypsin-treatment or integrin-blocking peptides. The endothelial cell activation was studied using the monocyte adhesion assay after the preeclamptic EVs exposure, with and/or without treatment with the integrin blocking peptide, YIGSR. The cellular mechanism of the uptake of the placental EVs was time, concentration and energy-dependent and both the phagocytosis and endocytosis were involved in this process. Additionally, proteins on the surface of the placental EVs, including integrins, were involved in the EV uptake process. Furthermore, inhibiting the uptake of preeclamptic EVs with YIGSR, reduced the endothelial cell activation. The interaction between the placental EVs and the recipient cells is mediated by integrins, and the cellular uptake is mediated by a combination of both phagocytosis and endocytosis.     

膜过滤法处理食用油生产碱炼洗涤废水的研究

食用油生产碱炼洗涤废水难以处理。在实验的基础上提出了膜过滤法处理食用油生产碱炼洗涤废水的新工艺 ,并就操作条件的变化对实验结果的影响进行了分析和探讨     

Epithelial Membrane Protein 2 Suppresses Non-Small Cell Lung Cancer Cell Growth by Inhibition of MAPK Pathway

Epithelial membrane proteins (EMP1-3) are involved in epithelial differentiation and carcinogenesis. Dysregulated expression of EMP2 was observed in various cancers, but its role in human lung cancer is not yet clarified. In this study, we analyzed the expression of EMP1-3 and investigated the biological function of EMP2 in non-small cell lung cancer (NSCLC). The results showed that lower expression of EMP1 was significantly correlated with tumor size in primary lung tumors (p = 0.004). Overexpression of EMP2 suppressed tumor cell growth, migration, and invasion, resulting in a G1 cell cycle arrest, with knockdown of EMP2 leading to enhanced cell migration, related to MAPK pathway alterations and disruption of cell cycle regulatory genes. Exosomes isolated from transfected cells were taken up by tumor cells, carrying EMP2-downregulated microRNAs (miRNAs) which participated in regulation of the tumor microenvironment. Our data suggest that decreased EMP1 expression is significantly related to increased tumor size in NSCLC. EMP2 suppresses NSCLC cell growth mainly by inhibiting the MAPK pathway. EMP2 might further affect the tumor microenvironment by regulating tumor microenvironment-associated miRNAs.     

Immunostimulatory Potential of Extracellular Vesicles Isolated from an Edible Plant,Petasites japonicus,via the Induction of Murine Dendritic Cell Maturation

Extracellular vesicles (EVs) have recently been isolated from different plants. Plant-derived EVs have been proposed as potent therapeutics and drug-delivery nanoplatforms for delivering biomolecules, including proteins, RNAs, DNAs, and lipids. Herein, Petasites japonicus-derived EVs (PJ-EVs) were isolated through a series of centrifugation steps and characterized using dynamic light scattering and transmission electron microscopy. Immunomodulatory effects of PJ-EVs were assessed using dendritic cells (DCs). PJ-EVs exhibited a spherical morphology with an average size of 122.6 nm. They induced the maturation of DCs via an increase in the expression of surface molecules (CD80, CD86, MHC-I, and MHC-II), production of Th1-polarizing cytokines (TNF-α and IL-12p70), and antigen-presenting ability; however, they reduced the antigen-uptake ability. Furthermore, maturation of DCs induced by PJ-EVs was dependent on the activation and phosphorylation of MAPK and NF-κB signal pathways. Notably, PJ-EV-treated DCs strongly induced the proliferation and differentiation of naïve T cells toward Th1-type T cells and cytotoxic CD8⁺ T cells along with robust secretion of IFN-γ and IL-2. In conclusion, our study indicates that PJ-EVs can be potent immunostimulatory candidates with an ability of strongly inducing the maturation of DCs.     

Human Mesenchymal Stem Cell-Derived Extracellular Vesicles Promote Neural Differentiation of Neural Progenitor Cells

Mesenchymal stem cells (MSCs) have been adopted in various preclinical and clinical studies because of their multipotency and low immunogenicity. However, numerous obstacles relating to safety issues remain. Therefore, MSC-derived extracellular vesicles (EVs) have been recently employed. EVs are nano-sized endoplasmic reticulum particles generated and released in cells that have similar biological functions to their origin cells. EVs act as cargo for bioactive molecules such as proteins and genetic materials and facilitate tissue regeneration. EVs obtained from adipose-derived MSC (ADMSC) also have neuroprotective and neurogenesis effects. On the basis of the versatile effects of EVs, we aimed to enhance the neural differentiation ability of ADMSC-derived EVs by elucidating the neurogenic-differentiation process. ADMSC-derived EVs isolated from neurogenesis conditioned media (differentiated EVs, dEVs) increased neurogenic ability by altering innate microRNA expression and cytokine composition. Consequently, dEVs promoted neuronal differentiation of neural progenitor cells in vitro, suggesting that dEVs are a prospective candidate for EV-based neurological disorder regeneration therapy.     

气相色谱同时测定食用油中甲草胺与腐霉利的残留量

建立了固相萃取-气相色谱-电子捕获检测器（SPE—GC—ECD）同时测定食用油中甲草胺和腐霉利的方法。样品经过乙腈提取、固相萃取柱净化,采用GC—ECD检测,外标法定量。在此条件下,甲草胺的回收率为92．9％-98．9％,相对标准偏差为2．52％-3．24％,腐霉利的回收率为92．8％-99．2％,相对标准偏差在3．42％-6．95％。该方法简便、快速,具有良好的灵敏度、重复性,可适用于食用油中甲草胺和腐霉利的残留检测。     

The Upregulation of Regenerative Activity for Extracellular Vesicles with Melatonin Modulation in Chemically Defined Media

Extracellular vesicles (EVs) derived from human mesenchymal stem cells (hMSCs) have been widely known to have therapeutic effects by representing characteristics of the origin cells as an alternative for cell-based therapeutics. Major limitations of EVs for clinical applications include low production yields, unknown effects from serum impurities, and relatively low bioactivities against dose. In this study, we proposed a cell modulation method with melatonin for human umbilical cord MSCs (hUCMSCs) cultured in serum-free chemically defined media (CDM) to eliminate the effects of serum-derived impurities and promote regeneration-related activities. miRNAs highly associated with regeneration were selected and the expression levels of them were comparatively analyzed among various types of EVs depending on culture conditions. The EVs derived from melatonin-stimulated hUCMSCs in CDM (CDM mEVs) showed the highest expression levels of regeneration-related miRNAs, and 7 times more hsa-let-7b-5p, 5.6 times more hsa-miR-23a-3p, and 5.7 times more hsa-miR-100-5p than others, respectively. In addition, the upregulation of various functionalities, such as wound healing, angiogenesis, anti-inflammation, ROS scavenging, and anti-apoptosis, were proven using in vitro assays by simulating the characteristics of EVs with bioinformatics analysis. The present results suggest that the highly regenerative properties of hUCMSC-derived EVs were accomplished with melatonin stimulation in CDM and provided the potential for clinical uses of EVs.     

Evaluation of Plant Ceramide Species-Induced Exosome Release from Neuronal Cells and Exosome Loading Using Deuterium Chemistry

The extracellular accumulation of aggregated amyloid-β (Aβ) in the brain leads to the early pathology of Alzheimer’s disease (AD). The administration of exogenous plant-type ceramides into AD model mice can promote the release of neuronal exosomes, a subtype of extracellular vesicles, that can mediate Aβ clearance. In vitro studies showed that the length of fatty acids in mammalian-type ceramides is crucial for promoting neuronal exosome release. Therefore, investigating the structures of plant ceramides is important for evaluating the potential in releasing exosomes to remove Aβ. In this study, we assessed plant ceramide species with D-erythro-(4E,8Z)-sphingadienine and D-erythro-(8Z)-phytosphingenine as sphingoid bases that differ from mammalian-type species. Some plant ceramides were more effective than mammalian ceramides at stimulating exosome release. In addition, using deuterium chemistry-based lipidomics, most exogenous plant ceramides were confirmed to be derived from exosomes. These results suggest that the ceramide-dependent upregulation of exosome release may promote the release of exogenous ceramides from cells, and plant ceramides with long-chain fatty acids can effectively release neuronal exosomes and prevent AD pathology.