Extracellular Vesicles RNA Research Articles

Characterization of Total RNA, CD44, FASN, and PTEN mRNAs from Extracellular Vesicles as Biomarkers in Gastric Cancer Patients.

Simple SummaryLiquid biopsy is an easily accessible and non-invasive method to gain information about tumor diseases. The purpose of our study was to determine the value of extracellular vesicle-derived mRNAs as biomarkers for the diagnosis of gastric cancer and the response to its treatment. In a cohort of 87 gastric cancer patients and a control group of 14 individuals, we analyzed the absolute RNA concentration from extracellular vesicles (EV) and the relative levels of FASN, PTEN, and CD44 mRNA, and their correlation with clinico-pathological features. These correlated with treatment, tumor grading, and the pathological subtype according to Laurén’s classification. This might reflect their potential as both diagnostic and therapeutic predictors.In-depth characterization has introduced new molecular subtypes of gastric cancer (GC). To identify these, new approaches and techniques are required. Liquid biopsies are trendsetting and provide an easy and feasible method to identify and to monitor GC patients. In a prospective cohort of 87 GC patients, extracellular vesicles (EVs) were isolated from 250 µL of plasma. The total RNA was isolated with TRIZOL. The total RNA amount and the relative mRNA levels of CD44, PTEN, and FASN were measured by qRT-PCR. The isolation of EVs and their contained mRNA was possible in all 87 samples investigated. The relative mRNA levels of PTEN were higher in patients already treated by chemotherapy than in chemo-naïve patients. In patients who had undergone neoadjuvant chemotherapy followed by gastrectomy, a decrease in the total RNA amount was observed after neoadjuvant chemotherapy and gastrectomy, while FASN and CD44 mRNA levels decreased only after gastrectomy. The amount of RNA and the relative mRNA levels of FASN and CD44 in EVs were affected more significantly by chemotherapy and gastrectomy than by chemotherapy alone. Therefore, they are a potential biomarker for monitoring treatment response. Future analyses are needed to identify GC-specific key RNAs in EVs, which could be used for the diagnosis of gastric cancer patients in order to determine their molecular subtype and to accompany the therapeutic response.

Marine pollutant exposures and human milk extracellular vesicle-microRNAs in a mother-infant cohort from the Faroe Islands

Background/AimsEarly life exposures to marine contaminants can adversely impact child health but modes of action are unclear. Human milk contains extracellular vesicles (EVs) that can transport biologically relevant cargo from mother to infant, including microRNAs (miRNAs), and may partly mediate the effects of pollutants on child health. However, the role of marine pollutants on miRNA expression in milk EVs is unexplored. MethodsWe isolated EV RNA from 333 milk samples collected between 2 and 74 days postpartum from a Faroese birth cohort born 1997–2000 and sequenced 2083 miRNAs using a targeted library preparation method. We quantified five perfluoroalkyl substances (PFAS), pesticide metabolite p,p’-dichlorodiphenyldichloroethylene (DDE), and the sum of three major polychlorinated biphenyls (ΣPCBs) in maternal serum at 34 weeks of gestation and maternal hair total mercury (Hg) at birth. We used negative binomial regressions to estimate associations between individual pollutants and 418 reliably expressed EV-miRNAs adjusted for potential confounders. We performed sparse principal components (PCs) analysis to derive the first four components of the EV-miRNA data and examined associations between pollutants and PCs using Bayesian kernel machine regression (BKMR). ResultsWe observed no associations between pollutants and individual EV-miRNA expression after controlling the false discovery rate at 0.1. However, BKMR suggested that Hg was positively associated with PC1 and negatively associated with PC3, while ΣPCBs was negatively associated with PC3, and two PFAS were associated with PC4. Exploration of PC loadings followed by pathway analyses suggested that miRNAs in PC1 (miR-200b-3p, miR-664a-3p, miR-6738-5p, miR-429, miR-1236-5p, miR-4464, and miR-30b-5p) may be related to Hg neurotoxicity, while remaining PCs require further research. ConclusionsOur findings suggest that groups of milk EV-miRNAs may better serve as environmental biomarkers than individual miRNAs. Future studies are needed to elucidate the role of milk EV-miRNAs in child health following prenatal exposures.

Trimester-specific associations between extracellular vesicle microRNAs and fetal growth

Objective Placenta-derived extracellular vesicles and their cargoes, especially microRNAs (EV-miRNAs), may contribute to fetal and placental development. During pregnancy, the levels of several maternal blood EV-miRNAs, including miRNAs of placental origin, vary among individuals and change throughout gestation. However, the effects of these miRNAs on fetal growth and trimester-specificity have not been fully elucidated. The purpose of this study is to test the hypothesis that the serum levels of two extracellular vesicles (EV)-miRNAs (miR-127-3p and miR-26b-5p), which may be involved in fetoplacental regulation, would be significantly associated with fetal growth in a trimester-specific manner. Materials and methods This is a single-center birth cohort of maternal serum samples obtained at both the second and third trimesters. To minimize the influence of confounding factors, the analysis was limited to singleton vaginal deliveries, resulting in 27 participants being included in this study. EV RNAs were isolated using a membrane affinity method, and the relative expression levels of miR-127-3p and miR-26b-5p were measured using the RT-qPCR method with miR-484 as control. The associations between the two EV-miRNAs and fetal and placental growth were evaluated using a linear regression model and compared between the two trimesters. Results EV-miR-127-3p levels tended to correlate inversely with the z-scores of birth weight for gestational age (BWGA) and placental weight for gestational age (PWGA) in the second trimester, but not in the third trimester. EV-miR-26b-5p levels were positively associated with birth weight in the second trimester, but this association was weakened in the third trimester. Conclusion Our results suggest a trimester-specific association of circulating miRNA levels with fetal and placental growth. The precise roles of EV-miR-127-3p and EV-miR-26b-5p in fetal and placental development warrant further investigation.

A Dual Marker for Monitoring MDR-TB Treatment: Host-Derived miRNAs and M. tuberculosis-Derived RNA Sequences in Serum.

BackgroundIn the absence of a late marker of treatment failure or relapse in MDR-TB patients, biomarkers based on host-miRNAs coupled with M. tuberculosis-RNAs evaluated in extracellular vesicles (EVs) are an alternative follow-up for MDR-TB disease. Characterization of EVs cargo to identify differentially expressed miRNAs before and after treatment, and to identify M. tuberculosis-derived RNA in serum EVs from resistant TB patients.MethodsEVs were isolated from serum of 26 drug-resistant TB (DR-TB) patients and 16 healthy subjects. Differential expression of miRNAs in pooled exosomes from both untreated and treated patients was assessed and individually validated at different time points during treatment. In addition, M. tuberculosis RNA was amplified in the same samples by qPCR.ResultsA multivariate analysis using miR-let-7e-5p, -197-3p and -223-3p were found to be a more sensitive discriminator between healthy individuals and those with TB for both DR-TB (AUC= 0.96, 95%, CI=0.907-1) and MDR-TB groups (AUC= 0.95, 95%, CI= 0.89-1). Upregulation of miR-let-7e-5p were observed at the time of M. tuberculosis negative culture T(3-5) for MDR-TB group or for long-term T(9-15) for MDR-TB group without diabetes (T2DM). A second pathogen-based marker based on 30kDa and 5KST sequences was detected in 33% of the MDR-TB patients after the intensive phase of treatment. The miR-let7e-5p is a candidate biomarker for long-term monitoring of treatment for the group of MDR-TB without T2DM. A dual marker of host-derived miR-let7e-5p and M. tuberculosis-derived RNA for monitoring-TB treatment based in serum EVs.ConclusionA dual marker consisting of host-derived miR-let7e-5p and M. tuberculosis-derived RNA, could be an indicator of treatment failure or relapse time after treatment was completed.

471: Short RNAs in extracellular vesicles secreted by human airway epithelial cells increase antibiotic sensitivity of Pseudomonas aeruginosa

471: Short RNAs in extracellular vesicles secreted by human airway epithelial cells increase antibiotic sensitivity of Pseudomonas aeruginosa

Extracellular RNA transfer from non-malignant human cholangiocytes can promote cholangiocarcinoma growth.

Extracellular vesicles (EV) within the cellular secretome are emerging as modulators of pathological processes involved in tumor growth through their ability to transfer donor‐derived RNA into recipient cells. While the effects of tumor and stromal cell EVs within the tumor microenvironment have been studied, less is known about the contributions of normal, nontransformed cells. We examined the impact of EVs within the cellular secretome from nonmalignant cells on transformed cell growth and behavior in cholangiocarcinoma cells. These effects were enhanced in the presence of the pro‐fibrogenic mediator TGF‐β. We identified miR‐195 as a TGF‐β responsive miRNA in normal cells that can be transferred via EV to tumor cells and regulate cell growth, invasion, and migration. The effects of miR‐195 involve modulation of the epithelial–mesenchymal transition through direct effects on the transcription factor Snail. These studies provide in vitro and in vivo evidence for the impact of normal cellular secretome on transformed cell growth, show the importance of EV RNA transfer, and identify mechanisms of EV‐mediated transfer of miRNA as a contributor to tumor development, which may provide new therapeutic opportunities for targeting human cholangiocarcinoma.

MP38-05 EXTRACELLULAR VESICLE RNA ISOLATED FROM URINE FOR DIAGNOSING HIGH-RISK NON-MUSCLE INVASIVE BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Epidemiology & Evaluation I (MP38)1 Sep 2021MP38-05 EXTRACELLULAR VESICLE RNA ISOLATED FROM URINE FOR DIAGNOSING HIGH-RISK NON-MUSCLE INVASIVE BLADDER CANCER Keita Minami, Takahiro Osawa, Hiroshi Harada, Toru Harabayashi, Norikata Takada, Akira Kashiwagi, Haruka Miyata, Kazushi Hirakawa, Yasuyuki Sato, Ryuji Matsumoto, Hiroshi Kikuchi, Takashige Abe, Sachiyo Murai, and Nobuo Shinohara Keita MinamiKeita Minami More articles by this author , Takahiro OsawaTakahiro Osawa More articles by this author , Hiroshi HaradaHiroshi Harada More articles by this author , Toru HarabayashiToru Harabayashi More articles by this author , Norikata TakadaNorikata Takada More articles by this author , Akira KashiwagiAkira Kashiwagi More articles by this author , Haruka MiyataHaruka Miyata More articles by this author , Kazushi HirakawaKazushi Hirakawa More articles by this author , Yasuyuki SatoYasuyuki Sato More articles by this author , Ryuji MatsumotoRyuji Matsumoto More articles by this author , Hiroshi KikuchiHiroshi Kikuchi More articles by this author , Takashige AbeTakashige Abe More articles by this author , Sachiyo MuraiSachiyo Murai More articles by this author , and Nobuo ShinoharaNobuo Shinohara More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002053.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Patients who are diagnosed with high grade Ta, T1, or any Tis are generally regarded as high-risk non-muscle invasive bladder cancer (HR-NMIBC) according to the several practice guidelines. Patients categorized as HR-NMIBC are exposed to higher risk of both recurrence and progression compared to those who are categorized as low-risk (LR) NMIBC (low grade Ta). Pathological specimen collected with transurethral resection (TUR) is crucial for diagnosing HR- or LR-NMIBC, because cystoscopy itself is not reliable for distinguish these two. Herein, we develop the new method to diagnose NMIBC, especially HR-NMIBC, both pre-operatively and non-invasively. METHODS: In this multi-center prospective study, 231 patients who were clinically diagnosed as NMIBC were enrolled prior to TUR and were followed up periodically. Urine specimens were collected every outpatient visit during the follow-up with cystoscopy examination. As we previously reported, we confirmed that urinary extracellular vesicle (EV) SLC2A1 (basal type), GPRC5A (luminal type) and KRT17(basal type) were elevated in HR-NMIBC as potential biomarkers as well as EV RNA markers (NET1, MDK, NONO). In this study, diagnostic performances of these new markers were evaluated and compared to urine cytology, NMP22 (Abbott, IL), and BTA stat (Polymedco, NY) tests by the area under the curve (AUC) in ROC curve analysis. RESULTS: Tumor stages of the 231 participants were low grade Ta (N=44), high grade Ta (N=51), T1 (N=39), any Tis (N=22), >pT2 (N=12), benign tumors or no tumor (N=139). In this cohort, we validated that urinary EV markers were elevated in HR-NMIBC compared to LR-NMIBC and benign/no-tumor controls. Individual markers such as NET1 and KRT17 were the most promising and able to identify HR-NMIBC with remarkable diagnostic performance as high as AUC = 0.860 (0.794–0.927). EV NET1 was useful even to detect LR-NMIBC with AUC 0.798 (0.654–0.941)). Support logistic regression analysis of multiple EV markers allowed us to detect HR- and LR-NMIBC with AUC 0.918 (0.878–0.959) and 0.928 (0.876–0.979), respectively (Table 1, Figure 1). CONCLUSIONS: Taken together, our findings suggest that the combination of these urinary EV mRNA were promising biomarkers for recurrence monitoring of NMIBC as well as preoperative precise risk stratification of NMIBC. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e693-e694 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Keita Minami More articles by this author Takahiro Osawa More articles by this author Hiroshi Harada More articles by this author Toru Harabayashi More articles by this author Norikata Takada More articles by this author Akira Kashiwagi More articles by this author Haruka Miyata More articles by this author Kazushi Hirakawa More articles by this author Yasuyuki Sato More articles by this author Ryuji Matsumoto More articles by this author Hiroshi Kikuchi More articles by this author Takashige Abe More articles by this author Sachiyo Murai More articles by this author Nobuo Shinohara More articles by this author Expand All Advertisement Loading ...

Small nucleolar RNAs in plasma extracellular vesicles and their discriminatory power as diagnostic biomarkers of Alzheimer's disease

The clinical diagnosis of Alzheimer's disease, at its early stage, remains a difficult task. Advanced imaging technologies and laboratory assays to detect Aβ peptides Aβ42 and Aβ40, total and phosphorylated tau in CSF provide a set of biomarkers of developing AD brain pathology and facilitate the diagnostic process. The search for biofluid biomarkers, other than in CSF, and the development of biomarker assays have accelerated significantly and now represent the fastest-growing field in AD research. The goal of this study was to determine the differential enrichment of noncoding RNAs (ncRNAs) in plasma-derived extracellular vesicles (EV) of AD patients and Cognitively Normal controls (NC). Using RNA-seq, we profiled four significant classes of ncRNAs: miRNAs, snoRNAs, tRNAs, and piRNAs. We report a significant enrichment of SNORDs – a group of snoRNAs, in AD samples compared to NC. To verify the differential enrichment of two clusters of SNORDs – SNORD115 and SNORD116, localized on human chromosome 15q11-q13, we used plasma samples of an independent group of AD patients and NC. We applied ddPCR technique and identified SNORD115 and SNORD116 with a high discriminatory power to differentiate AD samples from NC. The results of our study present evidence that AD is associated with changes in the enrichment of SNORDs, transcribed from imprinted genomic loci, in plasma EV and provide a rationale to further explore the validity of those SNORDs as plasma biomarkers of AD.

Characterization of RNA in Extracellular Vesicles

Extracellular vesicles (EVs) are important players in the communication between different kinds of cells by delivering their content, consisting of different types of RNA, proteins, bioactive lipids, or signaling nucleotides, into their target cells. Several types of EVs are distinguished: (1) exosomes with sizes from 30 to 150 nm originate from the endosomal pathway and form intracellular multivesicular bodies (MVBs), which fuse to the plasma membrane before their secretion. (2) EVs with sizes ranging from 100 to 1000 nm in diameter are formed during cell surface budding. (3) Apoptotic bodies with diameters from 500 to 2000 nm are released from blebbing of the cell membrane of apoptotic cells. It is well established that various RNA molecules such as coding RNAs and noncoding RNAs (long noncoding RNAs, microRNAs, circular RNAs, and rRNAs) are present in different amounts in EVs depending on the type and origin of EV. Here we will give an overview of methods to isolate different types of EVs and to quantify and characterize different RNA species.

Quantitative Capillary Electrophoresis for Analysis of Extracellular Vesicles (EVqCE)

Extracellular Vesicles (EVs) gained significant interest within the last decade as a new source of biomarkers for the early detection of diseases and a promising tool for therapeutic applications. In this work, we present Extracellular Vesicles Quantitative Capillary Electrophoresis (EVqCE) to measure an average mass of RNA in EVs, determine EV concentrations and the degree of EV degradation after sample handling. We used EVqCE to analyze EVs isolated from conditioned media of three cancer cell lines. EVqCE employs capillary zone electrophoresis with laser-induced fluorescent detection to separate intact EVs from free nucleic acids. After lysis of EVs with a detergent, the encapsulated nucleic acids are released. Therefore, the initial concentration of intact EVs is calculated based on a nucleic acid peak gain. EVqCE works in a dynamic range of EV concentrations from 108 to 1010 particles/mL. The quantification process can be completed in less than one hour and requires minimum optimization. Furthermore, the average mass of RNA was found to be in the range of 200–400 ag per particle, noting that more aggressive cancer cells have less RNA in EVs (200 ag per particle) than non-aggressive cancer cells (350 ag per particle). EVqCE works well for the degradation analysis of EVs. Sonication for 10 min at 40 kHz caused 85% degradation of EVs, 10 freeze-thaw cycles (from −80 °C to 22 °C) produced 40%, 14-day storage at 4 °C made 32%, and vortexing for 5 min caused 5% degradation. Presently, EVqCE cannot separate and distinguish individual EV populations (exosomes, microvesicles, apoptotic bodies) from each other. Still, it is tolerant to the presence of non-EV particles, protein-lipid complexes, and protein aggregates.

Liposome-mediated detection of SARS-CoV-2 RNA-positive extracellular vesicles in plasma.

Introductory paragraphPlasma SARS-CoV-2 RNA may represent a viable diagnostic alternative to respiratory RNA levels that rapidly decline after infection. RT-qPCR reference assays exhibit poor performance with plasma, likely reflecting dilution and degradation of viral RNA released into the circulation, but these issues could be addressed by analyzing viral RNA packaged into extracellular vesicles (EVs). Herein we describe an assay approach where EVs directly captured from plasma are fused with reagent-loaded liposomes to sensitively amplify and detect a SARS-CoV-2 gene target. This approach accurately diagnosed COVID-19 patients, including challenging cases missed by RT-qPCR. SARS-CoV-2-positive EVs were detected at day one post-infection, and plateaued from day six to the day 28 endpoint in a non-human primate model, while 20–60 day signal durations were observed in young children. This nanotechnology approach, which uses a non-infectious sample, could thus improve COVID-19 diagnosis by extending virus detection windows to identify COVID-19 cases missed by current assays.

MiR- 16 Regulates Crosstalk in NF-κB Inflammatory Signaling between Myeloma Cells and Bone Marrow Macrophages

Introduction: Multiple myeloma plasma cells (MM-PCs) home to and dynamically interact with the bone marrow (BM) microenvironment (BM-ME). Of the different factors secreted by the BM-ME, NF-κB-induced cytokines including IL-6 play a central role in MM-PC survival and resistance to treatments. Previously published data have shown that in the BM of MM patients the presence of CD163+ tumor associated macrophages (TAM) correlates with IL-6 production. The canonical NF-κB pathway is induced by most physiological stimuli to produce low levels of inflammatory cytokines, through the activation of the IκB kinase complex (IKKβ, IKKα, and IKKγ). This pathway seems constitutively activated in TAM, where instead the activation of the non-canonical pathway seems to play a critical role in the anti-cancer phenotype of BM macrophages (BM-Mϕ).Extracellular vesicles (EV) are membrane-covered cell fragments released by all cell types, and recently they have been shown to play a pivotal role in creating a tumor supportive BM-ME. Our group was the first to show that MM cells produce EV (Harshman et.al. 2013). Previous studies have shown that miR- 16 encapsulated inEV(EV-miR- 16) down-modulates IL-6 production in the BM-ME, and we showed that higher levels of circulating miR- 16 is a prognostic factor that correlates with better survival in newly diagnosed MM patients treated with proteasome inhibitor (PI) based therapy (Rocci et al. 2013). Here, we investigated whether MM derived EV can play a pivotal role in supporting monocyte to TAM differentiation and defined the molecular mechanisms of IL-6 modulation by miR- 16 .Methods: Monocyte to TAM differentiation was evaluated by flow cytometry analysis, light microscopy, q-RT-PCR and phagocytosis assays. EV were isolated via a serial centrifugation method. EV RNA content was analyzed by NanoString. EV isolated from producing cells (0.5x106cells/ml) were used to differentiate 1 monocyte (4:1 effector:target ratio). Cytokine arrays in the supernatant of BM-Mϕ and in the BM-Mϕ depleted fractions were performed. Cloning, Western blot and RT-PCR were used to confirm IKK complex targeting by miR- 16 . Single cell flow and NF-kB activation assays were conducted.Results: Our in vitro data show that EV isolated from the supernatant of MM cell lines (MM-EV) can promotemonocyte differentiation to CD163+ TAM. Mϕ differentiation was also observed when peripheral blood monocytes (PB-M) obtained from both MM and healthy donors were treated with EV isolated from the BM acellular fraction of MM patients (n=4). Limited differentiation was instead observed when the acellular fraction was EV-depleted (Fig. 1). Our data show that PB-M differentiation was partially reversed by the addition of an EV-miR- 16 mimic. We found that miR- 16 is dramatically down-regulated when PB-M is induced by macrophage colony stimulating factor to differentiate to TAM, and it is also down-regulated in primary BM-Mϕ as compared to PB-M of the same patients and to the BM-Mϕ of 3 age matched healthy donors. We learned that BM-Mϕ isolated from MM patients are the main source of NF - κB-induced cytokines including IL-6 and IL-10 under unstimulated conditions as well as IL-6, IL-8, IL-10, TNF-α and VEGF under Toll-like receptor stimulated conditions, as the Mϕ-depleted BM cellular fraction had significantly low levels. We also found that miR -16 ectopic expression in MM cells and in a human monocytic cell line downregulates both IKKβ/IKKα protein and mRNA levels, with subsequent downregulation of NF-κB activation signaling and decreased expression of inflammatory cytokines. Further, cell growth and apoptotic assays show that the attenuation of PI-based anti-MM activity by TAM is partially reversed by ectopic miR- 16 in both TAM and MM-PCs.Conclusions: MM-EV support monocyte differentiation to TAM, causing miR- 16 downregulation and an increase in NF-κB inflammatory signaling. We also show that this effect can be impaired by an ectopic miR- 16 mimic. Since constitutive activation of NF-κB decreases MM cells dependency on extrinsic growth factors, at the onset of the disease miR- 16 downregulation in MM-PCs can support NF-kB intrinsic activation and increased fitness of MM cells. Because NF-kB activation in MM-PCs counterbalances the anti-MM activity of PI, it is not surprising that miR- 16 expression can potentiate the killing effect of PI. We are exploring these hypotheses through use of miR -16 knock out mouse models. [Display omitted] DisclosuresHofmeister:Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Karyopharm: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Research Funding; Roche: Research Funding; Thrassos: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding. Krishnan:Takeda: Speakers Bureau; Onyx: Speakers Bureau; Sutro: Consultancy; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Equity Ownership, Speakers Bureau.

Extracellular mRNA transported to the nucleus exerts translation-independent function

RNA in extracellular vesicles (EVs) are uptaken by cells, where they regulate fundamental cellular functions. EV-derived mRNA in recipient cells can be translated. However, it is still elusive whether “naked nonvesicular extracellular mRNA” (nex-mRNA) that are not packed in EVs can be uptaken by cells and, if so, whether they have any functions in recipient cells. Here, we show the entrance of nex-mRNA in the nucleus, where they exert a translation-independent function. Human nex-interleukin-1β (IL1β)-mRNA outside cells proved to be captured by RNA-binding zinc finger CCCH domain containing protein 12D (ZC3H12D)-expressing human natural killer (NK) cells. ZC3H12D recruited to the cell membrane binds to the 3′-untranslated region of nex-IL1β-mRNA and transports it to the nucleus. The nex-IL1β-mRNA in the NK cell nucleus upregulates antiapoptotic gene expression, migration activity, and interferon-γ production, leading to the killing of cancer cells and antimetastasis in mice. These results implicate the diverse actions of mRNA.

Altered Expression of Extracellular Vesicles miRNAs from Primary Human Trabecular Meshwork Cells Induced by Transforming Growth Factor-β2.

Primary open-angle glaucoma (POAG) is tightly related with extracellular matrix (ECM) remodeling of human trabecular meshwork cells (HTMCs). Transforming growth factor-β2 (TGF-β2) can induce ECM remodeling. The aim of the study was to investigate the microRNAs (miRNAs) expression changes of extracellular vesicles (EVs) derived from HTMCs treated with TGF-β2. EVs were isolated from HTMCs supernatant cultured for 24 h with TGF-β2. The morphology of EVs pellets was examined by transmission electron microscopy. Nanoparticle tracking analysis used to demonstrate the particle size distribution. Total EVs RNAs were extracted for subsequent miRNA gene chip analysis to investigate differentially expressed miRNAs between the controls and treatment cells. Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to predict potential target and validate possible functions of the miRNAs. There were 23 miRNAs upregulated and 3 miRNAs downregulated and 469,102, and 94 GO terms involved in biological processes, cellular components, and molecular function for the possible functions of the 26 miRNAs. These findings indicate that TGF-β2 may alter EVs miRNAs expression to participate in the pathogenesis of POAG. They may provide significant information for potential biomarkers for POAG diagnosis and treatment.

Ultrasensitive detection of mRNA in extracellular vesicles using DNA tetrahedron-based thermophoretic assay

The analysis of messenger RNA (mRNA) in extracellular vesicles (EVs) may provide unprecedented opportunities for non-invasive and rapid diagnosis of cancers. However, clinical utility of EV mRNA is often hampered by the extremely low abundance of mRNA in EVs and the complicated procedures for mRNA detection. Here we address these challenges by developing an ultrasensitive assay for in situ measurement of EV mRNA, which combines FRET-based DNA tetrahedron (FDT) for efficient EV internalization and target mRNA detection, and size-selective thermophoretic accumulation for amplification of FRET signal in EVs. This DNA tetrahedron-based thermophoretic assay (DTTA) achieves a limit of detection (LoD) of 14 aM for detecting PSA mRNA in serum EVs, with resorting to RNA extraction and enzyme amplification. The DTTA showed that EV PSA mRNA outperforms serum PSA protein, the gold standard in PCa screening, for discrimination between prostate cancer and benign prostatic hyperplasia (area under the curve: 0.93 versus 0.74; 42 patients). This technology may greatly expand the applications of DNA nanostructure-enabled liquid biopsy.

Non-coding RNAs and lipids mediate the function of extracellular vesicles in cancer cross-talk.

Research on extracellular vesicles (EVs) has been expanded, especially in the field of cancer. The cargoes in EVs, especially those in small EVs such as exosomes include microRNAs (miRNAs), mRNA, proteins, and lipids, are assumed to work cooperatively in the tumor microenvironment. In 2007, it was reported that miRNAs were abundant among the non-coding RNAs present in exosomes. Since then, many studies have investigated the functions of miRNAs and have tried to apply these molecules to aid in the diagnosis of cancer. Accordingly, many reviews of non-coding RNAs in EVs have been published for miRNAs. This review focuses on relatively new cargoes, covering long noncoding (lnc) RNAs, circular RNAs, and repeat RNAs, among non-coding RNAs. These RNAs, regardless of EV or cell type, have newly emerged due to the innovation of sequencing technology. The poor conservation, low quantity, and technical difficulty in detecting these RNA types have made it difficult to elucidate their functions and expression patterns. We herein summarize a limited number of studies. Although lipids are major components of EVs, current research on EVs focuses on miRNA and protein biology, while the roles of lipids in exosomes have not drawn attention. However, several recent studies revealed that phospholipids, which are components of the EV membrane, play important roles in the intercommunication between cells and in the generation of lipid mediators. Here, we review the reported roles of these molecules, and describe their potential in cancer biology.

Isolation of intact extracellular vesicles from cryopreserved samples.

Extracellular vesicles (EVs) have emerged as promising candidates in biomarker discovery and diagnostics. Protected by the lipid bilayer, the molecular content of EVs in diverse biofluids are protected from RNases and proteases in the surrounding environment that may rapidly degrade targets of interests. Nonetheless, cryopreservation of EV-containing samples to -80°C may expose the lipid bilayer to physical and biological stressors which may result in cryoinjury and contribute to changes in EV yield, function, or molecular cargo. In the present work, we systematically evaluate the effect of cryopreservation at -80°C for a relatively short duration of storage (up to 12 days) on plasma- and media-derived EV particle count and/or RNA yield/quality, as compared to paired fresh controls. On average, we found that the plasma-derived EV concentration of stored samples decreased to 23% of fresh samples. Further, this significant decrease in EV particle count was matched with a corresponding significant decrease in RNA yield whereby plasma-derived stored samples contained only 47-52% of the total RNA from fresh samples, depending on the extraction method used. Similarly, media-derived EVs showed a statistically significant decrease in RNA yield whereby stored samples were 58% of the total RNA from fresh samples. In contrast, we did not obtain clear evidence of decreased RNA quality through analysis of RNA traces. These results suggest that samples stored for up to 12 days can indeed produce high-quality RNA; however, we note that when directly comparing fresh versus cryopreserved samples without cryoprotective agents there are significant losses in total RNA. Finally, we demonstrate that the addition of the commonly used cryoprotectant agent, DMSO, alongside greater control of the rate of cooling/warming, can rescue EVs from damaging ice formation and improve RNA yield.

ExoProK: A Practical Method for the Isolation of Small Extracellular Vesicles from Pleural Effusions.

Extracellular vesicles (EVs) are cell-secreted, lipid membrane-enclosed nanoparticles without functional nucleus. EV is a general term that includes various subtypes of particles named microvesicles, microparticles, ectosomes or exosomes. EVs transfer RNA, DNA and protein cargo between proximal and distant cells and tissues, thus constituting an organism-wide signal transduction network. Pathological tissues secrete EVs that differ in their cargo composition compared to their healthy counterparts. The detection of biomarkers in EVs from biological fluids may aid the diagnosis of disease and/or monitor its progression in a minimally invasive manner. Among biological fluids, pleural effusions (PEs) are integrated to clinical practice, as they accompany a wide variety of lung disorders. Due to the proximity with the pleura and the lungs, PEs are expected to be especially enriched in EVs that originate from diseased tissues. However, PEs are among the least studied biofluids regarding EV-specialized isolation methods and related biomarkers. Herein, we describe a practical EV isolation method from PEs for the screening of EV RNA biomarkers in clinical routine. It is based on a Proteinase K treatment step to digest contaminants prior to standard polyethylene-glycol precipitation. The efficiency of the method was confirmed by transmission electron microscopy, nanoparticle tracking analysis and Western blot. The reliability and sensitivity of the method towards the detection of EV-enriched RNA biomarkers from multiple PEs was also demonstrated.

Biological Characteristics and Roles of Noncoding RNAs in Milk-Derived Extracellular Vesicles

Extracellular vesicles (EVs) have diverse roles in the transport of proteins, lipids, and nucleic acids between cells, and they serve as mediators of intercellular communication. Noncoding RNAs (ncRNAs) that are present in EVs, including microRNAs, long noncoding RNAs, and circular RNAs, have been found to participate in complex networks of interactions and regulate a wide variety of genes in animals. Milk is an important source of nutrition for humans and other mammals. Evidence suggests that milk-derived EVs contain abundant ncRNAs, which are stable and can be transported to the offspring and other consumers. Current data suggest a strong link between milk EV ncRNAs and many biological processes, and these ncRNAs have been drawing increasing attention and might play an epigenetic regulatory role in recipients, though further research is still necessary to understand their precise roles. The present review introduces basic information about milk EV ncRNAs, summarizes their expression profiles, biological characteristics, and functions based on current knowledge, and discusses their biological roles, indeterminate issues, and perspectives. Our goal is to provide a deeper understanding of the physiological effects of milk EV ncRNAs on offspring and to provide a reference for future research in this field.

Skeletal Muscle Extracellular Vesicles Regulate Endothelial Cells in a Fiber Type Dependent Manner

Introduction Skeletal muscle regulates endothelial cells (EC) via the secretion of angiogenic signaling factors, including extracellular vesicles (EV). Muscle can be characterized as predominantly oxidative (OXI) or glycolytic (GLY) depending on the expression of specific fiber types. OXI fibers have greater capillary density and secrete greater quantities of several angiogenic factors and EV, however it is unknown if EV signaling is regulated by fiber type. We hypothesized that EV released from OXI muscle (OXI-EV) induce a greater angiogenic response in cultured EC compared to EV from GLY muscle (GLY-EV). Methods From male CL57BL/6 mice, OXI muscle was isolated from red portions of the gastrocnemius and soleus; GLY muscle was isolated from white portions of the gastrocnemius and tibialis anterior. EVs were isolated using ultrafiltration-size exclusion chromatography. EV number and RNA content were measured. Human umbilical vein endothelial cells (HUVEC) were treated with OXI-EV or GLY-EV (10 μg/ml) for 24 h; and HUVEC functional angiogenic responses and molecular pathways related to angiogenesis were analyzed. Significance was established at P ≤ 0.05. Results OXI muscle released more EV than GLY muscle (OXI: 8.1x106; GLY: 4.6x106 particles/ml/mg muscle). Selected angiogenic mRNA (vascular endothelial growth factor (VEGF) A and B, angiopoietin (ANG) 1 and 2) and miRNAs (15a, 126, 133a) were not different between OXI-EV and GLY-EV, although there was a trend towards greater VEGFA in OXI-EV. In HUVEC, OXI-EV promoted greater tube formation (# of tubes: +34%; length: +20%), lowered intracellular superoxide (-58%), and trended (P < 0.10) toward greater growth (+22%) with no difference in migration compared to GLY-EVs. Treatment with OXI-EV promoted greater HUVEC endothelial nitric oxide synthase (eNOS) phosphorylation (+40%) and gene expression (+42%) compared to GLY-EV. In HUVEC, there was no difference in gene expression of VEGFA, VEGFB, ANG1, or Ang2 between OXI-EV and GLY-EV treatments. Conclusions EV from oxidative muscle demonstrate greater angiogenic potential than EV from glycolytic muscle consistent with greater capillarization of oxidative muscle. Thus, greater oxidative vs. glycolytic muscle capillarization is likely regulated in part by the combination of greater release of EV and greater EV angiogenic potential from oxidative muscle fibers.