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Crossland, Rachel E., Jean Norden, Louis Bibby, Joanna Davis, and Anne M. Dickinson. "Validation of Isolation Methodology and Endogenous Control Selection for qRT-PCR Assessment of Microrna Expression in Serum and Urine Exosomes." Blood 124, no. 21 (December 6, 2014): 5793. http://dx.doi.org/10.1182/blood.v124.21.5793.5793.
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Abstract Introduction: MicroRNAs are short RNA molecules that control ~50% of genes by binding to the mRNA 3’ UTR and repressing translation. Recently, they have been detected within exosomes; small vesicles secreted by most cells and abundant in body fluids. Exosomes are highly enriched for specific microRNAs and have been proposed as the starting point for circulating biomarker studies. To increase the accuracy of microRNA assessment by qRT-PCR, endogenous controls are required to correct for variability factors. Exosomal microRNA studies can be problematic, as endogenous controls previously used in cellular samples may not be present. This study compared exosome isolation and RNA extraction methods from urine and serum samples and identified suitable endogenous controls for incorporation into qRT-PCR analysis. Methods and Results: For serum exosomes, specialist isolation reagents from System Biosciences (SBI) (ExoQuick Serum Exosome Precipitation Solution) and Life Technologies (Total Exosome Isolation Reagent) were compared, followed by RNA extraction (Norgen Biotek Total RNA Purification kit) and qRT-PCR assessment of 3 endogenous controls (HY3, RNU48 & U6). Superior exosomal RNA recovery was achieved using Life Technologies reagent, demonstrated by higher RNA concentration (Life Technologies ng/ul 4.4, 7.5 & 6.9 vs. SBI ng/ul 3.8, 5.0 & 2.7) and lower endogenous control Ct values (HY3: Life Technologies 25.56, 28.54 & 26.69 vs. SBI 27.48, 30.48 & 35.36. RNU48: Life Technologies 30.95, undetected & 34.45 vs. SBI 30.95, undetected & undetected. U6: Life Technologies 21.83, 24.72 & 22.59 vs. SBI 21.59, 27.55 & 32.71, respectively). Recovery of exosomes (30-150 nm) was verified by electron microscopy. Serum exosomal RNA recovery was further assessed by isolating exosomes then comparing three commercially available RNA extraction kits (SBI SeraMir Exosome RNA Purification Column kit, Norgen Biotek Total RNA Purification kit & Qiagen RNeasy Micro kit). The Norgen Biotek kit gave the highest RNA yield (SBI ng/ul 13.0, 10.9 & 6.7 vs. Norgen ng/ul 23.2, 22.6 & 33.2 vs. Qiagen ng/ul 0.3, 0.6 & 0.4) and expression of two endogenous controls (HY3 & U6) (HY3: Norgen 26.76, 29.37 & 27.66 vs. SBI 31.45, 29.43 & 33.38 vs. Qiagen 35.00, 35.12 & 33.99. U6: Norgen 21.38, 24.96 & 21.31 vs. SBI 25.95, 24.91 & 30.17 vs. Qiagen 26.48, 27.14 & 27.39). In each case, exosomal isolation was confirmed by electron microscopy. To validate the methodology to isolate urine exosomal RNA, a commercially available kit was compared to ultracentrifugation. The Urine Exosome RNA Isolation kit (Norgen Biotek) gave superior results compared to ultracentrifugation followed by RNA extraction using the Norgen Biotek Total RNA Purification kit. This was demonstrated by higher RNA quantity (Norgen ng/ul 6.6, 6.4 & 11.5 vs. ultracentrifugation ng/ul 3.3, 4.5 & 2.9) and endogenous control (HY3 & U6) expression (HY3: Norgen 25.31, 26.33 & 26.85 vs. ultracentrifugation 31.54, 29.21 & 29.36. U6: Norgen 31.66, 30.83 & 33.47 vs. ultracentrifugation 32.49, 33.46 & 33.30). Exosomes isolated by the Norgen kit were also visualised by electron microscopy for further validation. The stability of 8 endogenous controls (RNU6B, RNU19, RNU38B, RNU43, RNU48, HY3, U6 & miR-320) was assessed by qRT-PCR in a test serum (n=10) and urine (n=15) exosome cohort from healthy controls and hematopoietic stem cell transplantation (HSCT) patients. HY3 and U6 were selected as the optimal controls for serum exosome miRNA expression analysis, with the highest level of stability across the panel (HY3: S.D 1.77 & CoV 6.2%, U6: S.D 2.14 & CoV 8.6%). HY3 and RNU48 were selected as the optimal controls for urine exosome miRNA expression analysis panel (HY3: S.D 1.67 & CoV 6.4%, RNU48: S.D 1.85 & CoV 5.3%). Selected optimal controls were analysed in a clinical HSCT serum (n=55) and urine (n=50) cohort. Expression stability was acceptable for all controls (serum U6: S.D 2.93 & CoV 11.8%. HY3: S.D 2.22 & CoV 7.4%. Urine RNU48: S.D 2.26 & CoV 6.9%, HY3: S.D 2.42 & CoV 8.8%), indicating constitutive expression in clinical samples. Conclusions: Exosomal microRNA studies are in their infancy and the number of commercially available exosome and RNA isolation kits are increasing. This study identifies the optimal methods to isolate serum and urine exosomal RNA as well as suitable endogenous controls for incorporation into qRT-PCR studies. Disclosures No relevant conflicts of interest to declare.
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Liu, Keda, Nanjue Cao, Yuhe Zhu, and Wei Wang. "Exosome: A Novel Nanocarrier Delivering Noncoding RNA for Bone Tissue Engineering." Journal of Nanomaterials 2020 (August 14, 2020): 1–14. http://dx.doi.org/10.1155/2020/2187169.
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Bone growth and metabolism are mainly regulated by a series of intracellular molecules and extracellular stimuli. Exosome, as a nanoscale substance secreted to the outside of the cells, plays an extensive role in intercellular communication. This review provides theoretical references and evidences for further exploration of exosomes as noncoding RNA carriers to regulate bone tissue recovery through the following aspects: (1) basic characteristics of exosomes, (2) research progress of exosomal noncoding RNA in bone tissue engineering, (3) current status and advantages of engineering exosomes as nanocarriers for noncoding RNA delivery, and (4) problems and application prospects of exosome therapy in the field of orthopedics.
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Mańka, Rafał, Pawel Janas, Karolina Sapoń, Teresa Janas, and Tadeusz Janas. "Role of RNA Motifs in RNA Interaction with Membrane Lipid Rafts: Implications for Therapeutic Applications of Exosomal RNAs." International Journal of Molecular Sciences 22, no. 17 (August 30, 2021): 9416. http://dx.doi.org/10.3390/ijms22179416.
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RNA motifs may promote interactions with exosomes (EXO-motifs) and lipid rafts (RAFT-motifs) that are enriched in exosomal membranes. These interactions can promote selective RNA loading into exosomes. We quantified the affinity between RNA aptamers containing various EXO- and RAFT-motifs and membrane lipid rafts in a liposome model of exosomes by determining the dissociation constants. Analysis of the secondary structure of RNA molecules provided data about the possible location of EXO- and RAFT-motifs within the RNA structure. The affinity of RNAs containing RAFT-motifs (UUGU, UCCC, CUCC, CCCU) and some EXO-motifs (CCCU, UCCU) to rafted liposomes is higher in comparison to aptamers without these motifs, suggesting direct RNA-exosome interaction. We have confirmed these results through the determination of the dissociation constant values of exosome-RNA aptamer complexes. RNAs containing EXO-motifs GGAG or UGAG have substantially lower affinity to lipid rafts, suggesting indirect RNA-exosome interaction via RNA binding proteins. Bioinformatics analysis revealed RNA aptamers containing both raft- and miRNA-binding motifs and involvement of raft-binding motifs UCCCU and CUCCC. A strategy is proposed for using functional RNA aptamers (fRNAa) containing both RAFT-motif and a therapeutic motif (e.g., miRNA inhibitor) to selectively introduce RNAs into exosomes for fRNAa delivery to target cells for personalized therapy.
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Sasaki, Reina, Tatsuo Kanda, Osamu Yokosuka, Naoya Kato, Shunichi Matsuoka, and Mitsuhiko Moriyama. "Exosomes and Hepatocellular Carcinoma: From Bench to Bedside." International Journal of Molecular Sciences 20, no. 6 (March 20, 2019): 1406. http://dx.doi.org/10.3390/ijms20061406.
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As hepatocellular carcinoma (HCC) usually occurs in the background of cirrhosis, which is an end-stage form of liver diseases, treatment options for advanced HCC are limited, due to poor liver function. The exosome is a nanometer-sized membrane vesicle structure that originates from the endosome. Exosome-mediated transfer of proteins, DNAs and various forms of RNA, such as microRNA (miRNA), long noncoding RNA (lncRNA) and messenger RNA (mRNA), contributes to the development of HCC. Exosomes mediate communication between both HCC and non-HCC cells involved in tumor-associated cells, and several molecules are implicated in exosome biogenesis. Exosomes may be potential diagnostic biomarkers for early-stage HCC. Exosomal proteins, miRNAs and lncRNAs could provide new biomarker information for HCC. Exosomes are also potential targets for the treatment of HCC. Notably, further efforts are required in this field. We reviewed recent literature and demonstrated how useful exosomes are for diagnosing patients with HCC, treating patients with HCC and predicting the prognosis of HCC patients.
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Zhou, Cuiqi, Stephen Shen, Rosemary Moran, and Shlomo Melmed. "Pituitary Somatotroph Adenoma Cell-Derived Exosomes: Characterization of Novel Non-Hormonal Functions." Journal of the Endocrine Society 5, Supplement_1 (May 1, 2021): A652—A653. http://dx.doi.org/10.1210/jendso/bvab048.1331.
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Abstract Exosomes, small extracellular vesicles carrying lipids, proteins, DNA and RNA, enable intercellular communication. Pituitary-derived exosomes have not been well validated, and as no human pituitary cell lines are available, we characterized exosomes derived from rat somatotroph tumor cells (GH1 and GH3). Rat FR and H9C2 cells were used as non-pituitary controls. Exosomes were isolated from serum-free culture supernatants by combining ultrafiltration and ultracentrifugation to eliminate hormone contamination. Derived exosomes were analyzed by NanoSight to visualize, size, and count particles. Exosomal proteins were extracted and exosome markers including TSG101, ALIX, CD63, HSP70, HSP90 detected by Western Blot. The exosome inhibitor GW4869 (10 µM, 30 h) reduced exosome release (up to 81%), whereas treating cells with hydrocortisone (0.1 µM, 72 h) increased exosome production (up to 42%) in GH1 and GH3 cells. Exosomal shuttle RNA characterized by RNA-Seq showed distinct pituitary vs non-pituitary exosome RNA profiles. Selected miRNAs assessed in exosomes and corresponding cells by qRT-PCR validated exosomal RNA-seq and suggested that miRNA signatures in exosomes and in respective cells of origin were concordant. Next, we explored downstream signaling of GH1-derived exosomes (GH1-exo) in vitro and in vivo and studied biological actions in normal hepatocytes and in malignant cells. As evidenced by mRNA-seq, GH1-exo distinctly altered signaling pathways in rat primary hepatocytes, vs pathways elicited by GH or PRL (0.5 µg/mL, 24 h). GH1-exo, FR-exo or vehicle were intravenously injected to 4-week-old female Wistar rats twice weekly for 4 weeks (5*109 exo/200 g, n=3), and livers dissected for mRNA-seq. Among GH1-exo specifically regulated genes, EIF2AK/ATF4, involved in cAMP responses and amino acid biosynthesis, were attenuated. In hepatocytes, GH1-exo suppressed up to 65% of nascent protein synthesis and reduced forskolin (10 µM)-stimulated cAMP activity by 19%, while GH (0.01-1 µg/mL) did not affect this pathway. Notably, GH1-exo also attenuated malignant cell motility. Both GH1-exo incubation or GH1 cell co-culture (48 h) suppressed migration, invasion and wound healing of HCT116 cancer cells by up to 70%. In contrast, treatment with rGH (0.5 µg/mL) increased HCT116 motility. Intravenous administration of GH1-exo (1010 exo/mouse, twice a week for 5 weeks) decreased metastatic tumor volume by 40% in nude mice harboring splenic HCT116 implants (5*105 cells/mouse, n=10), and especially abrogated hepatic metastases. mRNA-seq of GH1-exo treated HCT116 cells vs controls indicated dysregulated p53 and MAPK pathways, which may partially explain mechanisms underlying motility attenuation. The results elucidate novel biological actions of somatotroph adenoma cell-derived exosomes and suggest exosomes as non-hormonal messengers produced by pituitary tumors.
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Schageman, Jeoffrey, Emily Zeringer, Mu Li, Tim Barta, Kristi Lea, Jian Gu, Susan Magdaleno, Robert Setterquist, and Alexander V. Vlassov. "The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo." BioMed Research International 2013 (2013): 1–15. http://dx.doi.org/10.1155/2013/253957.
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Exosomes are small (30–150 nm) vesicles containing unique RNA and protein cargo, secreted by all cell types in culture. They are also found in abundance in body fluids including blood, saliva, and urine. At the moment, the mechanism of exosome formation, the makeup of the cargo, biological pathways, and resulting functions are incompletely understood. One of their most intriguing roles is intercellular communication—exosomes function as the messengers, delivering various effector or signaling macromolecules between specific cells. There is an exponentially growing need to dissect structure and the function of exosomes and utilize them for development of minimally invasive diagnostics and therapeutics. Critical to further our understanding of exosomes is the development of reagents, tools, and protocols for their isolation, characterization, and analysis of their RNA and protein contents. Here we describe a complete exosome workflow solution, starting from fast and efficient extraction of exosomes from cell culture media and serum to isolation of RNA followed by characterization of exosomal RNA content using qRT-PCR and next-generation sequencing techniques. Effectiveness of this workflow is exemplified by analysis of the RNA content of exosomes derived from HeLa cell culture media and human serum, using Ion Torrent PGM as a sequencing platform.
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Morton, Derrick J., Emily G. Kuiper, Stephanie K. Jones, Sara W. Leung, Anita H. Corbett, and Milo B. Fasken. "The RNA exosome and RNA exosome-linked disease." RNA 24, no. 2 (November 1, 2017): 127–42. http://dx.doi.org/10.1261/rna.064626.117.
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Zhang, Liying, Yichen Ju, Si Chen, and Linzhu Ren. "Recent Progress on Exosomes in RNA Virus Infection." Viruses 13, no. 2 (February 8, 2021): 256. http://dx.doi.org/10.3390/v13020256.
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Recent research indicates that most tissue and cell types can secrete and release membrane-enclosed small vesicles, known as exosomes, whose content reflects the physiological/pathological state of the cells from which they originate. These exosomes participate in the communication and cell-to-cell transfer of biologically active proteins, lipids, and nucleic acids. Studies of RNA viruses have demonstrated that exosomes release regulatory factors from infected cells and deliver other functional host genetic elements to neighboring cells, and these functions are involved in the infection process and modulate the cellular responses. This review provides an overview of the biogenesis, composition, and some of the most striking functions of exosome secretion and identifies physiological/pathological areas in need of further research. While initial indications suggest that exosome-mediated pathways operate in vivo, the exosome mechanisms involved in the related effects still need to be clarified. The current review focuses on the role of exosomes in RNA virus infections, with an emphasis on the potential contributions of exosomes to pathogenesis.
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Sinha, Dona, Sraddhya Roy, Priyanka Saha, Nabanita Chatterjee, and Anupam Bishayee. "Trends in Research on Exosomes in Cancer Progression and Anticancer Therapy." Cancers 13, no. 2 (January 17, 2021): 326. http://dx.doi.org/10.3390/cancers13020326.
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Exosomes, the endosome-derived bilayered extracellular nanovesicles with their contribution in many aspects of cancer biology, have become one of the prime foci of research. Exosomes derived from various cells carry cargoes similar to their originator cells and their mode of generation is different compared to other extracellular vesicles. This review has tried to cover all aspects of exosome biogenesis, including cargo, Rab-dependent and Rab-independent secretion of endosomes and exosomal internalization. The bioactive molecules of the tumor-derived exosomes, by virtue of their ubiquitous presence and small size, can migrate to distal parts and propagate oncogenic signaling and epigenetic regulation, modulate tumor microenvironment and facilitate immune escape, tumor progression and drug resistance responsible for cancer progression. Strategies improvised against tumor-derived exosomes include suppression of exosome uptake, modulation of exosomal cargo and removal of exosomes. Apart from the protumorigenic role, exosomal cargoes have been selectively manipulated for diagnosis, immune therapy, vaccine development, RNA therapy, stem cell therapy, drug delivery and reversal of chemoresistance against cancer. However, several challenges, including in-depth knowledge of exosome biogenesis and protein sorting, perfect and pure isolation of exosomes, large-scale production, better loading efficiency, and targeted delivery of exosomes, have to be confronted before the successful implementation of exosomes becomes possible for the diagnosis and therapy of cancer.
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Makino, Debora Lika, and Elena Conti. "Structure determination of an 11-subunit exosome in complex with RNA by molecular replacement." Acta Crystallographica Section D Biological Crystallography 69, no. 11 (October 12, 2013): 2226–35. http://dx.doi.org/10.1107/s0907444913011438.
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The RNA exosome is an evolutionarily conserved multi-protein complex involved in the 3′ degradation of a variety of RNA transcripts. In the nucleus, the exosome participates in the maturation of structured RNAs, in the surveillance of pre-mRNAs and in the decay of a variety of noncoding transcripts. In the cytoplasm, the exosome degrades mRNAs in constitutive and regulated turnover pathways. Several structures of subcomplexes of eukaryotic exosomes or related prokaryotic exosome-like complexes are known, but how the complete assembly is organized to fulfil processive RNA degradation has been unclear. An atomic snapshot of aSaccharomyces cerevisiae420 kDa exosome complex bound to an RNA substrate in the pre-cleavage state of a hydrolytic reaction has been determined. Here, the crystallographic steps towards the structural elucidation, which was carried out by molecular replacement, are presented.
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Hamdan, Yousra, Loubna Mazini, and Gabriel Malka. "Exosomes and Micro-RNAs in Aging Process." Biomedicines 9, no. 8 (August 6, 2021): 968. http://dx.doi.org/10.3390/biomedicines9080968.
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Exosomes are the main actors of intercellular communications and have gained great interest in the new cell-free regenerative medicine. These nanoparticles are secreted by almost all cell types and contain lipids, cytokines, growth factors, messenger RNA, and different non-coding RNA, especially micro-RNAs (mi-RNAs). Exosomes’ cargo is released in the neighboring microenvironment but is also expected to act on distant tissues or organs. Different biological processes such as cell development, growth and repair, senescence, migration, immunomodulation, and aging, among others, are mediated by exosomes and principally exosome-derived mi-RNAs. Moreover, their therapeutic potential has been proved and reinforced by their use as biomarkers for disease diagnostics and progression. Evidence has increasingly shown that exosome-derived mi-RNAs are key regulators of age-related diseases, and their involvement in longevity is becoming a promising issue. For instance, mi-RNAs such as mi-RNA-21, mi-RNA-29, and mi-RNA-34 modulate tissue functionality and regeneration by targeting different tissues and involving different pathways but might also interfere with long life expectancy. Human mi-RNAs profiling is effectively related to the biological fluids that are reported differently between young and old individuals. However, their underlying mechanisms modulating cell senescence and aging are still not fully understood, and little was reported on the involvement of mi-RNAs in cell or tissue longevity. In this review, we summarize exosome biogenesis and mi-RNA synthesis and loading mechanism into exosomes’ cargo. Additionally, we highlight the molecular mechanisms of exosomes and exosome-derived mi-RNA regulation in the different aging processes.
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Qiu, Jun-Jun, Xiao-Jing Lin, Ting-Ting Zheng, Xiao-Yan Tang, Ying Zhang, and Ke-Qin Hua. "The Exosomal Long Noncoding RNA aHIF is Upregulated in Serum From Patients With Endometriosis and Promotes Angiogenesis in Endometriosis." Reproductive Sciences 26, no. 12 (February 26, 2019): 1590–602. http://dx.doi.org/10.1177/1933719119831775.
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Objective: The transfer of long noncoding RNAs (lncRNAs) via exosomes to modulate recipient cells represents an important mechanism for disease progression. Antisense hypoxia-inducible factor (aHIF) is a well-known angiogenesis-related lncRNA. Here, we aimed to investigate the clinical implications of aHIF and exosomal aHIF in endometriosis and the involvement of exosome-shuttled aHIF in endometriosis angiogenesis. Study Design: The distribution and expression of aHIF in ectopic, eutopic, and normal endometria was evaluated. Serum exosomal aHIF levels in patients with endometriosis were tested. The correlation between serum exosomal aHIF and aHIF expression in ectopic endometria was analyzed. Endometriotic cyst stromal cells (ECSCs)-derived exosomes were characterized. The internalization of exosomes by human umbilical vein endothelial cells (HUVECs) was observed. A series of in vitro assays were conducted to investigate the roles and mechanisms of exosomal aHIF in endometriosis angiogenesis. Results: Clinically, aHIF was highly expressed in ectopic endometria and serum exosomes in patients with endometriosis. Serum exosomal aHIF was significantly correlated to aHIF expression in matched ectopic endometria. In vitro, PKH67-labeled exosomes derived from aHIF high expression ECSCs were effectively internalized by recipient HUVECs. Notably, exosome-shuttled aHIF was transferred from ECSCs to HUVECs, which in turn elicited proangiogenic behavior in HUVECs by activating vascular endothelial growth factor (VEGF)-A, VEGF-D, and basic fibroblast growth factor, thereby facilitating endometriosis angiogenesis. Conclusion: Our study illustrates a potential cell–cell communication between ECSCs and HUVECs in an ectopic environment, provides a novel mechanistic model explaining how ECSCs induce angiogenesis from the perspective of the “exosomal transfer of aHIF,” and highlights the clinical value of circulating exosomal aHIF in endometriosis.
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Toh, Wei Seong, Ruenn Chai Lai, Bin Zhang, and Sai Kiang Lim. "MSC exosome works through a protein-based mechanism of action." Biochemical Society Transactions 46, no. 4 (July 9, 2018): 843–53. http://dx.doi.org/10.1042/bst20180079.
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Mesenchymal stem cell (MSC) exosome specifically defines the 50–200 nm vesicles that are secreted into the extracellular space when multivesicular bodies in the MSC fuse with the plasma membrane. However, the exosome is just one of several 50–200 nm extracellular vesicles (EVs) known to be secreted by cells. Nevertheless, the term ‘MSC exosome’ is often used to describe populations of 50–200 nm EVs that are prepared from culture medium conditioned by MSCs on the basis that these populations collectively exhibited typical exosome-associated proteins such as endosomal proteins, TSG101 and Alix, and tetraspanin proteins, CD9, CD63 and CD81. They also carry a rich diverse RNA cargo. MSC exosomes are increasingly implicated as the mediator of many of the MSC-associated therapeutic potencies. They elicit therapeutic activity by delivering their cargo of potentially therapeutic proteins and RNAs to the recipient cells. The therapeutic potency of MSC exosomes is usually rationalized on the presence of a biologically relevant protein or RNA in the MSC exosome. In the present paper, we expanded this rationale beyond a physical presence to include biologically relevant concentration, biochemical functionality and the potential to elicit an appropriate timely biochemical response. Based on these, we propose that MSC exosomes most probably work through the protein rather than the RNA.
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Wu, Yixing, Hongmei Zeng, Qing Yu, Huatian Huang, Beatrice Fervers, Zhe-Sheng Chen, and Lingeng Lu. "A Circulating Exosome RNA Signature Is a Potential Diagnostic Marker for Pancreatic Cancer, a Systematic Study." Cancers 13, no. 11 (May 24, 2021): 2565. http://dx.doi.org/10.3390/cancers13112565.
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Several exosome proteins, miRNAs and KRAS mutations have been investigated in the hope of carrying out the early detection of pancreatic cancer with high sensitivity and specificity, but they have proven to be insufficient. Exosome RNAs, however, have not been extensively evaluated in the diagnosis of pancreatic cancer. The purpose of this study was to investigate the potential of circulating exosome RNAs in pancreatic cancer detection. By retrieving RNA-seq data from publicly accessed databases, differential expression and random-effects meta-analyses were performed. The results showed that pancreatic cancer had a distinct circulating exosome RNA signature in healthy individuals, and that the top 10 candidate exosome RNAs could distinguish patients from healthy individuals with an area under the curve (AUC) of 1.0. Three (HIST2H2AA3, LUZP6 and HLA-DRA) of the 10 genes in exosomes had similar differential patterns to those in tumor tissues based on RNA-seq data. In the validation dataset, the levels of these three genes in exosomes displayed good performance in distinguishing cancer from both chronic pancreatitis (AUC = 0.815) and healthy controls (AUC = 0.8558), whereas a slight difference existed between chronic pancreatitis and healthy controls (AUC = 0.586). Of the three genes, the level of HIST2H2AA3 was positively associated with KRAS status. However, there was no significant difference in the levels of the three genes across the disease stages (stages I–IV). These findings indicate that circulating exosome RNAs have a potential early detection value in pancreatic cancer, and that a distinct exosome RNA signature exists in distinguishing pancreatic cancer from healthy individuals.
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Salomon, Carlos, Dominic Guanzon, Katherin Scholz-Romero, Sherri Longo, Paula Correa, Sebastian E. Illanes, and Gregory E. Rice. "Placental Exosomes as Early Biomarker of Preeclampsia: Potential Role of Exosomal MicroRNAs Across Gestation." Journal of Clinical Endocrinology & Metabolism 102, no. 9 (May 22, 2017): 3182–94. http://dx.doi.org/10.1210/jc.2017-00672.
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AbstractContextThere is a need to develop strategies for early prediction of patients who will develop preeclampsia (PE) to establish preventive strategies to reduce the prevalence and severity of the disease and their associated complications.ObjectiveThe objective of this study was to investigate whether exosomes and their microRNA cargo present in maternal circulation can be used as early biomarker for PE.Design, Setting, Patients, and InterventionsA retrospective stratified study design was used to quantify total exosomes and placenta-derived exosomes present in maternal plasma of normal (n = 32 per time point) and PE (n = 15 per time point) pregnancies. Exosomes present in maternal circulation were determined by nanoparticle tracking analysis. An Illumina TruSeq® Small RNA Library Prep Kit was used to construct a small RNA library from exosomal RNA obtained from plasma samples.ResultsIn presymptomatic women, who subsequently developed PE, the concentration of total exosomes and placenta-derived exosomes in maternal plasma was significantly greater than those observed in controls, throughout pregnancy. The area under the receiver operating characteristic curves for total exosome and placenta-derived exosome concentrations were 0.745 ± 0.094 and 0.829 ± 0.077, respectively. In total, over 300 microRNAs were identified in exosomes across gestation, where hsa-miR-486-1-5p and hsa-miR-486-2-5p were identified as the candidate microRNAs.ConclusionsAlthough the role of exosomes during PE remains to be fully elucidated, we suggest that the concentration and content of exosomes may be of diagnostic utility for women at risk for developing PE.
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Mao, Li, Panhong Liang, Wenliang Li, Shaohua Zhang, Maojun Liu, Leilei Yang, Jizong Li, et al. "Exosomes promote caprine parainfluenza virus type 3 infection by inhibiting autophagy." Journal of General Virology 101, no. 7 (July 1, 2020): 717–34. http://dx.doi.org/10.1099/jgv.0.001424.
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Caprine parainfluenza virus type 3 (CPIV3) is a novel important pathogen causing respiratory disease in goats, but the pathogenic mechanism is not clear yet. Evidence suggests that exosomes transfer biologically active molecules between cells. Viral infections can cause profound changes in exosome components, and exosomes have been involved in viral transmission and pathogenicity. In this study, we explored the characteristics and functions of exosomes purified from the supernatant of Madin–Darby bovine kidney (MDBK) cells inoculated with CPIV3. Infection of CPIV3 showed increased exosome secretion and the loading of viral proteins and RNA into exosomes. These exosomes were capable of transferring CPIV3 genetic materials to recipient cells to establish a productive infection and promote the viral replication. To explore the potential mechanism, small RNA deep sequencing revealed that CPIV3 exosomes contained a diverse range of RNA species, including miRNA and piRNA, in proportions different from exosomes isolated from mock-infected cells. Expression patterns of 11 differentially expressed miRNAs were subsequently validated by quantitative reverse transcriptase PCR (qRT-PCR). Targets of miRNAs were predicted and functional annotation analysis showed that the main pathways involved were autophagy signalling ways. Autophagy inhibited by the CPIV3-exosome was further verified, and miR-126–3 p_2 packaged in the vesicles was an important regulation factor in this process. Inhibition of autophagy may be one of the responsible reasons for promoting efficient replication of exosome-mediated CPIV3 infection. The study suggests that exosomes are key in pathogenesis or protection against CPIV3. Further understating of their role in CPIV3 infection may bring novel insight to the development of protection measures.
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Manda, Sasidhar Venkata, Yogesh Kataria, Babul Reddy Tatireddy, Balasubramaniam Ramakrishnan, Boola Gnana Ratnam, Rahul Lath, Alok Ranjan, and Amitava Ray. "Exosomes as a biomarker platform for detecting epidermal growth factor receptor–positive high-grade gliomas." Journal of Neurosurgery 128, no. 4 (April 2018): 1091–101. http://dx.doi.org/10.3171/2016.11.jns161187.
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OBJECTIVEHigh-grade glial brain tumors are often characterized by an elevated expression of the tumorigenic epidermal growth factor receptor variant III (EGFRvIII). The authors sought to establish a clinically adaptive protocol as a noninvasive diagnostic tool for EGFRvIII detection through serum exosomes.METHODSPurity of serum exosome/RNA was confirmed by electron microscopy and flow cytometry and through an RNA bioanalyzer profile. EGFRvIII amplification was initially established by semiquantitative polymerase chain reaction in tumor tissues and exosomes. Diagnostic performance of EGFRvIII transcript in tissue versus exosome was determined using a 2 × 2 clinical table approach. Overall survival was determined using Kaplan-Meier analysis.RESULTSThe EGFRvIII transcript was detected in 39.5% of tumor tissue samples and in 44.7% of their paired serum exosome samples; 28.1% of biopsy tumors coexpressed wild-type EGFR and EGFRvIII. Tissue EGFRvIII amplification served as the reference-positive control for its paired serum expression. The overall clinical sensitivity and specificity of semiquantitative exosome EGFRvIII polymerase chain reaction detection assay in serum were 81.58% (95% CI 65.67%–92.26%) and 79.31% (95% CI 66.65%–88.83%), respectively. Age, sex, tumor location, and side of the body on which the tumor was located had no effect on the detection rate of exosomal EGFRvIII transcript. EGFRvIII expression either in exosomes or tissue correlated with poor survival.CONCLUSIONSThe authors established a serum-based method for detection of EGFRvIII in high-grade brain tumors that might serve as an optimal noninvasive method for diagnosing EGFRvIII-positive high-grade gliomas.
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Andreeva, Olga E., Yuri Y. Shchegolev, Alexander M. Scherbakov, Ekaterina I. Mikhaevich, Danila V. Sorokin, Margarita V. Gudkova, Irina V. Bure, et al. "Secretion of Mutant DNA and mRNA by the Exosomes of Breast Cancer Cells." Molecules 26, no. 9 (April 25, 2021): 2499. http://dx.doi.org/10.3390/molecules26092499.
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Exosomes are the small vesicles that are secreted by different types of normal and tumour cells and can incorporate and transfer their cargo to the recipient cells. The main goal of the present work was to study the tumour exosomes’ ability to accumulate the parent mutant DNA or RNA transcripts with their following transfer to the surrounding cells. The experiments were performed on the MCF7 breast cancer cells that are characterized by the unique coding mutation in the PIK3CA gene. Using two independent methods, Sanger sequencing and allele-specific real-time PCR, we revealed the presence of the fragments of the mutant DNA and RNA transcripts in the exosomes secreted by the MCF7 cells. Furthermore, we demonstrated the MCF7 exosomes’ ability to incorporate into the heterologous MDA-MB-231 breast cancer cells supporting the possible transferring of the exosomal cargo into the recipient cells. Sanger sequencing of the DNA from MDA-MB-231 cells (originally bearing a wild type of PIK3CA) treated with MCF7 exosomes showed no detectable amount of mutant DNA or RNA; however, using allele-specific real-time PCR, we revealed a minor signal from amplification of a mutant allele, showing a slight increase of mutant DNA in the exosome-treated MDA-MB-231 cells. The results demonstrate the exosome-mediated secretion of the fragments of mutant DNA and mRNA by the cancer cells and the exosomes’ ability to transfer their cargo into the heterologous cells.
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Roberts, Douglas, Emily Mitsock, Olubode Ogunlusi, Seth Yu, and Johan Skog. "Biomarker screening for the early detection of prostate cancer using an exosomal-enrichment RNA liquid biopsy test." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e15553-e15553. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e15553.
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e15553 Background: Prostate cancer is one of the most common cancers in men, with approximately 10% of all new cancer cases and ~5% of all cancer deaths in 2019. The standard test for prostate cancer is the Prostate Serum Antigen (PSA) test. The PSA test suffers from low specificity (20-40%) in patients including ‘grey zone’ levels (4-10 ng/mL); moreover, the PSA test fails to identify patients with high-risk cancers. Previously we developed ExoDx Prostate Intelliscore (EPI), a urine-based exosome prostate cancer test optimized to rule out the need for a biopsy (risk stratification for high-grade prostate cancer). This study utilized a next generation exosome-based test that specifically enriches a subtype of prostate cancer exosomes from urine. Early detection of prostate cancer via a non-invasive method is desirable and the identification of patients with high-risk cancer is critical. Here we describe the development of a prostate-specific urinary exosome test for the identification of patients with prostate cancer. Methods: We have developed a prostate-specific enrichment method to isolate exosomes of prostate origin from urine samples. Using an affinity-based method against surface marker proteins found on prostate cells, we were able to selectively enrich for exosomes shed by the prostate gland with demonstrated specificity. Subsequent analysis of exosomal nucleic acids enables a promising panel of gene expression biomarkers capable of distinguishing patients with prostate cancer from healthy individuals. Results: RNA from prostate cancer enriched exosomes was compared to total exosomes from urine. Enrichment of prostate cancer specific exosomes significantly enhanced the RNA signature compared to total urine exosomes. Conclusions: Prostate cancer tests have recently been developed for RNA signatures in urine. Exosomes provide a source of nucleic acids as they are actively shed continuously from living cells as part of their normal life cycle. The urine exosomes can be used for total RNA transcriptome analysis and are therefore a very rich source of biomarkers for prostate cancer that can be tailored to different clinical indications. An affinity-based enrichment for tissue-specific exosomes allow for better resolution of the gene expression profile from the tissue of interest and reduces the RNA targets from non-relevant processes of the bladder and kidneys. The gene signature identified in this ongoing study could potentially provide a non-invasive molecular means for the early diagnosis of prostate cancer.
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Shrivastava, Shubham, Pradip Devhare, Nanthiya Sujijantarat, Robert Steele, Young-Chan Kwon, Ranjit Ray, and Ratna B. Ray. "Knockdown of Autophagy Inhibits Infectious Hepatitis C Virus Release by the Exosomal Pathway." Journal of Virology 90, no. 3 (November 18, 2015): 1387–96. http://dx.doi.org/10.1128/jvi.02383-15.
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ABSTRACTHepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. We showed previously that HCV induces autophagy for viral persistence by preventing the innate immune response. Knockdown of autophagy reduces extracellular HCV release, although the precise mechanism remains unknown. In this study, we observed that knockdown of autophagy genes enhances intracellular HCV RNA and accumulates infectious virus particles in cells. Since HCV release is linked with the exosomal pathway, we examined whether autophagy proteins associate with exosomes in HCV-infected cells. We observed an association between HCV and the exosomal marker CD63 in autophagy knockdown cells. Subsequently, we observed that levels of extracellular infectious HCV were significantly lower in exosomes released from autophagy knockdown cells. To understand the mechanism for reduced extracellular infectious HCV in the exosome, we observed that an interferon (IFN)-stimulated BST-2 gene is upregulated in autophagy knockdown cells and associated with the exosome marker CD63, which may inhibit HCV assembly or release. Taken together, our results suggest a novel mechanism involving autophagy and exosome-mediated HCV release from infected hepatocytes.IMPORTANCEAutophagy plays an important role in HCV pathogenesis. Autophagy suppresses the innate immune response and promotes survival of virus-infected hepatocytes. The present study examined the role of autophagy in secretion of infectious HCV from hepatocytes. Autophagy promoted HCV trafficking from late endosomes to lysosomes, thus providing a link with the exosome. Inhibition of HCV-induced autophagy could be used as a strategy to block exosome-mediated virus transmission.
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Taller, Daniel, Katherine Richards, Zdenek Slouka, Satyajyoti Senapati, Reginald Hill, David B. Go, and Hsueh-Chia Chang. "On-chip surface acoustic wave lysis and ion-exchange nanomembrane detection of exosomal RNA for pancreatic cancer study and diagnosis." Lab on a Chip 15, no. 7 (2015): 1656–66. http://dx.doi.org/10.1039/c5lc00036j.
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Hartung, Sophia, and Karl-Peter Hopfner. "Lessons from structural and biochemical studies on the archaeal exosome." Biochemical Society Transactions 37, no. 1 (January 20, 2009): 83–87. http://dx.doi.org/10.1042/bst0370083.
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The RNA exosome is a multisubunit exonuclease involved in numerous RNA maturation and degradation processes. Exosomes are found in eukaryotes and archaea and are related to bacterial polynucleotide phosphorylates. Over the past years structural and biochemical analysis revealed that archaeal exosomes have a large processing chamber with three phosphorolytic active sites that degrade RNA in the 3′→5′ direction in a highly processive manner. A narrow entry pore, framed by putative RNA-binding domains, could account for the high processivity and also prevent degradation of structured RNA. The phosphorolytic nuclease activity is reversible, leading to formation of heteropolymeric tails from nucleoside diphosphates as substrate. This reversibility is difficult to regulate, suggesting why, during evolution and emergence of stable poly(A) tails in eukaryotes, polyadenylation and nuclease activities in the human exosome and associated factors have been separated.
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Januszyk, Kurt, and Christopher D. Lima. "The eukaryotic RNA exosome." Current Opinion in Structural Biology 24 (February 2014): 132–40. http://dx.doi.org/10.1016/j.sbi.2014.01.011.
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Puno, M. Rhyan, Eva-Maria Weick, Mom Das, and Christopher D. Lima. "SnapShot: The RNA Exosome." Cell 179, no. 1 (September 2019): 282–282. http://dx.doi.org/10.1016/j.cell.2019.09.005.
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Hornick, Noah, Jianya Huan, Natalya A. Goloviznina, Amiee Potter, and Peter Kurre. "Hypoxia Regulates Exosomal Microrna Content, Trafficking and Function Of Key Elements In The AML Microenvironment." Blood 122, no. 21 (November 15, 2013): 742. http://dx.doi.org/10.1182/blood.v122.21.742.742.
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Abstract Small, non-coding micro RNA (miRNA) are recognized for their potent regulatory capacity. Several recent studies indicate the prognostic value of miRNA profiling in acute myelogenous leukemia (AML), although a more mechanistic understanding of the role miRNA play in AML biology is still lacking. We recently demonstrated that patient-derived AML blasts release exosomes (nanometer-size, extracellular vesicles) that traffic a non-random subset of miRNA to stromal bystander cells, eliciting changes in transcriptional activity and growth factor secretion (Huan et al., Cancer Res. 2013). Here we hypothesized that exosome miRNA provide a candidate mechanism for the adaptation of the bone marrow to a specialized leukemic niche. As oxygen levels in the bone marrow are substantially lower than those commonly used in tissue culture, we undertook a systematic study of miRNA incorporation and exosome trafficking in AML under physiological oxygen conditions. In carefully calibrated tissue culture conditions we initially observed an up to 7-fold net increase in exosome number released by Molm14 (Flt3-ITD+ AML cell line) leukemia cells at 1% O2versus 21% O2. Nanoparticle tracking analysis and RNA bioanalyzer tracings suggested that the decreased O2 did not alter vesicle composition, average RNA amount per exosome, or global RNA profiles. Further emphasizing the critical nature of appropriate compartmental oxygenation in exosome trafficking, both murine and human stromal cells demonstrated increased uptake of Molm14 exosomes under hypoxia. Low-oxygen conditions alter transcriptional profiles, phenotypic behavior and drug resistance in AML. Therefore, we next evaluated the miRNA expression of leukemic cells and their incorporation in exosomes at 1% versus 21% O2, utilizing the Affymetrix microarray platform containing >5,000 human (hsa) miRNA probesets, followed by select qRT-PCR validation. Array experiments showed broad differences between cellular and exosomal miRNA and revealed that certain miRNA were selectively regulated in an oxygen-responsive manner. For example, hematopoiesis relevant hsa-miR-124, -146a, and -155 increased an average of 4.6-, 5.5-, and 4.9-fold, respectively, in exosomes from hypoxia-conditioned cells. Intriguingly, several known, non-AML specific, hypoxia-responsive miRNA substantially increased in cells cultured at 1% O2 (e.g. miR-210 by 33-fold), but changed less than 2-fold in exosomes. Several recent reports show that leukemia cells actively convert the bone marrow microenvironment and contribute to the erosion of hematopoiesis by modulating hematopoietic-stromal interactions, in part via decrease in SDF1a, SCF, and Angpt1. We investigated the ability of AML-derived exosomes to regulate these transcripts, and found a 50% decrease in SCF and over 90% decrease in Angpt1 in murine stromal cells after in vitro exposure to leukemia exosomes, again with relatively greater differences for exosomes from hypoxia-conditioned AML cells. These experiments were complemented by observations of altered clonogenicity (CFU-C) of murine lin-negative hematopoietic cells after AML exosome exposure, whereby hypoxia conditioning prompted a decline in colony count to 46% from vesicle-free media baseline, compared with 31% decrease at 21 % O2. Exosomes equilibrate across biological fluids and can be recovered from serum. To translate our observations to an in vivo setting, we developed a xenograft model using Molm14 cells in immune-deficient NSG mice. Early after grafting animals, exosomes could be reproducibly isolated from as little as 20 microL serum and candidate miRNA (hsa-miR-146, -150, 155, 210) were amplified, allowing us to quantitatively track leukemia progression via a unique miRNA signature even before circulating leukemia cells were detectable in the peripheral blood. A comparison of leukemic animals to NSG controls bearing cord blood MNC grafts revealed that changes in circulating miRNA were disease specific and resembled those in the hypoxia setting in vitro. In sum, our work demonstrates that physiologic oxygen levels not only increase AML exosome trafficking between cells, but selectively alter the miRNA profile contained therein. These changes produce phenotypic alterations in stromal and hematopoietic bystander cells that correlate with the functional conversion of the bone marrow to a leukemic niche. Disclosures: No relevant conflicts of interest to declare.
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Haderk, Franziska, Laura Llao Cid, Murat Iskar, Ralph Schulz, Maria Goebel, Jan Duerig, Stephan Stilgenbauer, Marc Zapatka, Peter Lichter, and Martina Seiffert. "CLL Exosome-Derived Y RNA hY4 Induces TLR7/8-Mediated Inflammation and PD-L1 Expression in Monocytes." Blood 128, no. 22 (December 2, 2016): 3217. http://dx.doi.org/10.1182/blood.v128.22.3217.3217.
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Abstract Chronic lymphocytic leukemia (CLL) is a B-cell malignancy associated with an inflammatory milieu and impaired anti-tumor immunity. Both in human CLL samples and the Eµ-TCL1 mouse model of CLL, monocytes and macrophages were identified as key players in the involved processes, as they secrete immune regulatory cytokines and show enhanced expression of the immune checkpoint protein PD-L1 which is known to be involved in T-cell suppression. We recently showed that reactivation of T-cell activity using anti-PD-L1 antibodies controls leukemia development in mice and is associated with a normalization of CLL-associated immune defects. The current study aimed at unraveling the molecular mechanisms of CLL-induced changes in monocytes and macrophages and focused on CLL-derived exosomes and their role in the tumor microenvironment. Exosomes were isolated from blood plasma of CLL patients as well as culture supernatant of the CLL cell line MEC-1 by a serial centrifugation protocol. Characterization of isolated exosomes by electron microscopy, Nanoparticle Tracking Analysis (NTA), and Western blot analysis revealed vesicles, 30 to 350 nm in size, which were positive for various exosome marker proteins. Quantification of exosomes by NTA and ELISA indicated an enrichment of B-cell derived exosomes in plasma of CLL patients compared to healthy controls, although absolute exosome counts were not different. RNA sequencing and proteome analysis of CLL exosomes revealed a disease-specific composition and identified non-coding Y RNA hY4 as the most abundant exosomal RNA species. Transfer of CLL exosomes or hY4 RNA alone to monocytes triggered release of cytokines like CCL2, CCL3, CCL4, and IL-6, and increased expression of PD-L1. As these are key features associated with CLL, a novel role for exosomal Y RNAs in the tumor microenvironment of CLL is suggested. Of interest, exosome or hY4-induced responses in monocytes were significantly reduced by chloroquine treatment and completely abolished in TLR7-deficient monocytes. Therefore, exosomal hY4 was identified as novel ligand that activates Toll-like receptor 7/8 signaling pathway in monocytes. Tumor-derived exosomes as well as exosomal Y RNAs were detected in a number of malignancies, suggesting their more general contribution to cancer-related sterile inflammation and the formation of a tumor-supportive myeloid microenvironment. Ongoing studies will show whether these novel findings can be exploited in treatment approaches for CLL and other malignancies. Disclosures Stilgenbauer: Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding.
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Tollervey, David. "RNA surveillance and the exosome." RNA 21, no. 4 (March 16, 2015): 492–93. http://dx.doi.org/10.1261/rna.050989.115.
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Augimeri, Giuseppina, Giusi La Camera, Luca Gelsomino, Cinzia Giordano, Salvatore Panza, Diego Sisci, Catia Morelli, et al. "Evidence for Enhanced Exosome Production in Aromatase Inhibitor-Resistant Breast Cancer Cells." International Journal of Molecular Sciences 21, no. 16 (August 14, 2020): 5841. http://dx.doi.org/10.3390/ijms21165841.
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Aromatase inhibitors (AIs) represent the standard anti-hormonal therapy for post-menopausal estrogen receptor-positive breast cancer, but their efficacy is limited by the emergence of AI resistance (AIR). Exosomes act as vehicles to engender cancer progression and drug resistance. The goal of this work was to study exosome contribution in AIR mechanisms, using estrogen-dependent MCF-7 breast cancer cells as models and MCF-7 LTED (Long-Term Estrogen Deprived) subline, modeling AIR. We found that exosome secretion was significantly increased in MCF-7 LTED cells compared to MCF-7 cells. MCF-7 LTED cells also exhibited a higher amount of exosomal RNA and proteins than MCF-7 cells. Proteomic analysis revealed significant alterations in the cellular proteome. Indeed, we showed an enrichment of proteins frequently identified in exosomes in MCF-7 LTED cells. The most up-regulated proteins in MCF-7 LTED cells were represented by Rab GTPases, important vesicle transport-regulators in cancer, that are significantly mapped in “small GTPase-mediated signal transduction”, “protein transport” and “vesicle-mediated transport” Gene Ontology categories. Expression of selected Rab GTPases was validated by immunoblotting. Collectively, we evidence, for the first time, that AIR breast cancer cells display an increased capability to release exosomes, which may be associated with an enhanced Rab GTPase expression. These data provide the rationale for further studies directed at clarifying exosome’s role on endocrine therapy, with the aim to offer relevant markers and druggable therapeutic targets for the management of hormone-resistant breast cancers.
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Conigliaro, Alice, and Carla Cicchini. "Exosome-Mediated Signaling in Epithelial to Mesenchymal Transition and Tumor Progression." Journal of Clinical Medicine 8, no. 1 (December 27, 2018): 26. http://dx.doi.org/10.3390/jcm8010026.
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Growing evidence points to exosomes as key mediators of cell–cell communication, by transferring their specific cargo (e.g., proteins, lipids, DNA and RNA molecules) from producing to receiving cells. In cancer, the regulation of the exosome-mediated intercellular communication may be reshaped, inducing relevant changes in gene expression of recipient cells in addition to microenvironment alterations. Notably, exosomes may deliver signals able to induce the transdifferentiation process known as Epithelial-to-Mesenchymal Transition (EMT). In this review, we summarize recent findings on the role of exosomes in tumor progression and EMT, highlighting current knowledge on exosome-mediated intercellular communication in tumor-niche establishment, migration, invasion, and metastasis processes. This body of evidence suggests the relevance of taking into account exosome-mediated signaling and its multifaceted aspects to develop innovative anti-tumoral therapeutic approaches.
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Sterrett, Maria C., Liz Enyenihi, Sara W. Leung, Laurie Hess, Sarah E. Strassler, Daniela Farchi, Richard S. Lee, et al. "A budding yeast model for human disease mutations in the EXOSC2 cap subunit of the RNA exosome complex." RNA 27, no. 9 (June 23, 2021): 1046–67. http://dx.doi.org/10.1261/rna.078618.120.
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RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4. The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies.
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Li, Jian, Ting Fan, Yang Li, Yinyin Cao, Pengfei Guan, Lingjie Wu, Yunfeng Wang, and Jin Xu. "Effect and mechanism of miRNA on obstructive sleep apnea in children." Materials Express 10, no. 3 (March 1, 2020): 404–11. http://dx.doi.org/10.1166/mex.2020.1646.
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Exosomal microRNAs (miRNAs) have attracted increasing interest as biomarkers for the diagnosis of numerous human diseases; however, little is known about exosomal miRNAs in regard to pediatric obstructive sleep apnea syndrome (OSAS). The aim of this study was to identify exosomal miRNAs involved in OSAS and to determine their relative functions. Serum exosomal miRNA-expression patterns in pediatric OSAS patients and healthy donors were analyzed comprehensively via RNA sequencing, and differently expressed miRNAs were verified using quantitative reverse transcription polymerase chain reaction. The effect of the miRNAs on cell culture was measured by flow cytometry. Results revealed that 364 and 464 miRNAs were identified in normal and OSAS exosomes, respectively. Moreover, exosomal miR-664a-3p, miR-210, miR-21-3p, and miR-107 were significantly more downregulated in exosomes from OSAS than in those of healthy controls. These downregulated miRNAs were mainly involved in functions involving cell cycle, immune response, and cell proliferation and adhesion as well as pathways associated with mitogen-activated protein kinase, Wnt, and mammalian target rapamycin signaling. Furthermore, miR-107 promoted the arrest of the adenoid lymphocyte cycle at the G2/M phase. These data revealed several potential exosomal miRNA biomarkers and their associated functions and pathways. This study advanced the knowledge of OSAS exosome biology to facilitate the development of OSAS-specific exosome-based diagnostics and therapeutics.
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Zhou, Fang, Henry A. Paz, Mahrou Sadri, Juan Cui, Stephen D. Kachman, Samodha C. Fernando, and Janos Zempleni. "Dietary bovine milk exosomes elicit changes in bacterial communities in C57BL/6 mice." American Journal of Physiology-Gastrointestinal and Liver Physiology 317, no. 5 (November 1, 2019): G618—G624. http://dx.doi.org/10.1152/ajpgi.00160.2019.
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Exosomes and exosome-like vesicles participate in cell-to-cell communication in animals, plant, and bacteria. Dietary exosomes in bovine milk are bioavailable in nonbovine species, but a fraction of milk exosomes reaches the large intestine. We hypothesized that milk exosomes alter the composition of the gut microbiome in mice. C57BL/6 mice were fed AIN-93G diets, defined by their content of bovine milk exosomes and RNA cargos: exosome/RNA-depleted (ERD) versus exosome/RNA-sufficient (ERS) diets. Feeding was initiated at age 3 wk, and cecum content was collected at ages 7, 15, and 47 wk. Microbial communities were identified by 16S rRNA gene sequencing. Milk exosomes altered bacterial communities in the murine cecum. The abundance of three phyla, seven families, and 52 operational taxonomic units (OTUs) was different in the ceca from mice fed ERD and ERS ( P < 0.05). For example, at the phylum level, Tenericutes had more than threefold abundance in ERS mice at ages 15 and 47 wk compared with ERD mice ( P < 0.05). At the family level, Verrucomicrobiaceae were much less abundant in ERS mice compared with ERD mice age 47 wk ( P < 0.05). At the OTU level, four OTUs from the family of Lachnospiraceae were more than two times more abundant in ERS mice compared with ERD at age 7 and 47 wk ( P < 0.05). We conclude that exosomes in bovine milk alter microbial communities in nonbovine species, suggesting that exosomes and their cargos participate in the crosstalk between bacterial and animal kingdoms. NEW & NOTEWORTHY This is the first report that exosomes from bovine milk alter microbial communities in mice. This report suggests that the gut microbiome facilitates cell-to-cell communication by milk exosomes across species boundaries, and milk exosomes facilitate communication across animal and bacteria kingdoms.
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Pregnolato, Francesca, Lidia Cova, Alberto Doretti, Donatella Bardelli, Vincenzo Silani, and Patrizia Bossolasco. "Exosome microRNAs in Amyotrophic Lateral Sclerosis: A Pilot Study." Biomolecules 11, no. 8 (August 16, 2021): 1220. http://dx.doi.org/10.3390/biom11081220.
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The pathogenesis of amyotrophic lateral sclerosis (ALS), a lethal neurodegenerative disease, remains undisclosed. Mutations in ALS related genes have been identified, albeit the majority of cases are unmutated. Clinical pathology of ALS suggests a prion-like cell-to-cell diffusion of the disease possibly mediated by exosomes, small endocytic vesicles involved in the propagation of RNA molecules and proteins. In this pilot study, we focused on exosomal microRNAs (miRNAs), key regulators of many signaling pathways. We analyzed serum-derived exosomes from ALS patients in comparison with healthy donors. Exosomes were obtained by a commercial kit. Purification of miRNAs was performed using spin column chromatography and RNA was reverse transcribed into cDNA. All samples were run on the miRCURY LNATM Universal RT miRNA PCR Serum/Plasma Focus panel. An average of 29 miRNAs were detectable per sample. The supervised analysis did not identify any statistically significant difference among the groups indicating that none of the miRNA of our panel has a strong pathological role in ALS. However, selecting samples with the highest miRNA content, six biological processes shared across miRNAs through the intersection of the GO categories were identified. Our results, combined to those reported in the literature, indicated that further investigation is needed to elucidate the role of exosome-derived miRNA in ALS.
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Gonzalez-Villasana, Vianey, Mohammed H. Rashed, Yessica Gonzalez-Cantú, Recep Bayraktar, Jorge Luis Menchaca-Arredondo, Jose Manuel Vazquez-Guillen, Cristina Rodriguez-Padilla, Gabriel Lopez-Berestein, and Diana Resendez-Perez. "Presence of Circulating miR-145, miR-155, and miR-382 in Exosomes Isolated from Serum of Breast Cancer Patients and Healthy Donors." Disease Markers 2019 (February 12, 2019): 1–9. http://dx.doi.org/10.1155/2019/6852917.
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miR-145, miR-155, and miR-382 have been proposed as noninvasive biomarkers to distinguish breast cancer patients from healthy individuals. However, it is unknown if these three miRNAs are secreted by exosomes. Thus, we hypothesized that miR-145, miR-155, and miR-382 in breast cancer patients are present in exosomes. We isolated exosomes from serum of breast cancer patients and healthy donors, then we characterized them according to their shape, size, and exosome markers by scanning electron microscopy, atomic force microscopy, nanoparticle tracking analysis (NTA), and Western blot and determined the exosome concentration in all samples by NTA. Later, exosomal small RNA extraction was done to determine the expression levels of miR-145, miR-155, and miR-382 by qRT-PCR. We observed a round shape of exosomes with a mean size of 119.84 nm in breast cancer patients and 115.4 nm in healthy donors. All exosomes present the proteins CD63, Alix, Tsg, CD9, and CD81 commonly used as markers. Moreover, we found a significantly high concentration of exosomes in breast cancer patients with stages I, III, and IV compared to healthy donors. We detected miR-145, miR-155, and miR-382 in the exosomes isolated from serum of breast cancer patients and healthy donors. Our results show that the exosomes isolated from the serum of breast cancer patients and healthy donors contains miR-145, miR-155, and miR-382 but not in a selective manner in breast cancer patients. Moreover, our data support the association between exosome concentration and the presence of breast cancer, opening the possibility to study how miRNAs packaged into exosomes play a role in BC progression.
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Damanti, Carlotta C., Enrico Gaffo, Federica Lovisa, Anna Garbin, Piero Di Battista, Ilaria Gallingani, Anna Tosato, et al. "MiR-26a-5p as a Reference to Normalize MicroRNA qRT-PCR Levels in Plasma Exosomes of Pediatric Hematological Malignancies." Cells 10, no. 1 (January 8, 2021): 101. http://dx.doi.org/10.3390/cells10010101.
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Plasma exosomal microRNAs (miRNAs) are considered as valid circulating biomarkers for cancer diagnosis and prognosis. Quantitative real-time polymerase chain reaction (qRT-PCR), the most commonly used technique to assess circulating miRNA levels, requires a normalization step involving uniformly expressed endogenous miRNAs. However, there is still no consensus on reference miRNAs for plasma exosomal miRNA abundance normalization. In this study, we identified a panel of miRNAs with stable abundance by analyzing public plasma exosome RNA-seq data and selected miR-486-5p, miR-26a-5p, miR-423-5p and miR191-5p as candidate normalizers. Next, we tested the abundance variation of these miRNAs by qRT-PCR in plasma exosomes of healthy donors and pediatric patients with anaplastic large cell lymphoma, Burkitt lymphoma, Hodgkin lymphoma and mature B-cell acute lymphoblastic leukemia. MiR-486-5p and miR-26a-5p showed the most stable levels, both between healthy controls and patients and among the malignancies analyzed. In light of previous reports on miRNA stability in different exosome isolation methods, our data indicated that miR-26a-5p is a bona fide reference miRNA for qRT-PCR normalization to evaluate miRNA abundance from circulating plasma exosomes in studies of hematological malignancies.
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Damanti, Carlotta C., Enrico Gaffo, Federica Lovisa, Anna Garbin, Piero Di Battista, Ilaria Gallingani, Anna Tosato, et al. "MiR-26a-5p as a Reference to Normalize MicroRNA qRT-PCR Levels in Plasma Exosomes of Pediatric Hematological Malignancies." Cells 10, no. 1 (January 8, 2021): 101. http://dx.doi.org/10.3390/cells10010101.
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Plasma exosomal microRNAs (miRNAs) are considered as valid circulating biomarkers for cancer diagnosis and prognosis. Quantitative real-time polymerase chain reaction (qRT-PCR), the most commonly used technique to assess circulating miRNA levels, requires a normalization step involving uniformly expressed endogenous miRNAs. However, there is still no consensus on reference miRNAs for plasma exosomal miRNA abundance normalization. In this study, we identified a panel of miRNAs with stable abundance by analyzing public plasma exosome RNA-seq data and selected miR-486-5p, miR-26a-5p, miR-423-5p and miR191-5p as candidate normalizers. Next, we tested the abundance variation of these miRNAs by qRT-PCR in plasma exosomes of healthy donors and pediatric patients with anaplastic large cell lymphoma, Burkitt lymphoma, Hodgkin lymphoma and mature B-cell acute lymphoblastic leukemia. MiR-486-5p and miR-26a-5p showed the most stable levels, both between healthy controls and patients and among the malignancies analyzed. In light of previous reports on miRNA stability in different exosome isolation methods, our data indicated that miR-26a-5p is a bona fide reference miRNA for qRT-PCR normalization to evaluate miRNA abundance from circulating plasma exosomes in studies of hematological malignancies.
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Gartz, Melanie, Chien-Wei Lin, Mark A. Sussman, Michael W. Lawlor, and Jennifer L. Strande. "Duchenne muscular dystrophy (DMD) cardiomyocyte-secreted exosomes promote the pathogenesis of DMD-associated cardiomyopathy." Disease Models & Mechanisms 13, no. 11 (November 1, 2020): dmm045559. http://dx.doi.org/10.1242/dmm.045559.
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ABSTRACTCardiomyopathy is a leading cause of early mortality in Duchenne muscular dystrophy (DMD). There is a need to gain a better understanding of the molecular pathogenesis for the development effective therapies. Exosomes (exo) are secreted vesicles and exert effects via their RNA, lipid and protein cargo. The role of exosomes in disease pathology is unknown. Exosomes derived from stem cells have demonstrated cardioprotection in the murine DMD heart. However, it is unknown how the disease status of the donor cell type influences exosome function. Here, we sought to determine the phenotypic responses of DMD cardiomyocytes (DMD-iCMs) after long-term exposure to DMD cardiac exosomes (DMD-exo). DMD-iCMs were vulnerable to stress, evidenced by production of reactive oxygen species, the mitochondrial membrane potential and cell death levels. Long-term exposure to non-affected exosomes (N-exo) was protective. By contrast, long-term exposure to DMD-exo was not protective, and the response to stress improved with inhibition of DMD-exo secretion in vitro and in vivo. The microRNA (miR) cargo, but not exosome surface peptides, was implicated in the pathological effects of DMD-exo. Exosomal surface profiling revealed N-exo peptides associated with PI3K-Akt signaling. Transcriptomic profiling identified unique changes with exposure to either N- or DMD-exo. Furthermore, DMD-exo miR cargo regulated injurious pathways, including p53 and TGF-beta. The findings reveal changes in exosomal cargo between healthy and diseased states, resulting in adverse outcomes. Here, DMD-exo contained miR changes, which promoted the vulnerability of DMD-iCMs to stress. Identification of these molecular changes in exosome cargo and effectual phenotypes might shed new light on processes underlying DMD cardiomyopathy.This article has an associated First Person interview with the first author of the paper.
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Crouser, Elliott D., Mark W. Julian, Sabahattin Bicer, Vikas Ghai, Taek-Kyun Kim, Lisa A. Maier, May Gillespie, Nabeel Y. Hamzeh, and Kai Wang. "Circulating exosomal microRNA expression patterns distinguish cardiac sarcoidosis from myocardial ischemia." PLOS ONE 16, no. 1 (January 26, 2021): e0246083. http://dx.doi.org/10.1371/journal.pone.0246083.
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ObjectiveCardiac sarcoidosis is difficult to diagnose, often requiring expensive and inconvenient advanced imaging techniques. Circulating exosomes contain genetic material, such as microRNA (miRNA), that are derived from diseased tissues and may serve as potential disease-specific biomarkers. We thus sought to determine whether circulating exosome-derived miRNA expression patterns would distinguish cardiac sarcoidosis (CS) from acute myocardial infarction (AMI).MethodsPlasma and serum samples conforming to CS, AMI or disease-free controls were procured from the Biologic Specimen and Data Repository Information Coordinating Center repository and National Jewish Health. Next generation sequencing (NGS) was performed on exosome-derived total RNA (n = 10 for each group), and miRNA expression levels were compared after normalization using housekeeping miRNA. Quality assurance measures excluded poor quality RNA samples. Differentially expressed (DE) miRNA patterns, based upon >2-fold change (p< 0.01), were established in CS compared to controls, and in CS compared to AMI. Relative expression of several DE-miRNA were validated by qRT-PCR.ResultsDespite the advanced age of the stored samples (~5–30 years), the quality of the exosome-derived miRNA was intact in ~88% of samples. Comparing plasma exosomal miRNA in CS versus controls, NGS yielded 18 DE transcripts (12 up-regulated, 6 down-regulated), including miRNA previously implicated in mechanisms of myocardial injury (miR-92, miR-21) and immune responses (miR-618, miR-27a). NGS further yielded 52 DE miRNA in serum exosomes from CS versus AMI: 5 up-regulated in CS; 47 up-regulated in AMI, including transcripts previously detected in AMI patients (miR-1-1, miR-133a, miR-208b, miR-423, miR-499). Five miRNAs with increased DE in CS included two isoforms of miR-624 and miR-144, previously reported as markers of cardiomyopathy.ConclusionsMiRNA patterns of exosomes derived from CS and AMI patients are distinct, suggesting that circulating exosomal miRNA patterns could serve as disease biomarkers. Further studies are required to establish their specificity relative to other cardiac disorders.
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Wu, Mengxi, Yingshi Ouyang, Zeyu Wang, Rui Zhang, Po-Hsun Huang, Chuyi Chen, Hui Li, et al. "Isolation of exosomes from whole blood by integrating acoustics and microfluidics." Proceedings of the National Academy of Sciences 114, no. 40 (September 18, 2017): 10584–89. http://dx.doi.org/10.1073/pnas.1709210114.
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Exosomes are nanoscale extracellular vesicles that play an important role in many biological processes, including intercellular communications, antigen presentation, and the transport of proteins, RNA, and other molecules. Recently there has been significant interest in exosome-related fundamental research, seeking new exosome-based biomarkers for health monitoring and disease diagnoses. Here, we report a separation method based on acoustofluidics (i.e., the integration of acoustics and microfluidics) to isolate exosomes directly from whole blood in a label-free and contact-free manner. This acoustofluidic platform consists of two modules: a microscale cell-removal module that first removes larger blood components, followed by extracellular vesicle subgroup separation in the exosome-isolation module. In the cell-removal module, we demonstrate the isolation of 110-nm particles from a mixture of micro- and nanosized particles with a yield greater than 99%. In the exosome-isolation module, we isolate exosomes from an extracellular vesicle mixture with a purity of 98.4%. Integrating the two acoustofluidic modules onto a single chip, we isolated exosomes from whole blood with a blood cell removal rate of over 99.999%. With its ability to perform rapid, biocompatible, label-free, contact-free, and continuous-flow exosome isolation, the integrated acoustofluidic device offers a unique approach to investigate the role of exosomes in the onset and progression of human diseases with potential applications in health monitoring, medical diagnosis, targeted drug delivery, and personalized medicine.
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Zhao, Guiping, Anni Zhou, Xiao Li, Shengtao Zhu, Yongjun Wang, Shutian Zhang, and Peng Li. "The Significance of Exosomal RNAs in the Development, Diagnosis, and Treatment of Gastric Cancer." Genes 12, no. 1 (January 8, 2021): 73. http://dx.doi.org/10.3390/genes12010073.
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Gastric cancer (GC) is one of the most common malignancies in the world. Exosomes, a subset of extracellular vesicles with an average diameter of 100 nm, contain and transfer a variety of functional macromolecules such as proteins, lipids, and nucleic acids. A large number of studies indicated that exosomes can play a significant role in the initiation and development of GC via facilitating intercellular communication between gastric cancer cells and microenvironment. Exosomal RNAs, one of the key functional cargos, are involved in the pathogenesis, development, and metastasis of GC. In addition, recent studies elucidated that exosomal RNAs may serve as diagnostic and prognostic biomarkers or therapeutic targets for GC. In this review, we summarized the function of exosomal RNA in the tumorigenesis, progression, diagnosis, and treatment of GC, which may further unveil the functions of exosome and promote the potentially diagnostic and therapeutic application of exosomes in GC.
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Zhao, Guiping, Anni Zhou, Xiao Li, Shengtao Zhu, Yongjun Wang, Shutian Zhang, and Peng Li. "The Significance of Exosomal RNAs in the Development, Diagnosis, and Treatment of Gastric Cancer." Genes 12, no. 1 (January 8, 2021): 73. http://dx.doi.org/10.3390/genes12010073.
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Gastric cancer (GC) is one of the most common malignancies in the world. Exosomes, a subset of extracellular vesicles with an average diameter of 100 nm, contain and transfer a variety of functional macromolecules such as proteins, lipids, and nucleic acids. A large number of studies indicated that exosomes can play a significant role in the initiation and development of GC via facilitating intercellular communication between gastric cancer cells and microenvironment. Exosomal RNAs, one of the key functional cargos, are involved in the pathogenesis, development, and metastasis of GC. In addition, recent studies elucidated that exosomal RNAs may serve as diagnostic and prognostic biomarkers or therapeutic targets for GC. In this review, we summarized the function of exosomal RNA in the tumorigenesis, progression, diagnosis, and treatment of GC, which may further unveil the functions of exosome and promote the potentially diagnostic and therapeutic application of exosomes in GC.
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Crenshaw, Brennetta J., Linlin Gu, Brian Sims, and Qiana L. Matthews. "Exosome Biogenesis and Biological Function in Response to Viral Infections." Open Virology Journal 12, no. 1 (September 28, 2018): 134–48. http://dx.doi.org/10.2174/1874357901812010134.
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Introduction: Exosomes are extracellular vesicles that originate as intraluminal vesicles during the process of multivescular body formation. Exosomes mediate intercellular transfer of functional proteins, lipids, and RNAs. The investigation into the formation and role of exosomes in viral infections is still being elucidated. Exosomes and several viruses share similar structural and molecular characteristics. Explanation: It has been documented that viral hijacking exploits the exosomal pathway and mimics cellular protein trafficking. Exosomes released from virus-infected cells contain a variety of viral and host cellular factors that are able to modify recipient host cell responses. Recent studies have demonstrated that exosomes are crucial components in the pathogenesis of virus infection. Exosomes also allow the host to produce effective immunity against pathogens by activating antiviral mechanisms and transporting antiviral factors between adjacent cells. Conclusion: Given the ever-growing roles and importance of exosomes in both host and pathogen response, this review will address the impact role of exosome biogenesis and composition after DNA, RNA virus, on Retrovirus infections. This review also will also address how exosomes can be used as therapeutic agents as well as a vaccine vehicles.
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Han, Jeong-Sun, Sung Eun Kim, Jun-Qing Jin, Na Ri Park, Ji-Young Lee, Hong Lim Kim, Seong-Beom Lee, Suk-Woo Yang, and Dong-Jun Lim. "Tear-Derived Exosome Proteins Are Increased in Patients with Thyroid Eye Disease." International Journal of Molecular Sciences 22, no. 3 (January 23, 2021): 1115. http://dx.doi.org/10.3390/ijms22031115.
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Exosomes contain proteins, lipids, RNA, and DNA that mediate intercellular signaling. Exosomes can contribute to the pathological processes of various diseases, although their roles in ocular diseases are unclear. We aimed to isolate exosomes from tear fluids (TF) of patients with Thyroid eye disease (TED) and analyze the exosomal proteins. TFs were collected from eight patients with TED and eight control subjects. The number of TF exosomes were measured using nanoparticle-tracking analysis. The expression of specific proteins in the purified exosome pellets were analyzed using a Proteome Profiler Array Kit. Cultured normal orbital fibroblasts were incubated with TF exosomes from patients with TED and control subjects, and changes in inflammatory cytokine levels were compared. TF exosomes from TED patients showed more exosomes than the control subjects. The expression levels of exosomal proteins vitamin D-binding (VDB) protein, C-reactive protein (CRP), chitinase 3-like 1 (CHI3L1), matrix metalloproteinase-9 (MMP-9), and vascular adhesion molecule-1 (VCAM-1) were significantly increased in patients with TED, compared to those of controls. Orbital fibroblasts exposed to TF exosomes from patients with TED showed significantly higher levels of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) production than those treated with control TF exosomes. Specific proteins showed higher expression in exosomes from TED patients, implying that they may play keys roles in TED pathogenesis.
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Heusermann, Wolf, Justin Hean, Dominic Trojer, Emmanuelle Steib, Stefan von Bueren, Alexandra Graff-Meyer, Christel Genoud, et al. "Exosomes surf on filopodia to enter cells at endocytic hot spots, traffic within endosomes, and are targeted to the ER." Journal of Cell Biology 213, no. 2 (April 25, 2016): 173–84. http://dx.doi.org/10.1083/jcb.201506084.
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Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery.
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Niederberger, Theresa, Sophia Hartung, Karl-Peter Hopfner, and Achim Tresch. "Processive RNA decay by the exosome." RNA Biology 8, no. 1 (January 2011): 55–60. http://dx.doi.org/10.4161/rna.8.1.14067.
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Chlebowski, Aleksander, Michał Lubas, Torben Heick Jensen, and Andrzej Dziembowski. "RNA decay machines: The exosome." Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1829, no. 6-7 (June 2013): 552–60. http://dx.doi.org/10.1016/j.bbagrm.2013.01.006.
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Zhang, Duo, Heedoo Lee, Ziwen Zhu, Jasleen K. Minhas, and Yang Jin. "Enrichment of selective miRNAs in exosomes and delivery of exosomal miRNAs in vitro and in vivo." American Journal of Physiology-Lung Cellular and Molecular Physiology 312, no. 1 (January 1, 2017): L110—L121. http://dx.doi.org/10.1152/ajplung.00423.2016.
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Exosomes are nanovesicles secreted by cells and contain various molecules including protein, lipid, and DNA/RNA. They are crucial mediators of the intercellular communication and serve as promising vehicles for drug delivery and gene therapy. Recently, accumulating evidence suggests that microRNAs (miRNAs) may serve as new and potentially powerful targets for therapeutic interventions against various human diseases. However, steadily and effectively delivering miRNA mimics or inhibitors to target cells remains a major obstacle. To enhance the efficacy of exosome-mediated delivery of miRNA molecules, it is crucial to develop a convenient and efficient method to enrich specific miRNAs or antisense oligos in isolated exosomes. Here we report a novel method to prepare specific miRNA molecule-loaded exosomes. Using a modified calcium chloride-mediated transfection method, we successfully enhanced the designated miRNA mimics or inhibitors in isolated exosomes directly, instead of transfecting their mother cells. We also compared this method with direct transfection of exosomes using electroporation. Both methods confirmed that exosomes can serve as cargos to deliver a robustly increased amount of selected miRNA mimic(s) or inhibitor(s) to the recipient cells. Delivery of these miRNA molecule enriched-exosomes subsequently results in highly efficient overexpression or deletion of the designated miRNAs in the recipient cells both in vivo and in vitro. Additionally, we confirmed that exosome-delivered miRNA mimics or inhibitors are functional in the recipient cells. Collectively, we developed a novel protocol to conveniently manipulate exosomal miRNAs with high efficiency and successfully deliver the exosomal miRNA molecules to recipient cells.
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Wang, Zhuo, Zhong Deng, Nadia Dahmane, Kevin Tsai, Pu Wang, Dewight R. Williams, Andrew V. Kossenkov, et al. "Telomeric repeat-containing RNA (TERRA) constitutes a nucleoprotein component of extracellular inflammatory exosomes." Proceedings of the National Academy of Sciences 112, no. 46 (November 2, 2015): E6293—E6300. http://dx.doi.org/10.1073/pnas.1505962112.
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Telomeric repeat-containing RNA (TERRA) has been identified as a telomere-associated regulator of chromosome end protection. Here, we report that TERRA can also be found in extracellular fractions that stimulate innate immune signaling. We identified extracellular forms of TERRA in mouse tumor and embryonic brain tissue, as well as in human tissue culture cell lines using RNA in situ hybridization. RNA-seq analyses revealed TERRA to be among the most highly represented transcripts in extracellular fractions derived from both normal and cancer patient blood plasma. Cell-free TERRA (cfTERRA) could be isolated from the exosome fractions derived from human lymphoblastoid cell line (LCL) culture media. cfTERRA is a shorter form (∼200 nt) of cellular TERRA and copurifies with CD63- and CD83-positive exosome vesicles that could be visualized by cyro-electron microscopy. These fractions were also enriched for histone proteins that physically associate with TERRA in extracellular ChIP assays. Incubation of cfTERRA-containing exosomes with peripheral blood mononuclear cells stimulated transcription of several inflammatory cytokine genes, including TNFα, IL6, and C-X-C chemokine 10 (CXCL10) Exosomes engineered with elevated TERRA or liposomes with synthetic TERRA further stimulated inflammatory cytokines, suggesting that exosome-associated TERRA augments innate immune signaling. These findings imply a previously unidentified extrinsic function for TERRA and a mechanism of communication between telomeres and innate immune signals in tissue and tumor microenvironments.
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Huang, Haobo, Jinfeng Zhu, Liping Fan, Qiuyan Lin, Danhui Fu, Biyu Wei, and Shijin Wei. "MicroRNA Profiling of Exosomes Derived from Red Blood Cell Units: Implications in Transfusion-Related Immunomodulation." BioMed Research International 2019 (June 13, 2019): 1–10. http://dx.doi.org/10.1155/2019/2045915.
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Purpose. To elucidate the microRNAs existent in exosomes derived from stored red blood cell (RBC) unit and their potential function. Materials and Methods. Exosomes were isolated from the supernatant derived from stored RBC units by sequential centrifugation. Isolated exosomes were characterized by TEM (transmission electron microscopy), western blotting, and DLS (dynamic light scattering). MicroRNA (miRNA) microarray was performed to detect the expression of miRNAs in 3 exosome samples. Results revealed miRNAs that were simultaneously expressed in the 3 exosome samples and were previously reported to exist in mature RBCs. Functions and potential pathways of some detected miRNAs were illustrated by bioinformatic analysis. Validation of the top 3 abundant miRNAs was carried out by qRT-PCR (quantitative reverse transcription‐polymerase chain reaction). Results. TEM and DLS revealed the mean size of the exosomes (RBC-derived) as 64.08 nm. These exosomes exhibited higher abundance of short RNA than the long RNA. 78 miRNAs were simultaneously detected in 3 exosome samples and mature RBCs. Several biological processes might be impacted by these miRNAs, through their target gene(s) enriched in a particular signalling pathway. The top 3 (abundant) miRNAs detected were as follows: miR-125b-5p, miR-4454, and miR-451a. qRT-PCR revealed higher abundance of miR-451a than others. Only miR-4454 and miR-451a abundance tended to increase with increasing storage time. Conclusion. Exosomes derived from stored RBC units possessed multiple miRNAs and, hence, could serve various functions. The function of exosomes (RBC-derived) might be implemented partly by the predominantly enriched miR-451a.
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Milligan, Laura, Laurence Decourty, Cosmin Saveanu, Juri Rappsilber, Hugo Ceulemans, Alain Jacquier, and David Tollervey. "A Yeast Exosome Cofactor, Mpp6, Functions in RNA Surveillance and in the Degradation of Noncoding RNA Transcripts." Molecular and Cellular Biology 28, no. 17 (June 30, 2008): 5446–57. http://dx.doi.org/10.1128/mcb.00463-08.
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ABSTRACT A genome-wide screen for synthetic lethal (SL) interactions with loss of the nuclear exosome cofactors Rrp47/Lrp1 or Air1 identified 3′→5′ exonucleases, the THO complex required for mRNP assembly, and Ynr024w (Mpp6). SL interactions with mpp6Δ were confirmed for rrp47Δ and nuclear exosome component Rrp6. The results of bioinformatic analyses revealed homology between Mpp6 and a human exosome cofactor, underlining the high conservation of the RNA surveillance system. Mpp6 is an RNA binding protein that physically associates with the exosome and was localized throughout the nucleus. The results of functional analyses demonstrated roles for Mpp6 in the surveillance of both pre-rRNA and pre-mRNAs and in the degradation of “cryptic” noncoding RNAs (ncRNAs) derived from intergenic regions and the ribosomal DNA spacer heterochromatin. Strikingly, these ncRNAs are also targeted by other exosome cofactors, including Rrp47, the TRAMP complex (which includes Air1), and the Nrd1/Nab3 complex, and are degraded by both Rrp6 and the core exosome. Heterochromatic transcripts and other ncRNAs are characterized by very rapid degradation, and we predict that functional redundancy is an important feature of ncRNA metabolism.