Exosomal miRNA Expression Profile Research Articles

Abstract 571: Atherogenic Macrophages Transfer microRNA-containing Exosomes to Naïve Cells and Promote Macrophage Migration

Atherosclerosis, a maladaptive inflammatory disease, is driven by the interplay between excess cholesterol accumulation in the vessel wall and the immune system. Macrophages are fundamental to the propagation of atherosclerosis due to their capacity to engulf modified lipoproteins and induce a pro-inflammatory state. Macrophages and other immune cells can secrete miRNAs in exosomes as a form of extracellular communication during inflammation and infection. miRNAs are small noncoding RNAs that post-transcriptionally regulate gene expression by degradation or translational inhibition of target mRNAs and can repress gene expression in recipient cells when transferred via exosomes or other vesicles. Here, we hypothesize that atherogenic macrophages secrete miRNAs in exosomes to mediate cell-cell communication, which regulates the atherogenic response. Results: We examined the miRNA expression profile of exosomes from control and cholesterol-loaded (oxLDL-treated) mouse macrophages. Among the 88 differentially expressed miRNAs, 68 were down-regulated whereas 20 were up-regulated in exosomes isolated from cholesterol-loaded macrophages relative to control. The enrichment of several miRNAs such as miR-146a, miR-128, miR-185 and miR-503 in exosomes from atherogenic macrophages was confirmed by qPCR. Bioinformatic pathway analysis suggests that atherogenic exosomal miRNAs regulate cell migration and adhesion pathways via targeting the 3’UTR of migration/adhesion genes (i.e. integrin α3, VE-cadherin, CXCR4). Interestingly, live cell imaging demonstrated that fluorescent-labeled exosomes secreted from control or oxLDL-loaded macrophages were internalized by naïve macrophages and exogenous miRNA (from C.elegans) can transfer from normal/atherogenic macrophage exosomes to naïve cells. Further, naïve macrophages treated with extracellular components from atherogenic but not control macrophages promoted the migration of naïve cells towards a chemokine stimulus (1.5-fold increase in migration relative to control). Thus, miRNAs secreted from atherogenic macrophages may promote atherosclerosis by regulating macrophage migration in the vessel wall and we are now testing the function of these exo-miRNAs in vitro and in vivo.

Role of Extracellular MicroRNAs in Atherosclerosis

Atherosclerosis is a maladaptive inflammatory disease driven by the interplay between excess cholesterol accumulation in the arterial wall and the immune system. Macrophages are fundamental to the propagation of atherosclerosis due to their capacity to engulf modified lipoproteins and induce a pro‐inflammatory state. Macrophages can secrete miRNAs in exosomes as a form of intercellular communication during inflammation and infection. miRNAs are small noncoding RNAs that post‐transcriptionally regulate gene expression. Herein, we hypothesize that atherogenic macrophages secrete miRNAs in exosomes to mediate cell‐cell communication, which regulates the atherogenic response. To test this hypothesis, we first examined the miRNA expression profile of exosomes from control and atherogenic macrophages. Among the 88 differentially expressed miRNAs, 68 were down‐regulated whereas 20 were up‐regulated in exosomes from cholesterol‐loaded macrophages as compared with the control. qPCR confirmed the enrichment of several miRNAs such as miR‐146a, miR‐128, miR‐185, and miR‐503 in exosomes derived from atherogenic macrophages, whereas miR‐150 was downregulated in these exosomes. Bioinformatic pathway analysis suggests that atherogenic exosomal miRNAs may regulate cell migration and adhesion pathways via targeting the 3′UTR of migration/adhesion genes (i.e. integrin α3, VE‐cadherin, CXCR4). Interestingly, live cell imaging showed that exosomes secreted from cholesterol‐loaded macrophages were internalized by naïve macrophages and exogenous C. elegans miR‐39 could transfer from atherogenic macrophages to naïve cells. Thus, miRNAs secreted from atherogenic macrophages may promote atherosclerosis by regulating macrophage migration in the vessel wall and the role of these exo‐miRNAs will be tested in vitro and in vivo.

Vps4A functions as a tumor suppressor by regulating the secretion and uptake of exosomal microRNAs in human hepatoma cells.

The deregulation of microRNAs (miRNAs) plays an important role in human hepatocarcinogenesis. In this study, we highlight exosomes as mediators involved in modulating miRNA profiles in hepatocellular carcinoma (HCC) cells. First, we examined the different miRNA expression profiles in HCC cells and HCC cell-derived exosomes. Next, coculture experiments indicated that HCC cell-derived exosomes promoted the cell growth, migration, and invasion of HCC cells and had the ability to shuttle miRNAs to recipient cells. Further, our data showed that Vps4A, a key regulator of exosome biogenesis, was frequently down-regulated in HCC tissues. The reduction of Vps4A in HCC tissues was associated with tumor progression and metastasis. In vitro studies revealed that Vps4A repressed the growth, colony formation, migration, and invasion of HCC cells. We further investigated the role and involvement of Vps4A in suppressing the bioactivity of exosomes and characterized its ability to weaken the cell response to exosomes. By small RNA sequencing, we demonstrated that Vps4A facilitated the secretion of oncogenic miRNAs in exosomes as well as accumulation and uptake of tumor suppressor miRNAs in cells. A subset of Vps4A-associated miRNAs was identified. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that the phosphatidylinositol-3-kinase/Akt signaling pathway was the most likely candidate pathway for modulation by these miRNAs. Indeed, we proved that the phosphatidylinositol-3-kinase/Akt pathway was inactivated by Vps4A overexpression. Exosome-mediated miRNA transfer is an important mechanism of self-modulation of the miRNA expression profiles in HCC cells, and Vps4A may function as a tumor suppressor, which utilizes exosomes as mediators to regulate the secretion and uptake of miRNAs in hepatoma cells; these observations provide new insights into the development of HCC.

Exosomal MicroRNA Clusters Are Important for the Therapeutic Effect of Cardiac Progenitor Cells

Although current evidence indicates that cardiac progenitor cells (CPCs) may improve cardiac repair after ischemic injury, the exact mechanism for this cardioprotective effect to date remains undefined. In this issue of Circulation Research , Gray et al1 report on the use of systems biology to link the change in exosomal microRNAs (miRNA) to the beneficial effects of CPCs during ischemic injury in mice. Expression analysis showed that exosomes from hypoxic CPCs contain a higher level of a subset of miRNAs than exosomes coming from normoxic CPCs. To connect the relevance of the change in miRNA levels to the biological effect of exosomes, the authors performed a principle component analysis of the miRNA expression profiles in exosomes, which identified 4 unique miRNA clusters, which after further mathematical modeling seemed to correlate to a biological effect related to the therapeutic benefit. Instead of studying the biological function of a single miRNA, computational modeling can be applied to integrate multiple variants to gain more insight into the overall relationship and interaction between the different datasets. Gray et al1 show that especially for miRNA biology, where a miRNA can target up to hundreds of genes in a parallel, systems biology could turn out to be a suitable approach for predicting a biological outcome or therapeutic benefit. Article, see p 255 Cells communicate with each other via direct cell–cell contact and secretion of soluble factors. Soluble factors that are gaining an increasing amount of attention are extracellular membrane vesicles. The ability of vesicles to transport different molecules, such as proteins, peptides, mRNA, and microRNAs, from one cell to the other and their general body-wide distribution make them attractive candidates for horizontal information transfer between cells.2 Although the release of vesicles to influence other cells seems to be straightforward, multiple mechanisms, size distributions, …

MicroRNA expression profile in exosome discriminates extremely severe infections from mild infections for hand, foot and mouth disease

BackgroundChanges of miRNAs in exosome have been reported in different disease diagnosis and provided as potential biomarkers. In this study, we compared microRNA profile in exosomes in 5 MHFMD and 5 ESHFMD as well as in 5 healthy children.MethodsDifferent expression of miRNAs in exosomes across all the three groups were screened using miRNA microarray method. Further validated test was conducted through quantitative real-time PCR assays with 54 exosome samples (18 ESHFMD, 18 MHFMD, and 18 healthy control). The judgment accuracy was then estimated by the receiver operating characteristic (ROC) curve analysis; and the specificity and sensitivity were evaluated by the multiple logistic regression analysis.ResultsThere were 11 different miRNAs in exosomes of MHFMD and ESHFMD compared to healthy children, of which 4 were up-regulated and 7 were down-regulated. Further validation indicated that the 4 significant differentially expressed candidate miRNAs (miR-671-5p, miR-16-5p, miR-150-3p, and miR-4281) in exosome showed the same changes as in the microarray analysis, and the expression level of three miRNAs (miR-671-5p, miR-16-5p, and miR-150-3p) were significantly different between MHFMD or ESHFMD and the healthy controls. The accuracy of the test results were high with the under curve (AUC) value range from 0.79 to 1.00. They also provided a specificity of 72%-100% and a sensitivity of 78%-100%, which possessed ability to discriminate ESHFMD from MHFMD with the AUC value of 0.76-0.82.ConclusionsThis study indicated that the exosomal miRNA from patients with different condition of HFMD express unique miRNA profiles. Exosomal miRNA expression profiles may provide supplemental biomarkers for diagnosing and subtyping HFMD infections.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2334-14-506) contains supplementary material, which is available to authorized users.

Expression profiling of exosomal miRNAs derived from human esophageal cancer cells by Solexa high-throughput sequencing.

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

Exosome-derived miRNAs and ovarian carcinoma progression.

The objective of this study was to analyze the expression, biological role and clinical relevance of exosomal microRNAs (miRNAs) from ovarian carcinoma (OC) effusion supernatants. Exosomal miRNA expression profiling was performed using miRNA Taqman arrays. Selected miRNAs were validated using quantitative PCR in 86 OC effusion supernatants. The role of exosomal miRNA in this cancer was further studied using in vitro and in vivo models. miRNA profiling identified 99 miRNAs with high expression levels in exosomes from OC effusion supernatants. Quantitative PCR validation of 11 miRNAs showed significant associations with effusion site (peritoneum versus pleura) and International Federation of Gynecology and Obstetrics stage. In univariate survival analysis, high levels of miRNAs 21, 23b and 29a were associated with poor progression-free survival (P = 0.01, P = 0.015 and P = 0.009, respectively), whereas high expression of miRNA 21 correlated with poor overall survival (P = 0.017). The latter association was retained in Cox multivariate analysis (P = 0.001). Exposure of LP9 mesothelial cells and ES2 OC cells to OC effusion-derived exosomes inhibited tumor spheroid expansion and reduced mesothelial clearance area. Treatment of severe combined immunodeficiency mice with exosomes from OC effusions prior to injection of tumor cells was associated with larger tumor load, more infiltrative tumors and shorter survival. Patient-derived OC effusion exosomes contain multiple miRNAs, of which some may have clinical relevance. In experimental models, OC exosomes affect both tumor cells and cells in the tumor microenvironment and induce more aggressive disease. Collectively, these data demonstrate the central role of miRNAs and their content in the biology of this cancer.

Abstract 3085: Expression profiling of exosomal miRNAs derived from human esophageal cancer cells by Solexa high-through sequencing.

Abstract Background: Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment on fusion of multivesicular bodies (MVB) with the plasma membrane. Studies showed that inside exosomes, cellular gene products including proteins, mRNAs, and microRNAs (miRNAs) are packaged and these molecules can be transferred to recipient cells to deliver their molecular signaling. Objective: To explore the miRNA expression profile and distribution characteristics of exosomes derived from human esophageal cancer cells by Solexa high-throughput sequencing. Methods: Exosomes were extracted from cell culture supernatants by ultracentrifugation. Exosomes were identified by transmission electronic microscopy and western blot assay with anti-CD63 antibody. The types and abundance of known and novel miRNAs in cells and exosomes were identified by Solexa high-throughput sequencing. Results:There were 56421(2.94%) unique sequences in cells and 7727(0.63%) in exosomes matched the known miRNAs(Table 1). 64 in cells and 32 in exosomes novel miRNAs were predicted. Further analysis revealed extensive sequence variations (isomiRs) of cell and exosome miRNAs. The known miRNAs showed an increased expression in cells compared with exosomes. The expression level of hsa-miR-chr15_4885 and hsa-miR-chr18_7463 in exosomes were significantly higher than cells, with an average fold-change of 10 while hsa-miR-chr7_20613 and hsa-miR-chr19_8205 in cells were significantly higher than exosomes, with an average fold-change of thousands. Conclusion: The miRNA expression profile in exosomes is different from that in cells. It suggested that the significantly enriched miRNAs in exosomes may be the result of an active uptaken by exosomes. These specific miRNAs expressed in exosomes may play an important role in intercellular communication. Table 1. Distribution of sequence counts for the different classes of small RNAs in cells and exosomes Classification Unique RNA in cells Percentage (%) in cells Unique RNA in exosomes Percentage (%) in exosomes miRNA 56,421 2.94 7,727 0.63 mRNA 59,655 3.11 5,988 0.49 Repbase 31,421 1.64 5,066 0.41 rRNA 260,307 13.56 85,303 6.95 tRNA 80,635 4.20 119,500 9.74 snRNA 27,935 1.45 1,823 0.15 Unknown RNA 1,403,576 73.10 1,001,499 81.63 Total 1,919,950 100.00 1,226,905 100.00 Citation Format: Juan Liao, Ran Liu, Lihong Yin. Expression profiling of exosomal miRNAs derived from human esophageal cancer cells by Solexa high-through sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3085. doi:10.1158/1538-7445.AM2013-3085 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 5279: Expression of exosomal microRNAs in malignant ascites, peritoneal lavage fluid and cell culture supernatant of gastric cancer.

Abstract Background. A microRNA (miRNA) is non-coding RNA molecules of 21-25nt. miRNAs are post-transcriptional regulators that bind to messenger RNA transcripts, resulting in translational repression of target degradation and gene silencing. miRNAs are wrapped in exosome and secreted stably in body fluid, which can prevent RNase from degrading the miRNAs. Therefore several methods for miRNA-based early cancer detection using serum, plasma, and urine are reported. However there is no report to detect peritoneal dissemination of gastric cancer using malignant ascitis. Aim. To detect predictive factors for peritoneal dissemination of gastric cancer, global analysis of exosomal miRNAs in gastric malignant ascites, intraoperative peritoneal lavage fluid and cell culture supernatant were performed. Materials and Methods. A total of 6 gastric malignant ascites, 20 peritoneal lavage fluids and 2 human gastric carcinoma cell culture supernatants (OCUM-2M and its sub line with high potential for metastasis to the peritoneal cavity named OCUM-2MD3) were included in this study. The amount of 10 ml samples underwent a final ultracentrifugation at 120,000g twice to pellet the exosomes. miRNA was extracted and analyzed using Bioanalyzer. Exosomal miRNA expression profiling was performed using the Agilent Human miRNA Microarrays. To quantitatively detect mature miRNAs, TaqMan MicroRNA Assays was used. Statistical tests by spearman's rank-correlation coefficient were performed to investigate correlation of exosomal miRNAs that were expressed between malignant ascites and cell culture supernatant. Results. miRNAs were detected in each gastric malignant ascites at concentrations ranging from 61.7 ng to 220 ng, however no or very low amounts of ribosomal RNA (18S and 28S). Microarray analysis detected 327 miRNAs in malignant ascites, 389 in OCUM-2M and 414 in OCUM-2MD3. miRNA expression of Malignant ascites correlated that of cell culture supernatants; OCUM-2M(r=0.774, p<0.01) and OCUM-2MD3(r=0.776, r<0.001). Total of 140 miRNAs in OCUM-2MD3 showed a higher expression level (>2-fold change) than in OCUM-2M. Among these 140 miRNAs, 7 miRNAs detected at high expression in malignant ascites were selected for confirmation by qRT-PCR. All 7 miRNA was detectable both malignant ascites and intraoperative peritoneal lavage fluids by qRT-PCR. Conclusions. Exosomal miRNAs were highly detectable in malignant ascites and peritoneal lavage fluids. In this study 7 miRNAs related to peritoneal dissemination were identified. This result may provide a new diagnostic approach of peritoneal dissemination. Citation Format: Motohiko Tokuhisa, Yasushi Ichikawa, Takashi Kosaka, Satoshi Hasegawa, Hirotoshi Akiyama, Takashi Ishikawa, Nobuyoshi Kosaka, Takahiro Ochiya, Masakazu Yashiro, Kousei Hirakawa, Chikara Kunisaki, Ayumu Goto, Noritoshi Kobayashi, Itaru Endo. Expression of exosomal microRNAs in malignant ascites, peritoneal lavage fluid and cell culture supernatant of gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5279. doi:10.1158/1538-7445.AM2013-5279 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Lactation-Related MicroRNA Expression Profiles of Porcine Breast Milk Exosomes

Breast milk is the primary source of nutrition for newborns, and is rich in immunological components. MicroRNAs (miRNAs) are present in various body fluids and are selectively packaged inside the exosomes, a type of membrane vesicles, secreted by most cell types. These exosomal miRNAs could be actively delivered into recipient cells, and could regulate target gene expression and recipient cell function. Here, we analyzed the lactation-related miRNA expression profiles in porcine milk exosomes across the entire lactation period (newborn to 28 days after birth) by a deep sequencing. We found that immune-related miRNAs are present and enriched in breast milk exosomes (p<10−16, χ 2 test) and are generally resistant to relatively harsh conditions. Notably, these exosomal miRNAs are present in higher numbers in the colostrums than in mature milk. It was higher in the serum of colostrum-only fed piglets compared with the mature milk-only fed piglets. These immune-related miRNA-loaded exosomes in breast milk may be transferred into the infant body via the digestive tract. These observations are a prelude to in-depth investigations of the essential roles of breast milk in the development of the infant’s immune system.