Exosomal miRNA Expression Research Articles

Exosomal miRNAs as Potential Biomarkers to Monitor Phosphodiesterase 5 Inhibitor Induced Anti-Fibrotic Effects on CCl4 Treated Rats.

MicroRNAs (miRNAs) are short, non-coding RNA species that are important post-transcriptional regulators of gene expression and play an important role in the pathogenesis of non-alcoholic fatty liver disease. Here, we investigated the phosphodiesterase 5 (PDE5) inhibitor induced effects on hepatic and plasma exosomal miRNA expression in CCl4-treated rats. In the present study, hepatic miRNA profiling was conducted using the Nanostring nCounter technology and mRNA profiling using RNA sequencing from PDE5 treated rats in the model of CCl4-induced liver fibrosis. To evaluate if the PDE5 inhibitor affected differentially expressed miRNAs in the liver can be detected in plasma exosomes, qRT-PCR specific assays were used. In livers from CCl4-treated rats, the expression of 22 miRNAs was significantly increased (>1.5-fold, adj. p < 0.05), whereas the expression of 16 miRNAs was significantly decreased (>1.5-fold, adj. p < 0.05). The majority of the deregulated miRNA species are implicated in fibrotic and inflammatory processes. The PDE5 inhibitor suppressed the induction of pro-fibrotic miRNAs, such as miR-99b miR-100 and miR-199a-5p, and restored levels of anti-fibrotic miR-122 and miR-192 in the liver. In plasma exosomes, we observed elevated levels of miR-99b, miR-100 and miR-142-3p after treatment with the PDE5-inhibitor compared to CCl4/Vehicle-treated. Our study demonstrated for the first time that during the development of hepatic fibrosis in the preclinical model of CCl4-induced liver fibrosis, defined aspects of miRNA regulated liver pathogenesis are influenced by PDE5 treatment. In conclusion, miRNA profiling of plasma exosomes might be used as a biomarker for NASH progression and monitoring of treatment effects.

Tumor-Derived Exosomal miR-620 as a Diagnostic Biomarker in Non-Small-Cell Lung Cancer.

Background Evidence has suggested the functional role of exosomal miRNAs in cancer diagnosis. This study aimed to determine whether the serum exosomal biomarkers can improve the diagnosis of patients with non-small-cell lung cancer (NSCLC). Materials and Methods The exosomes were extracted from the serum of NSCLC patients (n = 235) and healthy donors (n = 231) using ultracentrifugation and then were evaluated by using transmission electron microscopy, qNano, and western blotting. The serum exosomal miRNA expression was validated using qPCR. Results Exosomal miR-620 was significantly reduced in NSCLC and early-stage NSCLC patients (P < 0.0001) when compared to that of healthy controls, with an area under the curve (AUC) of 0.728 and 0.707, respectively. Exosomal miR-620 expression showed an association with drinking (P=0.008) and distant metastasis (P=0.037). Additionally, the downregulated exosomal miR-620 showed association with chemotherapeutic effect (P=0.044). Conclusion These findings suggest the serum exosomal miR-620 as a promising diagnostic and prognostic noninvasive biomarker in NSCLC patients.

Comprehensive analysis of liver and blood miRNA in precancerous conditions

Streptozotocin administration to mice (STZ-mice) induces type I diabetes and hepatocellular carcinoma (HCC). We attempted to elucidate the carcinogenic mechanism and the miRNA expression status in the liver and blood during the precancerous state. Serum and liver tissues were collected from STZ-mice and non-treated mice (CTL-mice) at 6, 10, and 12 W. The exosome enriched fraction extracted from serum was used. Hepatic histological examination and hepatic and exosomal miRNA expression analysis were serially performed using next-generation sequencing (NGS). Human miRNA expression analysis of chronic hepatitis liver tissue and exosomes, which were collected before starting the antiviral treatment, were also performed. No inflammation or fibrosis was found in the liver of CTL-mice during the observation period. In STZ-mice, regeneration and inflammation of hepatocytes was found at 6 W and nodules of atypical hepatocytes were found at 10 and 12 W. In the liver tissue, during 6–12 W, the expression levels of let-7f-5p, miR-143-3p, 148a-3p, 191-5p, 192-5p, 21a-5p, 22-3p, 26a-5p, and 92a-3p was significantly increased in STZ-mice, and anti-oncogenes of their target gene candidates were down-regulated. miR-122-5p was also significantly down-regulated in STZ-mice. Fifteen exosomal miRNAs were upregulated in STZ-mice. Six miRNAs (let-7f-5p, miR-10b-5p, 143-3p, 191-5p, 21a-5p, and 26a-5p) were upregulated, similarly to human HCC cases. From the precancerous state, aberrant expression of hepatic miRNAs has already occurred, and then, it can promote carcinogenesis. In exosomes, the expression pattern of common miRNAs between mice and humans before carcinogenesis was observed and can be expected to be developed as a cancer predictive marker.

Association of exosomal microRNAs in human ovarian follicular fluid with oocyte quality

The postponement of childbearing by women has led to an increase in infertility. The reproductive aging process leads to a decrease in both the quantity and quality of oocytes. The aim of this study was to investigate exosomal microRNAs in human ovarian follicular fluid and explore their potential association with oocyte quality. We collected ovarian follicle fluid from 68 patients and assigned the patients to A (superior oocyte quality) or B (poor oocyte quality) group according to their oocyte quality. Exosomal miRNAs were extracted, library constructed and sequenced using the Illumina HiSeq platform. Subsequently, we analyzed exosomal miRNA expression, predicted the miRNA target genes, and enriched Gene Ontology terms using GOSeq. Kyoto Encyclopedia of Genes and Genomes pathway analysis was performed using miRanda. A total of 47 miRNAs were found to be significantly differentially expressed between group A and group B (p < 0.05). Among nine differentially expressed miRNAs that were previously known, seven were upregulated in group B. In silico analysis indicated that several of these exosomal miRNAs were involved in pathways implicated in oocyte quality. Analysis of the expression of exosomal miRNAs in human ovarian follicular fluid showed that they were critical for maintaining oocyte quality. Our findings provide the basis for further investigations of the functions of exosomal miRNAs in the ovarian microenvironment and suggest that these exosomal miRNAs may be potential biomarkers for evaluating oocyte quality. The findings are potentially important to maintain oocyte quality in clinical settings.

Human Periodontal Ligament Stem Cell-Derived Exosomes Promote Bone Regeneration by Altering MicroRNA Profiles.

The role and underlying mechanism of exosomes derived from human periodontal ligament stem cells (PDLSC) in osteogenesis are unclear. In the present study, we identified the exosomes derived from PDLSCs and found that osteogenic induction can enhance the osteogenic ability of PDLSC-derived exosomes in promoting the osteogenic differentiation of rat bone marrow stem cells (BMSCs). To investigate the underlying mechanism, we analyzed the exosomal miRNA expression profiles of undifferentiated and osteogenic differentiated PDLSCs by RNA sequencing. The results showed that seventy-two miRNAs were upregulated and thirty-five miRNAs were downregulated after osteogenic induction. The results of Gene Ontology analysis and pathway analysis demonstrated that the target genes of differentially expressed exosomal miRNAs participate in the regulation of a variety of biological processes, such as catalytic activity, protein binding, metabolic processes, cell development, and differentiation, and are enriched in osteogenic differentiation-related pathways, such as MAPK signaling, AMPK signaling, and insulin signaling pathways. Our results reveal for the first time that the exosomal miRNAs derived from osteogenic differentiated PDLSCs may promote the osteogenic differentiation of BMSCs, which provides a basis for further research on the regulatory function of exosomal miRNA of PDLSCs during osteogenesis.

Abstract 15839: Patients With Abdominal Aortic Aneurysms Have Reduced Levels of MicroRNA 122-5p in Circulating Exosomes

Introduction: There are currently no specific biomarkers to screen and monitor disease progression in abdominal aortic aneurysms (AAA), which are the 15 th leading cause of death in the United States. Circulating exosomes contain microRNAs (miRNA) that are potential biomarkers of disease. This study aimed to characterize the exosomal miRNA expression profile of patients with AAA in order to identify novel markers of disease. Methods: A total of 109 patients scheduled to undergo a duplex ultrasound (US) for screening or surveillance of AAA were screened to participate in the study. Eleven patients with AAA (max aortic diameter &gt;3cm) and 15 non-aneurysmal controls (max aortic diameter &lt;3cm on screening US) were enrolled. Circulating plasma exosomes were isolated using Cushioned-Density Gradient Ultracentrifugation and total RNA was extracted. Next Generation Sequencing was performed on the Illumina HiSeq4000 SE50 after NEBNext Multiplex Small RNA Library Prep. Differential miRNA expression analysis was performed using DESeq2 software with a Benjamini-Hochberg correction. MicroRNA expression profiles were validated by Quantitative Real-Time PCR. Results: Aortic diameter, as measured by US, was significantly larger in the AAA group (mean maximum diameter 5.2 vs 2.3 cm, p =2.84x10 -6 ). Aneurysm patients were more likely to suffer from CAD (5/11 vs 1/15, p = 0.05) but had similar levels of PAD (4/11 vs 2/15, p =0.35) and COPD (4/11 vs 4/15, p =0.68) compared to controls. A total of 40 miRNAs were differentially expressed ( p &lt;0.05). Of these, 18 miRNAs were up-regulated and 22 were down-regulated in controls compared to AAA. After false discovery rate (FDR) adjustment, only miR-122-5p was expressed at significantly different levels in AAA compared to controls (fold change = 5.03 controls vs AAA; raw p = 1.8x10 -5 ; FDR p =0.02). Conclusions: Plasma exosomes from AAA patients have significantly reduced levels of miRNA-122-5p compared to controls.

Functional Implication of Exosomal miR-217 and miR-23b-3p in the Progression of Prostate Cancer.

ObjectiveThe microRNA expression profile of plasma exosomes in prostate cancer (PCa) is of critical importance in the disease exploration. This study aimed to explore the clinical application of exosomal miRNAs as biomarkers for PCa.MethodsExosome-like vesicles of PCa patients and healthy controls were purified by differential centrifugation. The purified vesicles within the ranges of 50 and 100 nm were classified as exosomes according to the results of transmission electron microscopy and Western blot. Both, in vitro and in vivo, validations were performed by small RNA sequencing, CCK8, RT-qPCR, flow cytometry, Western blot, transwell and immunofluorescent staining assays.ResultsHigh-throughput sequencing identified that 94 miRNAs were differentially expressed in PCa patients in comparison with healthy controls (P<0.01; fold change ≥2). Among them, 64 miRNAs were upregulated, and 30 miRNAs were downregulated. In comparison to the healthy controls, the expression levels of miR-217 were significantly upregulated, while miR-23b-3p were significantly downregulated in the exosomes and serum collected from PCa patients. Both, in vitro and in vivo, studies revealed that exosomes secreted by PCa cells with up-regulated miR-217 levels promoted cell proliferation and invasion; meanwhile, the exosomes with up-regulated miR-23b-3p levels inhibited cell proliferation and invasion. The epithelial–mesenchymal transition process may have been involved in the above-mentioned regulation.ConclusionThis study identified the dysregulated expression of exosomal miRNAs in PCa patients, including miR-217 and miR-23b-3p, by validating their function on proliferation and invasion in PCa cells. This regulation may have been affected by the epithelial–mesenchymal transition process, suggesting that they can be used as potential targets in the diagnosis and treatment of PCa.

Differential expression profile of plasma exosomal microRNAs in women with polycystic ovary syndrome

To examine different expression profiles of plasma exosomal microRNA (miRNA) in polycystic ovary syndrome (PCOS) patients and controls, and their potential roles in PCOS pathogenesis. Experimental study. Center for reproductive medicine. Seventy-five PCOS patients and 75 age-matched controls. Plasma exosomes miRNAs sequenced from 15 PCOS patients and 15 controls. Plasma exosomal miRNA expression and the correlation between PCOS phenotypes and miRNA expression. The sequenced plasma exosomes miRNAs were further determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in a larger cohort, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Correlation analysis and receiver operating characteristic (ROC) curve analysis were used to determine the association between PCOS phenotypes and miRNA expression. The miRNA sequencing revealed 34 exosomal miRNAs were differentially expressed between PCOS patients and controls. Via qRT-PCR, five differentially expressed miRNAs (miR-126-3p, miR-146a-5p, miR-20b-5p, miR-106a-5p, and miR-18a-3p) were identified. The GO and KEGG analyses predicted their target functions included axon guidance, mitogen-activated protein kinase (MAPK) signaling, endocytosis, circadian rhythms, and cancer pathways. The expression of these miRNAs correlated with menstrual cycle, antral follicle count, hormone level, and combined yielded a ROC curve area of 0.781 in discriminating PCOS patients from the controls. Differential expression of plasma exosomal miRNAs may confer a risk of PCOS and may be helpful in distinguishing PCOS patients from controls. Certain miRNA expression may associated to the disease progression, which could help in an epigenetic understanding of the pathophysiology of PCOS.

Next-generation sequencing analysis of exosomal microRNAs: &lt;i&gt;Fusobacterium&lt;/i&gt;&lt;i&gt;nucleatum&lt;/i&gt; regulates the expression profiling of exosomal microRNAs in human colorectal cancer cells

Colon cancer is one of the most common malignant tumors, but there are still a few validated biomarkers of colon cancer. Exosome-mediated microRNAs (miRNAs) have been recognized as potential biomarkers in cancers, and miRNAs can regulate a variety of genes. Recently, Fusobacterium nucleatum was discovered in the tissues of human colon cancer patients. Its role in colon cancer was highlighted. F. nucleatum may contribute to the progression of colon cancer through the mechanism of exosome-mediated miRNAs transfer. However, the exosomal miRNAs regulation mechanism by F. nucleatum in colon cancer is not well known. Thus, we performed next-generation sequencing to investigate the overall pattern of exosomal miRNAs expression in the colon cancer cell culture supernatant. We have confirmed the alterations of various exosomal miRNAs. In addition, to investigate the function of exosomal miRNAs, a Kyoto Encyclopedia of Genes and Genomes analysis was performed on the target genes of changed miRNAs. Potential target genes were associated with a variety of signaling pathways, and one of these pathways was related to colorectal cancer. These findings suggested that F. nucleatum can alter exosomal miRNAs released from colorectal cancer cells. Furthermore, exosomal miRNAs altered by F. nucleatum could be potential biomarkers for the diagnosis and therapy of colon cancer.

Plasma Exosomal miRNA Expression Profile as Oxaliplatin-Based Chemoresistant Biomarkers in Colorectal Adenocarcinoma.

Background: Chemotherapy is one of the most common therapies used in the treatment of colorectal cancer (CRC), but chemoresistance inevitably occurs. It is challenging to obtain an immediate and accurate diagnosis of chemoresistance. The potential of circulating exosomal miRNAs as oxaliplatin-based chemoresistant biomarkers in CRC patients was investigated in this study.Methods: Plasma exosomal miRNAs in sensitive and resistant patients were analyzed by miRNA microarray analysis, followed by verification with a quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assay in two independent cohorts. The diagnostic accuracy was determined by ROC curve analysis. Logistic regression analysis and Spearman's rank correlation test were also performed. Finally, bioinformatics was used to preliminarily explore the potential molecular mechanism of the selected miRNAs in chemoresistance.Results: miRNA microarray analysis identified four upregulated miRNAs and 20 downregulated miRNAs in chemoresistant patients compared to chemosensitive patients. Twelve markedly dysregulated miRNAs were selected for further investigation, of which six (miR-100, miR-92a, miR-16, miR-30e, miR-144-5p, and let-7i) were verified to be significantly and consistently dysregulated (>1.5-fold, P < 0.05). The combination of the six miRNAs had the highest AUC (0.825, 95% CI, 0.753–0.897). The expression level of these 6 miRNAs was not correlated with tumor location, stage, or chemotherapy program. Only miR-100 was significantly upregulated in low histological grade. GO analysis and KEGG pathway analysis showed that miRNAs were related to RNA polymerase II transcription and enriched in the PI3K-AKT signaling pathway, AMPK signaling pathway, and FoxO signaling pathway.Conclusions: We identified a panel of plasma exosomal miRNAs, containing miR-100, miR-92a, miR-16, miR-30e, miR-144-5p, and let-7i, that could significantly distinguish chemoresistant patients from chemosensitive patients. The detection of circulating exosomal miRNAs may serve as an effective way to monitor CRC patient responses to chemotherapy. Targeting these miRNAs may also be a promising strategy for CRC treatment.

INFLAMMATORY MACROPHAGES DERIVED EXOSOMAL MIR-222 PROMOTED THECA CELL STEROIDOGENESIS BY MODULATING MYLIP EXPRESSION

To explore the role of M1 macrophages (M1M) in the linkage between low-grade inflammation and ovarian dysfunction in obesity. In vitro experimental laboratory study. Raw264.7 macrophage was treated with lipopolysaccharide (LPS) to establish a model of M1M. Exosome (Exo) secreted by macrophage was isolated by ultracentrifugation and identified by transmission electron microscopy (TEM), western blot, and flow nanoanalyzer. The primary murine ovary theca cell (TC) was co-incubated with PKH67 fluorescence-labeled Exo to detect whether Exo secreted by macrophage could be uptaken by TC. The expression of CYP11A1 and CYP17A1 were analyzed by qPCR and western blot. Medium testosterone and progesterone level were analyzed. High-throughput sequencing was performed to analyze the expression of exosomal miRNA (miRNA-seq). In order to select key differential exosomal miRNA, the GSE97652 (species: mouse) was downloaded from the GEO DataBases. The data set contains 7 replications in both experimental group and control group. The experimental group is obese mouse adipose tissue macrophage (ATM)-derived Exo, and the control group is control mouse ATM -derived Exo. The overlap between miRNA-seq and GSE97652 differentially expressed miRNA was analyzed according to the Fold change > 2, p < 0.01 standard, which determined miR-222 and MYLIP for subsequent experiments. Next, gain-of-function experiments were performed to examine their role in TC steroidogenesis function with the involvement of the cholesterol metabolism pathway. TEM, Flow NanoAnalyzer and western blot analysis confirmed the successful extraction of Exo. After co-cultured TC with PKH67-labeled Exo, green fluorescence could be detected in TC which demonstrated that TC was able to ingest Exo secreted by the macrophage. M1M increased the expression of CYP11A1 and the production of progesterone in TC which is dependent upon M1-Exo. The result of miRNA-seq showed that there were 30 upregulated miRNA and 112 down-regulated miRNA in M1M derived Exo compared with control Exo according to the standard of Fold change > 2(p < 0.05). A total of two differentially expressed miRNA, namely miR-222 and miR-155, were selected from the venn diagram of miRNA-seq and GSE97652. QPCR verified that miR-222 in both M1M derived Exo and M1M derived Exo treated TC were significantly upregulated. Subsequently, we predicted the target gene of miR-222 through five different databases. MYLIP, with the highest target score was selected, which has been identified as a key factor promoting LDLR degradation. M1M derived Exo was verified to display a high expression level of miR-222, and M1M derived Exo mediated TC steroidogenesis depended miR-222. The overexpression of miR-222 resulted in the down-regulation of MYLIP expression and up-regulation of LDLR expression, followed by increased cholesterol influx, CYP11A1 overexpression and progesterone overproduction. M1M derived Exo can be ingested by TC. M1M promote TC CYP11A1 overexpression and progesterone overproduction through Exo by delivering miR-222 into TC.

48P Macrophage-derived exosomal microRNAs regulate macrophage-cancer communications

Macrophages are a type of phagocyte, which play a critical role in the immune system. They are involved in both innate and adaptive immunity by either removing pathogens or antigen presentation. It has been shown that macrophages are polarized to subgroups with distinct functions, M1 and M2. M1 polarization is characterized by pro-inflammatory and anti-cancer functions, whereas M2 macrophages promote immune suppression and tumor growth. As small noncoding RNAs, miRNAs have been shown to regulate macrophage polarization. In this study, we further investigate that exosomal miRNAs from macrophages play roles in cancer progression. Specifically, we demonstrated that macrophages could modulate cancer immune microenvironment through upregulation and release of miRNAs. To investigate macrophage and cancer communication in a cancer-immune microenvironment, human macrophage differentiation was performed and co-cultured with human melanoma. Homo sapiens small RNA sequencing was performed by exosome purification from co-cultured human macrophages. The whole transcript expression array was executed in co-cultured human melanoma. M1 polarized macrophage has been shown that pro-inflammatory and anti-cancer functions, whereas M2 macrophages promoted tumor growth and migration. According to small RNA sequencing data,differential exosomal miRNA expression profiles depend on macrophage differentiation and these exosomal miRNAs play roles in cancer progression. The whole transcript expression array represents target genes changed by exosomal miRNA. In this report, we demonstrated exosomal miRNA mediated-communication between cancer and macrophages. Interestingly, M1 or M2 macrophage showed differential exosomal miRNA expression profiles. Specifically, we found that let-7i and miR-19a derived from M1 macrophage induces anti-cancer effect. Due to its anti-cancer efficacy, let-7i and miR-19a could be candidates for therapeutics.

Urinary Exosomal MiRNA-4534 as a Novel Diagnostic Biomarker for Diabetic Kidney Disease.

Urinary exosomal miRNAs can reflect the physiological and possibly pathophysiological state of cells lining the kidney and participate in the regulation of transcription and translation of proteins, which are playing an important role in the pathogenesis of diabetic kidney disease. In the present study, urine was collected from DM and DKD patients with a duration more than 10 years and urinary exosomal miRNA profiling was conducted in urinary exosomes obtained from three patients with type 2 diabetes (DM) and three patients with type 2 diabetic kidney disease (DKD) using Exiqon's microRNA arrays. In total, the expression of 14 miRNAs (miR-4491, miR-2117, miR-4507, miR-5088-5P, miR-1587, miR-219a-3p, miR-5091, miR-498, miR-4687-3p, miR-516b-5p, miR-4534, miR-1275, miR-5007-3p, and miR-4516) was up-regulated (>2-fold) in DKD patients compared to healthy controls and DM patients. We used qRT-PCR based analysis of these 14 miRNAs in urinary exosomes from 14 DKD to 14 DM patients in confirmation cohort, among which seven miRNAs were consistent with the microarray results. The expressions of miR-4534 and miR-516b-5p correlated with trace proteinuria levels in the confirmation cohort. In conclusion, it has been confirmed that the expression of urinary exosomal miRNA in patients with type 2 diabetes DKD has changed. Mir-4534 might affect the FoxO signaling pathway by targeting BNIP3, and is expected to become a new biomarker for the progression of type 2 DKD disease, which will provide further research on the pathogenesis of DKD.

Abstract 1536: MiRNA expression profile of exosomes derived from breast cancer cells following induction of the epithelial-to-mesenchymal transition

Abstract Breast cancer-derived exosomes are small extracellular vesicles that play a crucial role in the metastatic progression of breast cancer due to their ability to transport a variety of biologically active molecules (e.g., miRNAs) to local and distant sites in the body. The molecular cargo transported by breast cancer-derived exosomes alters the transcriptome of recipient cells at these sites to stimulate pro-metastatic events, including the epithelial-to-mesenchymal transition (EMT). Importantly, the molecular cargo loaded into exosomes can vary depending on cell state. However, it is unclear whether the expression of miRNAs in breast cancer-derived exosomes is altered following inducement of the EMT. Regulation of exosomal miRNAs following the EMT may enhance the ability of circulating tumor cells to target specific tissue sites or improve formation and maintenance of metastatic tumors compared to miRNAs packaged into exosomes secreted from cancer cells without induction of the EMT. To address this knowledge gap, we hypothesized that the EMT alters expression of miRNAs in exosomes derived from EMT-induced MDA-MB-468 breast cancer cells. To induce the EMT, MDA-MB-468 cells were treated with exogenous epidermal growth factor. MDA-MB-468 cells treated with epidermal growth factor exhibited greater mRNA expression of the mesenchymal marker Vimentin compared to non-treated MDA-MB-468 cells by qRT-PCR. Subsequently, exosomes were isolated from the conditioned cell culture medium of MDA-MB-468 cells, with and without induction of the EMT, using ExoQuick exosome isolation reagent. Molecular size analysis of isolated exosomes revealed that they were similar in size to the typical size range reported for exosomes (40 - 150nm). Moreover, a common exosomal marker protein, CD9, was enriched in the MDA-MB-468-derived exosome fractions compared to whole cell lysates by Western blot. Furthermore, next generation sequencing and bioinformatic analysis were performed to determine the miRNA expression profiles of these exosomes. Results from this study reveal expression of miRNAs in breast cancer cell-derived exosomes before and after activation of the EMT and may indicate how exosomes secreted from advanced stage breast cancer enhance metastatic tumor progression. Citation Format: Christian A. Showalter, Yinan Huang, Monica M. Burdick. MiRNA expression profile of exosomes derived from breast cancer cells following induction of the epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1536.

Abstract 6446: Liquid biopsy in colon cancer utilizing exosomal miRNA biomarkers: An initial analysis

Abstract Traditional clinicopathologic variables do not predict recurrence and disease progression in colorectal cancer (CRC) accurately. Development of a liquid biopsy as a means to obtain real-time, clinically meaningful biomarkers to assess progression and response to treatment is critical. Current liquid biopsy techniques utilizing circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have significant limitations. Exosomes are emerging as a promising tool for liquid biopsy due to their stability and role in cancer cell to cell communication. To that end, the purpose of this study is to describe patterns of highly abundant microRNA (miRNA) found in serum exosomes of patients with metastatic colon cancer as a step toward developing an exosomal miRNA signature for liquid biopsy. Next generation sequencing was performed on exosomes isolated from serum of stage IV colon cancer patients (n=9) to determine the miRNA read counts. miRNA data from primary tumors (n=53) of stage IV colon cancer patients and available paired peritumoral tissue (n=8) were obtained from The Cancer Genome Atlas (TCGA) database for qualitative comparison. There were 515 total miRNA shared in common between the serum exosomes and primary tumors and 277 miRNA uniquely present in serum exosomes. In our initial analysis, we identified five miRNA, two in the common pool (mir-486-1 and mir-486-2) and three in the unique group (miR-486-5p, miR-92a-3p, and miR-146a-5p) that were most abundant in serum exosomes and may serve as biomarkers. mir-486-1 and mir-486-2 were the most highly abundant miRNAs in exosomes, but were only expressed at low levels in the tissue. This inverse relationship has been demonstrated by other investigators, with decreased levels of tissue mir-486 associated with more advanced stages of disease and poorer survival. Exosomal miR-92a-3p has been shown to correlate with tumor grade and stage in CRC patients. Delivery of miR-146a-5p by exosomes has been shown to promote tumorigenesis and generate immunosuppressive microenvironment in CRC. In general, the top 10 most abundant exosomal miRNA were not among the most abundant tissue level miRNAs. Our group is currently analyzing the differential expression of serum exosomal miRNA from 52 additional patients with non-metastatic colon cancer (n=14), liver metastases (n=16) and PC (n=12) in comparison to healthy volunteer serum (n=10) to better characterize miRNA patterns at different stages of disease. In summary, highly abundant miRNA found in serum exosomes of patients with metastatic colon cancer demonstrate a different pattern of expression than miRNA found in the primary colon cancer itself. Further analysis of these miRNA patterns from serum exosomes of colon cancer patients with non-metastatic disease, visceral metastases and PC compared to normal volunteers will aid in the development of a serum exosomal miRNA signature. Citation Format: Audrey H. Choi, Paul A. Vallejos, Mei Li M. Kwong, Janviere Kabagwira, Matthew J. Selleck, Nathan R. Wall, Maheswari Senthil. Liquid biopsy in colon cancer utilizing exosomal miRNA biomarkers: An initial analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6446.

Serum exosomal microRNA-766-3p expression is associated with poor prognosis of esophageal squamous cell carcinoma.

The aim was to analyze the association between exosomal microRNA (miR)‐766‐3p expression levels in serum and the prognosis of esophageal squamous cell carcinoma (ESCC). The serum global exosomal miRNA expression of ESCC patients was measured by microRNA microarray. Quantitative real‐time PCR was used to analyze the expression levels of candidate miRNAs in both serum and tissues from ESCC patients. Wilcoxon tests were applied to evaluate clinical characteristics and their association with serum levels of exosomal miR‐766‐3p. A Cox regression model was used to identify prognostic factors. The effects of miR‐766‐3p expression on cell migration and invasion were examined using Transwell assays, and CCK‐8 assays were carried out to measure cell proliferation. The TNM stage was associated with high serum exosomal miR‐766‐3p levels of ESCC patients (P = .030). Higher serum exosomal miR‐766‐3p expression levels were associated with poor prognosis (for overall survival, hazard ratio [HR] [95% confidence interval (CI)], 2.21 [1.00, 4.87]; for disease‐free survival, HR [95% CI], 2.15 [1.01, 4.59]). However, we found no association between the expression of miR‐766‐3p in tissue and ESCC prognosis. In vitro results showed that miR‐766‐3p promotes cell migration and invasion, but not cell proliferation. By using dual‐luciferase reporter assay, HOXA13 was confirmed as a direct target gene of miR‐766‐3p. The ESCC patients with highly expressed serum exosomal miR‐766‐3p had a significantly worse survival. Therefore, serum exosomal miR‐766‐3p could serve as a prognostic marker for the assessment of ESCC.

Ultraviolet B irradiation enhances the secretion of exosomes by human primary melanocytes and changes their exosomal miRNA profile

ObjectiveMelanocytes play a central role in skin homeostasis. In this study, we focus on the function of melanocyte releasing exosomes as well as exosomal microRNAs (miRNAs) and investigate whether ultraviolet B (UVB) irradiation exerts an impact on it.Materials and methodsExosomes derived from human primary melanocytes were isolated through differential centrifugation and were identified in three ways, including transmission electron microscopy observation, nanoparticle tracking analysis, and Western blot analysis. Melanocytes were irradiated with UVB for the indicated time, and then melanin production and exosome secretion were measured. The exosomal miRNA expression profile of melanocytes were obtained by miRNA sequencing and confirmed by real-time PCR.ResultsExosomes derived from human primary melanocytes were verified. UVB irradiation induced melanin production and increased the exosome release by the melanocytes. In total, 15 miRNAs showed higher levels in UVB-irradiated melanocyte-derived exosomes compared with non-irradiated ones, and the top three upregulated exosomal miRNAs were miR-4488, miR-320d, and miR-7704 (fold change > 4.0).ConclusionIt is verified for the first time that UVB irradiation enhanced the secretion of exosomes by melanocytes and changed their exosomal miRNA profile. This findings open a new direction for investigating the communication between melanocytes and other skin cells, and the connection between UVB and skin malignant initiation.

Exosomal microRNA expression profiles of cerebrospinal fluid in febrile seizure patients

PurposeFebrile seizures (FS) are the most common seizures found in pediatric patients. Recently, microRNA (miRNA) have been used as a novel biomarker for the diagnosis of various diseases. This study aims to explore the exosomal miRNA expression profile of the cerebrospinal fluid (CSF) in atypical FS patients. MethodsThis is a case-control study including CSF specimens of 41 pediatric patients. The CSF specimens were categorized into FS and a control group. Microarray assays were performed to evaluate the CSF exosomal miRNA expression profile. Quantitative PCR (qPCR) assays were conducted to validate the microarray assay result. Bioinformatic analysis was performed to analyze the result. ResultsThirteen (62%) patients in the FS group experienced complex FS. A total of 96 miRNAs were significantly expressed in the CSF study samples and 95 amongst them, exhibited higher expression in FS than in the control group. Further validation qPCR test indicated that the top 5 highly expressed miRNA (miR-4486, miR-6850-5p, miR-642b-3p, miR-7107-5p, miR-4281) showed same results as in the microarray assay. Bioinformatic analysis identified 455 target genes in the FS group. ConclusionFS patients displayed higher CSF exosomal miRNA profiles than the control. These altered miRNA profiles appeared to be related to complex FS.

Exosomal MicroRNAs in Military Personnel with Mild Traumatic Brain Injury: Preliminary Results from the Chronic Effects of Neurotrauma Consortium Biomarker Discovery Project.

Chronic symptoms after mild traumatic brain injury (mTBI) are common among veterans and service members, and represent a significant source of morbidity, with those who sustain multiple mTBIs at greatest risk. Exosomal micro-RNAs (miRNAs), mediators of intercellular communication, may be involved in chronic TBI symptom persistence. Exosomal miRNA (exomiR) was extracted from 153 participants enrolled in the Chronic Effect of Neurotrauma Consortium (CENC) longitudinal study (no TBI, n = 35; ≥ 3 mTBIs (rTBI), n = 45; 1-2 mTBIs, n = 73). Analyses were performed with nCounter® Human miRNA Expression Panels and Ingenuity Pathway Analysis (IPA) for identification of gene networks associated with TBI. Generalized linear models were used to analyze the predictive value of exomiR dysregulation and remote neurobehavioral symptoms. Compared with controls, there were 17 dysregulated exomiRs in the entire mTBI group and 32 dysregulated exomiRs in the rTBI group. Two miRNAs, hsa-miR-139-5p and hsa-miR-18a-5p, were significantly differentially expressed in the rTBI and 1-2 mTBI groups. IPA analyses showed that these dysregulated exomiRs correlated with pathways of inflammatory regulation, neurological disease, and cell development. Within the rTBI group, exomiRs correlated with gene activity for hub-genes of tumor protein TP53, insulin-like growth factor 1 receptor, and transforming growth factor beta. TBI history and neurobehavioral symptom survey scores negatively and significantly correlated with hsa-miR-103a-3p expression. Participants with remote mTBI have distinct exomiR profiles, which are significantly linked to inflammatory and neuronal repair pathways. These profiles suggest that analysis of exosomal miRNA expression may provide novel insights into the underlying pathobiology of chronic TBI symptom persistence.

Analysis of exosomal lncRNA, miRNA and mRNA expression profiles and ceRNA network construction in endometriosis.

Aim: To investigate exosomal RNAs (long noncoding RNAs (lncRNAs), microRNAs (miRNAs) and messenger RNAs (mRNAs)) profiling and their related networks in endometriosis (EMs). Materials & methods: RNA sequence was performed in exosomes from ovarian endometriomas (EC), eutopic endometria (EU) and normal endometria (Control) stromal cells. The bioinformatics algorithms evaluated competing endogenous RNA (ceRNA) networks. The top-ranked ceRNA networks were confirmed by RT-PCR. Results: Overlapped differentially expressed 938 lncRNAs, 39 miRNAs and 1449 mRNAs were identified. 13 co-expression modules and 61 ceRNA networks were constructed. Conclusion: This study for the first time shows exosomal RNA biomarkers and lncRNA-related networks in EMs, which reveals a novel molecular mechanism of EMs and provides new resources for EM diagnosis and treatment.