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A microRNA signature in circulating exosomes is superior to exosomal glypican-1 levels for diagnosing pancreatic cancer.

ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy that often presents clinically at an advanced stage and that may be confused with chronic pancreatitis (CP). Conversely, CP may be misdiagnosed as PDAC leading to unwarranted pancreas resection. Therefore, early PDAC diagnosis and clear differentiation between PDAC and CP are crucial for improved care. Exosomes are circulating microvesicles whose components can serve as cancer biomarkers. We compared exosomal glypican-1 (GPC1) and microRNA levels in normal control subjects and in patients with PDAC and CP. We report that exosomal GPC1 is not diagnostic for PDAC, whereas high exosomal levels of microRNA-10b, (miR-10b), miR-21, miR-30c, and miR-181a and low miR-let7a readily differentiate PDAC from normal control and CP samples. By contrast with GPC1, elevated exosomal miR levels decreased to normal values within 24 h following PDAC resection. All 29 PDAC cases exhibited significantly elevated exosomal miR-10b and miR-30c levels, whereas 8 cases had normal or slightly increased CA 19-9 levels. Thus, our exosomal miR signature is superior to exosomal GPC1 or plasma CA 19-9 levels in establishing a diagnosis of PDAC and differentiating between PDAC and CP.

SUBMITTER: Lai X

PROVIDER: S-EPMC5386003 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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A microRNA signature in circulating exosomes is superior to exosomal glypican-1 levels for diagnosing pancreatic cancer.

Lai Xianyin X Wang Mu M McElyea Samantha Deitz SD Sherman Stuart S House Michael M Korc Murray M

Cancer letters 20170220

Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy that often presents clinically at an advanced stage and that may be confused with chronic pancreatitis (CP). Conversely, CP may be misdiagnosed as PDAC leading to unwarranted pancreas resection. Therefore, early PDAC diagnosis and clear differentiation between PDAC and CP are crucial for improved care. Exosomes are circulating microvesicles whose components can serve as cancer biomarkers. We compared exosomal glypican-1 (GPC1) and mi ...[more]

PMID: 28232049

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Project description:PurposeRenal cell carcinoma (RCC) is the most common type of kidney cancer in adults. Exosomes are membrane-enclosed extracellular vesicles, and exosomal RNA can be a biomarker for cancer diagnosis and prognosis in RCC patients. We aim to identify differences in miRNA expression profiles in peripheral blood exosomes between RCC patients and healthy subjects as well as to investigate novel markers of RCC.MethodsWe performed exosomal miRNA sequencing of plasma samples obtained from five RCC patients and five control subjects, subsequently 22 RCC patients and 16 control subjects were investigated using qPCR to confirm the differential miRNA which from plasma exosomal RNA sequencing. ROC curves were constructed to assess the diagnostic accuracy of exosomal miRNAs as diagnostic biomarkers of RCC.ResultsExosomes were isolated with the exoeasy maxi kit and confirmed using TEM and NTA. They have a spherical structure with a diameter of approximately 40-180 nm. The exosomal miRNA sequence results showed that a total of 2357 miRNAs were detected, and 245 miRNAs were differentially expressed between RCC patients and healthy controls (p<0.001, average counts >5, log|fc|>1). Further analysis revealing that, versus the control, 17 miRNAs are up-regulated and 5 miRNAs are down-regulated under selection conditions with average miRNAs counts >100. qPCR was performed using 38 subjects-the results showed that the expression levels of hsa-mir-149-3p and hsa-mir-424-3p were upregulated; the expression levels of hsa-mir-92a-1-5p were significantly downregulated in the plasma exosomes of RCC. For diagnosis of RCC, the AUC of hsa-mir-92a-1-5p, hsa-mir-149-3p and hsa-mir-424-3p was 0.8324, 0.7188 and 0.7727, with the sensitivity of 0.875, 0.750 and 0.750, and the specificity of 0.773,0.727 and 0.818, respectively, at the best cutoff value.ConclusionOur study revealed that the expression levels of hsa-mir-92a-1-5p, hsa-mir-149-3p and hsa-mir-424-3p were significantly abnormal in RCC patients, which may be novel biomarkers for RCC diagnosis.

Project description:BackgroundThe diagnostic performance and prognostic value of serum exosomal glypican 1 (GPC-1) in pancreatic ductal adenocarcinoma (PDAC) remain controversial. In this study, we detected serum exosomal GPC-1 using enzyme-linked immunosorbent assay (ELISA) and determined whether it serves as a predictor of diagnosis and recurrence for early-stage PDAC.MethodsSerum samples were obtained from patients with 50 PDAC, 6 benign pancreatic tumor (BPT), or 9 chronic pancreatitis (CP) and 50 healthy controls (HCs). Serum exosomes were isolated using an exosome isolation kit. Exosomal and serum GPC-1 levels were measured using ELISA. The freeze-thaw process was carried out to analyze the stability of GPC-1. Receiver operating characteristic (ROC) analysis was employed to assess the diagnostic value of GPC-1. Kaplan-Meier and multivariate Cox analyses were used to evaluate the prognostic value of GPC-1.ResultsThe average concentrations of serum exosomal and serum GPC-1 were 1.5 and 0.8 ng/ml, respectively. GPC-1 expression levels were stable under repeated freezing and thawing (d1-5 freeze-thaw cycles vs. d0 P > 0.05). Serum exosomal and serum GPC-1 were significantly elevated in patients with PDAC compared with HCs (P < 0.0001) but were slightly higher compared with that in patients with CP and BPT (P > 0.05). The expression levels of exosomal and serum GPC-1 were elevated 5 days after surgery in patients with PDAC, CP, and BPT (P < 0.05). Patients with high levels of exosomal and serum GPC-1 had a shorter relapse-free survival (RFS) (P = 0.006, and P = 0.010). Multivariate analyses showed that serum exosomal and serum GPC-1 were independent prognostic indicators for early RFS (P = 0.008, and P = 0.041).ConclusionELISA is an effective and sensitive method to detect exosomal and serum GPC-1. The detection of GPC-1 was stable under repeated freezing and thawing cycles and could distinguish early-stage PDAC from HCs but not CP and BPT. Exosomal and serum GPC-1 may be good independent predictors of early recurrence in early-stage PDAC.

Project description:The heterogeneity of exosome populations presents a great challenge to their study. The current study was designed to investigate the potential heterogeneity miRNA contents in circulating exosomes purified via different exosomal markers. In this study, exosomes from the serum of C57BL/6 mice after cecum ligation and perforation (CLP) or sham operation were isolated by precipitation using ExoQuick-TC and affinity purified with anti-Rab5b, anti-CD9, anti-CD31, and anti-CD44 antibodies using the Exo-Flow Exosome Capture kit to collect exosome subpopulations. RNA extracted from the exosomes isolated by ExoQuick-TC were profiled by next-generation sequencing (NGS). Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was also employed to determine the expression profiles of four representative exosomal miRNAs (mmu-miR-486-5p, mmu-miR-10a-5p, mmu-miR-143-3p, and mmu-miR-25-3p) selected from the NGS analysis. The results revealed that the expression patterns of these miRNAs in exosomes isolated by ExoQuick-TC as determined by RT-qPCR and NGS were similar, showing upregulation of mmu-miR-10a-5p and mmu-miR-143-3p but downregulation of mmu-miR-25-3p and mmu-miR-486-5p following CLP when compared to the levels in exosomes from sham control mice. However, their expression levels in the antibody-captured exosome subpopulations varied. The miRNAs in the exosomes captured by anti-Rab5b or anti-CD9 antibodies were more similar to those isolated by ExoQuick-TC than to those captured by anti-CD44 antibodies. However, there were no significant differences in these four miRNAs in CD31-captured exosomes. This study demonstrated that purification with different exosomal markers allows the collection of different exosome subpopulations with various miRNA contents. The results of this study demonstrate the heterogeneity of circulating exosomes and suggest the importance of stratifying exosome subpopulations when using circulating exosomes as biomarkers or investigating exosome function. In addition, this study also emphasized the necessity of using a consistent exosome marker across different samples as detecting biomarkers.

Project description:ObjectiveThis study aimed to characterize the circulating exosomal microRNA (miRNA) profiles associated with acute soft tissue injury.Materials and methodsIn this experimental study, a total of 12 rats were randomly divided into control group and model group (n=6 for each group). The rats in the model group were used to establish an acute soft tissue injury following the mechanical injury of the leg. The exosomes from the peripheral blood of all the rats were isolated and then characterized by Nanosight NS300 particle size analyser (NTA), transmission electron microscopy (TEM) and western blot. Next, the exosomal miRNAs in the control and model groups were sequenced, and the differentially expressed miRNAs (DE-miRNAs) were identified using the DESeq algorithm. Functional analyses were performed using Gene Ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases. Finally, quantitative reverse-transcription polymersa chain reaction (qRT-PCR) was used to verify the expression of the DE-miRNAs.ResultsTEM, NTA and western blot results showed that the exosomes were approximately 100 nm in size and exhibited cup-shaped morphology. A total of 628 miRNAs were obtained by sequencing. After that, 28 DE miRNAs (DEmiRNAs) were identified, including seven down-regulated miRNAs and 21 up-regulated miRNAs. These DE-miRNAs were linked to 7539 target genes with GO. Also, KEGG analyses demonstrated that these genes were enriched for phosphorylation, VEGF signaling pathway, and MAPK signaling pathway. Additionally, the consistency rate between the qRT-PCR and sequencing results was 83.33%, which showed a high relative reliability of the sequencing results.ConclusionThese findings suggest that these 28 exosomal miRNAs may be involved in the regulation of acute soft tissue injury, by one of critical biological processes (BP), phosphorylation. The findings provide valuable clues by utilizing exosomes as therapeutic targets for the effective treatment of acute soft tissue injury.

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A microRNA signature in circulating exosomes is superior to exosomal glypican-1 levels for diagnosing pancreatic cancer.

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A microRNA signature in circulating exosomes is superior to exosomal glypican-1 levels for diagnosing pancreatic cancer.

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