Dataset Information

Non-Invasive Approach for Evaluation of Pulmonary Hypertension Using Extracellular Vesicle-Associated Small Non-Coding RNA.

ABSTRACT: Extracellular vesicles are released by numerous cell types of the human body under physiological but also under pathophysiological conditions. They are important for cell-cell communication and carry specific signatures of peptides and RNAs. In this study, we aimed to determine whether extracellular vesicles isolated from patients with pulmonary hypertension show a disease specific signature of small non-coding RNAs and thus have the potential to serve as diagnostic and prognostic biomarkers. Extracellular vesicles were isolated from the serum of 23 patients with chronic thromboembolic pulmonary hypertension (CTEPH) and 23 controls using two individual methods: a column-based method or by precipitation. Extracellular vesicle- associated RNAs were analyzed by next-generation sequencing applying molecular barcoding, and differentially expressed small non-coding RNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). We identified 18 microRNAs and 21 P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) or piRNA clusters that were differentially expressed in CTEPH patients compared with controls. Bioinformatic analysis predicted a contribution of these piRNAs to the progression of cardiac and vascular remodeling. Expression levels of DQ593039 correlated with clinically meaningful parameters such as mean pulmonary arterial pressure, pulmonary vascular resistance, right ventricular systolic pressure, and levels of N-terminal pro-brain natriuretic peptide. Thus, we identified the extracellular vesicle- derived piRNA, DQ593039, as a potential biomarker for pulmonary hypertension and right heart disease.

SUBMITTER: Lipps C

PROVIDER: S-EPMC6920761 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Non-Invasive Approach for Evaluation of Pulmonary Hypertension Using Extracellular Vesicle-Associated Small Non-Coding RNA.

Lipps Christoph C Northe Philipp P Figueiredo Ricardo R Rohde Manfred M Brahmer Alexandra A Krämer-Albers Eva-Maria EM Liebetrau Christoph C Wiedenroth Christoph B CB Mayer Eckhard E Kriechbaum Steffen D SD Dörr Oliver O Nef Holger H Hamm Christian W CW Keller Till T Troidl Christian C

Biomolecules 20191029 11

Extracellular vesicles are released by numerous cell types of the human body under physiological but also under pathophysiological conditions. They are important for cell-cell communication and carry specific signatures of peptides and RNAs. In this study, we aimed to determine whether extracellular vesicles isolated from patients with pulmonary hypertension show a disease specific signature of small non-coding RNAs and thus have the potential to serve as diagnostic and prognostic biomarkers. Ex ...[more]

PMID: 31671920

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Project description:BackgroundThe identification of indeterminate pulmonary nodules (IPNs) following a low-dose computed tomography (LDCT) is a major challenge for early diagnosis of lung cancer. The inadequate assessment of IPNs' malignancy risk results in a large number of unnecessary surgeries or an increased risk of cancer metastases. However, limited studies on non-invasive diagnosis of IPNs have been reported.MethodsIn this study, we identified and evaluated the diagnostic value of circulating small extracellular vesicle (sEV) microRNAs (miRNAs) in patients with IPNs that had been newly detected using LDCT scanning and were scheduled for surgery. Out of 459 recruited patients, 109 eligible patients with IPNs were enrolled in the training cohort (n = 47) and the test cohort (n = 62). An external cohort (n = 99) was used for validation. MiRNAs were extracted from plasma sEVs, and assessed using Small RNA sequencing. 490 lung adenocarcinoma samples and follow-up data were used to investigate the role of miRNAs in overall survival.ResultsA circulating sEV miRNA (CirsEV-miR) model was constructed from five differentially expressed miRNAs (DEMs), showing 0.920 AUC in the training cohort (n = 47), and further identified in the test cohort (n = 62) and in an external validation cohort (n = 99). Among five DEMs of the CirsEV-miR model, miR-101-3p and miR-150-5p were significantly associated with better overall survival (p = 0.0001 and p = 0.0069). The CirsEV-miR scores were calculated, which significantly correlated with IPNs diameters (p < 0.05), and were able to discriminate between benign and malignant PNs (diameter ≤ 1 cm). The expression patterns of sEV miRNAs in the benign, adenocarcinoma in situ/minimally invasive adenocarcinoma, and invasive adenocarcinoma subgroups were found to gradually change with the increase in aggressiveness for the first time. Among all DEMs of the three subgroups, five miRNAs (miR-30c-5p, miR-30e-5p, miR-500a-3p, miR-125a-5p, and miR-99a-5p) were also significantly associated with overall survival of lung adenocarcinoma patients.ConclusionsOur results indicate that the CirsEV-miR model could help distinguish between benign and malignant PNs, providing insights into the feasibility of circulating sEV miRNAs in diagnostic biomarker development.Trial registrationChinese Clinical Trials: ChiCTR1800019877. Registered 05 December 2018, https://www.chictr.org.cn/showproj.aspx?proj=31346 .

Project description:Small extracellular vesicles (EVs) are 50-200 nm sized mediators in intercellular communication that reflect both physiological and pathophysiological changes of their parental cells. Thus, EVs hold great potential for biomarker detection. However, reliable purification methods for the downstream screening of the microRNA (miRNA) cargo carried within urinary EVs by small RNA sequencing have yet to be established. To address this knowledge gap, RNA extracted from human urinary EVs obtained by five different urinary EV purification methods (spin column chromatography, immunoaffinity, membrane affinity, precipitation and ultracentrifugation combined with density gradient) was analyzed by small RNA sequencing. Urinary EVs were further characterized by nanoparticle tracking analysis, Western blot analysis and transmission electron microscopy. Comprehensive EV characterization established significant method-dependent differences in size and concentration as well as variances in protein composition of isolated vesicles. Even though all purification methods captured enough total RNA to allow small RNA sequencing, method-dependent differences were also observed with respect to library sizes, mapping distributions, number of miRNA reads and diversity of transcripts. Whereas EVs obtained by immunoaffinity yielded the purest subset of small EVs, highly comparable with results attained by ultracentrifugation combined with density gradient, precipitation and membrane affinity, sample purification by spin column chromatography indicated a tendency to isolate different subtypes of small EVs, which might also carry a distinct subset of miRNAs. Based on our results, different EV purification methods seem to preferentially isolate different subtypes of EVs with varying efficiencies. As a consequence, sequencing experiments and resulting miRNA profiles were also affected. Hence, the selection of a specific EV isolation method has to satisfy the respective research question and should be well considered. In strict adherence with the MISEV (minimal information for studies of extracellular vesicles) guidelines, the importance of a combined evaluation of biophysical and proteomic EV characteristics alongside transcriptomic results was clearly demonstrated in this present study.

Dataset Information

Non-Invasive Approach for Evaluation of Pulmonary Hypertension Using Extracellular Vesicle-Associated Small Non-Coding RNA.

Publications

Non-Invasive Approach for Evaluation of Pulmonary Hypertension Using Extracellular Vesicle-Associated Small Non-Coding RNA.

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