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have been described as potential biomarkers of AA and MDS.9,10 There is evidence that miRNAs are crucial in con- trolling and modulating immunity, promoting survival and growth of malignant cells, and in cancer metastasis.11,12 Some studies have demonstrated the presence of cancer- specific miRNAs in plasma or serum, suggesting utility of these molecules as potential biomarkers of diseases.13,14
Exosomes are small extracellular vesicles directly released into the extracellular space by all cell types;15 RNAs (protected from degradation16) and proteins are con- tained within these vesicles, allowing the transfer of genetic material and signaling proteins to recipient cells.17,18 Indeed, exosomal miRNAs influence biological functions, and they have been proposed as specific and reliable biomarkers of many malignant disorders, includ- ing colon cancer, melanoma, and AML, and to track mini- mal residual diseases in hematologic malignancies.19-27
In the current work, we investigated plasma exosomal miRNAs as potential minimally-invasive biomarkers of AA and MDS. Detection of specific exosomal miRNAs in these diseases might aid in understanding the pathophys- iology of BM failure as well as diagnosis and prognosis.
Methods
Human plasma samples
Whole PB was collected in ethylenediaminetetraacetic acid (EDTA) tubes from patients and healthy subjects after informed consent was obtained in accordance with the Declaration of Helsinki28 and protocols approved by the National Heart, Lung, and Blood Institute Institutional Review Board [National Institutes of Health (NIH), Bethesda, MD, USA]. All patients had a diagnosis of severe AA (SAA) or MDS according to standard criteria.29,30 Healthy controls were recruited from donors of the NIH Clinical Center Department of Transfusion Medicine.
A discovery set (n=42), which was used for an initial screening of an exosomal miRNA signature, included 16 healthy controls, 16 SAA patients, and 10 MDS patients. For a validation set (n=99), 36 healthy controls, 54 SAA patients, and 20 MDS patients were recruited. Additionally, 10 patients from the discovery set and 30 SAA patients from the validation set were screened for exosomal miRNA expression before and after six months of IST. Clinical characteristics are summarized in Online Supplementary Table S1. Specimens were collected at the time of diagnosis and after six months of IST. After centrifugation at 2000 RPM for 10 min, plas- ma was collected and stored at -80°C until use.
RNA extraction and cDNA synthesis
To obtain high-quality RNA, a bead-based RNA purification protocol was performed using the Direct-zolTM -96 MagBead RNA kit (Zymo Research, Irvine, CA, USA) according to the manufac- turer’s instructions with minor modifications. Purified RNA was subjected to cDNA synthesis with the miScript® II RT kit (Qiagen, Hilden, Germany), and undiluted cDNA stored at -20°C
until further use. To confirm that RNA was confined within exo- somes, exosomes extracted from several samples were treated with RNase A, as described in the Online Supplementary Appendix.
miRNA profiling
Initial screening of 372 miRNAs and 12 miRNA controls was performed in the discovery set using the miScript® miRNA PCR Array Human Serum & Plasma 384HC array (MIHS-106ZE, Qiagen) and the miScript® SYBR® Green PCR kit (Qiagen). Data were analyzed using the real-time thermal cycler 7900HT Fast Real-Time PCRs (Applied Biosystems, Thermo Fisher Scientific). A custom miScript miRNA PCR array (CMIHS02531E, Qiagen) including 42 candidate miRNAs and 6 controls was designed for validation.
Statistical analysis
Data were analyzed using Prism (v.7.02; GraphPad software, La Jolla, CA, USA). miScript miRNA PCR Array Data Analysis soft- ware was utilized to analyze data from miRNA PCR arrays. Ingenuity Pathway Analysis (v.33559992, Qiagen Bioinformatics)32 and miRWalk 2.033 software were used for pathway and/or predic- tion analyses.
Results
Exosomal miRNA content profiling shows a distinct signature in SAA and MDS
Previous studies investigated circulating miRNA expres- sion levels in the plasma of AA and MDS patients,9,10 but no characteristic exosomal miRNAs have been described to date in these diseases. We first screened a large number of miRNAs (372 miRNAs associated with serum and plas- ma and 12 control miRNAs; miScript® miRNA PCR Array MIHS-3106Z; http://www.sabiosciences. com/genetable.php?pcatn=MIHS-3106Z) in plasma exosome samples in the discovery set by principal component analysis (PCA). We found different miRNAs in SAA (7 up- and 4 down-regulated) and MDS (15 up- and 7 down-reg- ulated) (Figure 1A and B). Exosomal miRNA expression profiles were compared between SAA and MDS, resulting in 42 miRNAs (21 and 21 up-regulated in MDS and SAA, respectively) (Figure 1C).
Validation of miRNA content profiles
For validation of results obtained in the discovery set, 42 candidate miRNAs were selected and examined in a larger cohort of SAA, MDS, and healthy controls (n=99) (see Online Supplementary Table S1 for clinical characteristics and Online Supplementary Table S2 for selected miRNAs). To assess reaction quality and for data normalization, 6 control miRNAs (miRTC, PPC, SNORD61, miR-339-3p, miR-211-5p, and miR-30c-5p) were included in the cus- tom PCR array plate, and analysis was performed as described in the Online Supplementary Appendix.
PCA of the validation set revealed 9 miRNAs present in SAA (6 up- and 3 down-regulated) (Figure 2A). When MDS patients were compared to healthy controls, 15 and 5 miRNAs were significantly up-regulated and down-reg- ulated, respectively (Figure 2B). In addition, MDS patients were compared to the SAA group, and 20 miRNAs were found to be differentially expressed (Figure 2C). Hierarchical clustering displayed differences in exosomal miRNA signatures between SAA and MDS (Figure 2D).
Relative expression (Log2FC) values of 42 selected
Exosome extraction
Isolation of exosomes from 800 mL of plasma was performed using the PureExo Exosome Isolation kit (101Bio, Palo Alto, CA, USA) according to the manufacturer’s instructions. Flow-through was collected and stored at -80°C until further use. To confirm the presence of exosomes in the flow-through, protein content, parti- cle size, CD63 expression, and transmission electron microscopy were assessed (Online Supplementary Appendix and Online Supplementary Figure S1).31
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