Published Ahead of Print on September 22, 2016, as doi:10.3324/haematol.2016.151076.

Copyright 2016 Ferrata Storti Foundation.

A plasma microRNA signature as a biomarker for acquired

aplastic anemia

by Kohei Hosokawa, Sachiko Kajigaya, Xingmin Feng, Marie J. Desierto,

Maria del Pilar Fernandez Ibanez, Olga Rios, Barbara Weinstein, Phillip Scheinberg,
Danielle M. Townsley, and Neal S. Young

Haematologica 2016 [Epub ahead of print]

Citation: Hosokawa K, Kajigaya S, Feng X, Desierto MJ, del Pilar Fernandez Ibanez M, Rios O,
Weinstein B, Scheinberg P, Townsley DM, and Young NS. A plasma microRNA signature as a biomarker
for acquired aplastic anemia. Haematologica. 2016; 101:xxx
doi:10.3324/haematol.2016.151076

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Original Article

A plasma microRNA signature as a biomarker for acquired aplastic anemia

Kohei Hosokawa, Sachiko Kajigaya, Xingmin Feng, Marie J. Desierto, Maria del Pilar

Fernandez Ibanez, Olga Rios, Barbara Weinstein, Phillip Scheinberg, Danielle M. Townsley,

Neal S. Young

1
Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda,

Maryland 20892-1202, USA.

Running heads: Circulating microRNA profile in aplastic anemia

Key words: bone marrow failure, aplastic anemia, circulating microRNA, biomarker

Corresponding Author:

Kohei Hosokawa, MD, Ph.D

Cell Biology Section, Hematology Branch,

National Heart, Lung, and Blood Institute, National Institutes of Health

Building 10-CRC, Room 3E-5140, 10 Center Drive, Bethesda MD 20892

email: kohei.hosokawa@nih.gov,

phone: 301-496-5093, fax: 301-496-8396

Abstract: 259 words

Text: 3791 words

Table Count: 4

1
Figure Count: 5

Supplemental Table Count: 6

Supplemental Figure Count: 5

Clinicaltrials.gov identifier: #NCT00260689, #NCT00217594, #NCT00961064

Acknowledgments

The authors would like to thank Kinneret Broder for assistance in obtaining healthy volunteer

samples; Camilo Canel and Barbara R. Gould for designing the custom plate. This research

was supported by the Intramural Research Program of the NIH, National Heart, Lung, and

Blood Institute.

2
Abstract

Aplastic anemia is an acquired bone marrow disease characterized by marrow hypoplasia, a

paucity of hematopoietic stem and progenitor cells, and pancytopenia of the peripheral blood,

due to immune attack on the bone marrow. In aplastic anemia, a major challenge is to develop

immune biomarkers to monitor the disease. We analyzed circulating miRNAs in plasma

samples of aplastic anemia patients in order to identify disease-specific miRNAs. A total of

179 miRNAs were analyzed in 35 plasma samples from 13 aplastic anemia, 11

myelodysplastic syndrome, and 11 healthy controls using the Serum/Plasma Focus

microRNA PCR Panel. Subsequently, 19 miRNAs from the discovery set were investigated in

the 108 plasma samples from 41 aplastic anemia, 24 myelodysplastic syndrome, and 43

healthy controls for validation, confirming that three miRNAs could be validated as

dysregulated (> 1.5 fold change) in aplastic anemia, compared to healthy controls.

MiR-150-5p (induction of T-cell differentiation) and miR-146b-5p (involvement in the

feedback regulation of innate immune response) were elevated in aplastic anemia plasma,

whereas miR-1 was decreased in aplastic anemia. By receiver-operating characteristic curve

analysis, we developed a logistic model with these three miRNAs that enabled us to predict

the probability of a diagnosis of aplastic anemia with an area under the curve of 0.86.

Dysregulated expression levels of the miRNAs were normalized after immunosuppressive

therapy at 6 months. Specifically, miR-150-5p expression was significantly reduced after

successful immunosuppressive therapy, but did not change in non-responders. We propose

three novel plasma biomarkers in AA, in which miR-150-5p, miR-146b-5p, and miR-1 can

serve for diagnosis and miR-150-5p for disease monitoring.

Clinicaltrials.gov identifier: NCT00260689, NCT00217594, NCT00961064

3
Introduction

The disease, aplastic anemia (AA) in most patients is caused by immune-mediated

destruction of hematopoietic stem and progenitor cells (HSPCs), resulting in trilineage

marrow hypoplasia and pancytopenia of the peripheral blood. Responsiveness of a significant

proportion of AA patients to immunosuppressive therapies (IST) is the best evidence of an

underlying immune pathophysiology: the majority of patients show hematologic

improvement after only transient T-cell depletion by antithymocyte globulins (ATGs).1, 2

Although the immune pathophysiology of AA is well characterized,3-5 there are no

biomarkers that would allow a better understanding of the immunological status of an

individual AA patient, including disease severity and response to therapy.6 Reliable

biomarkers that correlated with disease severity or response would be used for individual

treatment decisions and in clinical trials.

MicroRNAs (miRNAs) are a group of small, conserved, non-coding RNA molecules that

primarily modulate gene expression, post-transcriptionally by hybridization to

complementary sequences in the 3 untranslated region of corresponding mRNAs.7 MiRNAs

′

contribute to the pathophysiology of important human diseases.8, 9 Emerging evidence

supports that miRNAs have important roles in controlling and modulating immunity.10

Dysregulation of miRNAs can lead to autoimmune diseases, such as rheumatoid arthritis and

multiple sclerosis (MS).11, 12 Although miRNA regulation of each target results in small

changes in gene expression, the network activity of miRNAs affecting hundreds of genes

simultaneously can effect dramatic changes in cell behavior.13 We recently reported that

downregulation of miR-126-3p and miR-145-5p promotes CD4+ and CD8+ T cell activation

by increasing MYC and PIK3R2 expression levels in AA patients.14

MiRNAs can also be detected outside the cell. Extracellular miRNAs are cell-free

circulating molecules residing in microvesicles, exosomes, and microparticles.15 These

4
circulating miRNAs can be detected and quantified in biofluids, such as serum, plasma, urine,

and saliva.16 Circulating miRNAs mirror physiological and pathophysiological conditions and

have high stability in stored patient samples, allowing them to serve as biomarkers for

various diseases.17 In particular, detection of miRNA levels in blood plasma and serum has

the potential for early cancer diagnosis and to predict prognosis and response to therapy.18, 19

Recent studies have also identified circulating miRNAs as biomarkers to monitor the disease

state in T cell mediated autoimmune diseases, such as MS and myasthenia gravis (MG).20, 21

However, miRNAs have yet to be explored in the serum or plasma of AA. The purpose of our

study was to analyze the circulating miRNA profile in the plasma of AA patients and to assess

whether specific miRNAs could serve as new biomarkers for AA.

5
Methods

Patients and treatment

The study population was 183 human subjects who were enrolled on clinical protocols

between 2006 and 2015 at NIH the National Heart, Lung, and Blood Institute (Bethesda, MD,

USA). All human subjects were collected after obtaining written informed consent for

participation in accordance with the Declaration of Helsinki through clinical protocols

approved by the NHLBI Institutional Review Board. EDTA anticoagulated plasma samples

were obtained from patients and age-matched healthy blood donors. Standard criteria were

used for diagnosis of AA and assessment of disease severity.22 All AA patients were

diagnosed as severe AA and none had received IST at the time of sampling. All AA patients

received IST [horse-ATG + cyclosporine (CsA) or rabbit-ATG + CsA] on a clinical research

protocol (clinicaltrials.gov, #NCT00260689).2, 23 EDTA plasma samples from

myelodysplastic syndrome (MDS) were used for comparison (clinicaltrials.gov,

#NCT00217594 or NCT00961064).24 Healthy controls (HC) were recruited from donors of

the National Institutes of Health Clinical Center Department of Transfusion Medicine.

A discovery set (n= 35) included 13 AA patients without IST at the time of sampling, 11

MDS patients, and 11 blood donors as HC. A validation set (n = 108) consisted of 41

treatment naïve AA patients, 24 MDS patients, and 43 HC. Demographics of the discovery

and validation sets are shown in Supplemental Table 1. To assess the effect of IST, 40 out of

41 AA patients with plasma samples available both before and after 6 months of IST were

further analyzed.

RNA isolation and cDNA synthesis

Blood samples collected in EDTA tubes were centrifuged and stored at -80°C until use. RNA

isolation from 200 µl EDTA plasma was performed using the miRCURY RNA Isolation Kit -

6
Biofluids (Exiqon, Vedbaek, Denmark), according to the manufacturer’s instructions. RNA

isolation efficiency was monitored with three synthetic RNA spike-ins at different

concentrations (UniSp 2, UniSp 4, and UniSp5). Isolated RNA samples were employed for

cDNA synthesis with the Universal cDNA Synthesis Kit II (Exiqon). Two synthetic RNA

spike-ins in different concentrations (UniSp6 and cel-miR-39-3p) were used to check for

reverse transcription reactions and PCR inhibitors. Prepared cDNAs were stored at -20°C

until use.

MiRNA profiling using the Plasma Focus microRNA PCR Panel

Initial miRNA detection screening with the discovery set (n = 35) was performed using the

Serum/Plasma Focus microRNA PCR Panel, 384 well (V4.M) and the Exilent SYBR Green

master mix (Exiqon). This panel allows analysis of 179 human miRNAs and was used to

profile the discovery set of 13 AA, 11 MDS, and 11 HC (Supplemental Table 2). The

information for validation of the miRNA profiling by a custom PCR panel is shown in

Supplemental Table 3 and Supplemental Methods.

Statistics

Statistical analysis was performed using the GenEx6 software (Exiqon), SPSS 23.0 software,

and graphs were generated using GraphPad PRISM version 6.0 (GraphPad Software, Inc., La

Jolla, CA, USA).

More detailed information is provided in Supplemental Experimental Methods.

7
Results

A distinct circulating miRNA profile in AA, compared to MDS and HC

To examine whether there were unique AA-associated miRNAs, we first analyzed 35

discovery-set plasma samples (13 untreated AA, 11 MDS, and 11 HC) without hemolysis

(∆CT < 7) using the Serum/Plasma Focus microRNA PCR Panel (179 miRNAs). Of 179

miRNAs, 178 miRNAs showed amplification in more than 60% of the samples. When

compared between AA and HC, 14 miRNAs displayed more than 1.5 FC (P < 0.05 by t-test;

Figure 1A and Table 1): 7 miRNAs were significantly upregulated (miR-501-3p,

miR-146b-5p, miR-150-5p, let-7b-5p, miR-200a-3p, miR-1260a, and miR-424-5p) and 7

miRNAs were significantly downregulated (miR-1, miR-29b-3p, miR-30e-5p, miR-143-3p,

let-7e-5p, let-7c-5p, and let-7f-5p) in AA, compared to HC. Hierarchical clustering showed

that the plasma miRNA signature distinguished AA from HC (Figure 1B). Comparison of

different groups by one-way ANOVA revealed multiple differentially expressed circulating

miRNAs (P < 0.05): 12, 47, and 36 miRNAs distinguished AA from HC, MDS from HC, and

AA from MDS, respectively (Supplemental Table 4).

To further address differences of plasma miRNA levels between individual groups, a

principal component analysis (PCA) of differentially expressed miRNAs was performed,

resulting in distinct profiles between AA and MDS vs HC and between AA vs MDS (Figure

1C-E). The PCA plots clearly visualized potential distinct grouping of the compared groups,

providing the basis to validate results of the discovery set in a separate cohort. Supplemental

Figure 1 summarizes all analysis steps for the candidate miRNAs.

Validation of miRNA expression profiles

To validate findings in the discovery set, 19 miRNAs including 17 candidate miRNAs from

the discovery set (Supplemental Table 4) were selected to investigate in a separate cohort of

AA, MDS, and HC (n = 108; Supplemental Table 1) using the custom PCR array plate

8
(Supplemental Table 3): 12 miRNAs (let-7f-5p, miR-1, miR-1260a, miR-143-3p,

miR-146b-5p, miR-150-5p, miR-20a-5p, miR-21-5p, miR-26b-5p, miR-29b-3p, miR-30e-5p,

and miR-501-3p) differentially expressed between AA and HC (P < 0.05 by one-way

ANOVA); 5 miRNAs (let-7g-5p, miR-181a-5p, miR-22-3p, miR-29c-3p, and miR-424-5p)

significantly different by 3 group comparison; and additional 2 miRNAs (let-7a-5p and

miR-144-5p) reported previously to distinguish MDS and HC.19, 25 Box plots of 19 miRNA

expression levels in the discovery set are shown in Supplemental Figure 2. As a hemolysis

test (miR-23a-3p-miR-451a) was > 7 in three HC and three patient plasma samples (one AA

and two MDS), these six samples were excluded from further analysis. All of the selected 19

miRNAs were amplified by qRT-PCR in > 60% of the samples, then subjected to further

analysis. Of the five reference genes, miR-106a-5p and miR-320a were selected to calculate

∆CT and relative expression, as these two miRNAs displayed stable amplification in all of the

individual samples with good expression (CT ≤ 30, Supplemental Figure 3).

Group comparison revealed distinct expression profiles at statistical significance (> 1.5

FC, P < 0.05): upregulated miR-150-5p and miR-146b-5p and downregulated miR-1 in AA,

compared to HC (Figure 2A, D); upregulated miR-146b-5p and miR-22-3p in MDS,

compared to HC (Figure 2B, D); and downregulated miR-1, miR-22-3p, and miR-424-5p in

AA, compared to MDS (Figure 2C-D). Summary data (AA and MDS vs HC, and AA vs

MDS) obtained from the validation set are shown in Table 2. When AA was compared to HC,

six miRNAs showed significant association with AA: miR-1 (P = 0.004), let-7g-5p (P = 0.04),

miR-146b-5p (P = 0.0001), miR-26b-5p (P = 0.049), miR-150-5p (P = 0.015), and

miR-181a-5p (P = 0.029). Further, these six miRNAs were subjected to multivariate analysis

to determine which miRNA associated with AA: miR-1 [odds ratio (OR) = 0.34, adjusted P =

0.005], miR-146b-5p (OR = 3.82, adjusted P = 0.03), and miR-150-5p (OR = 2.19, adjusted

9
P = 0.04), that were differentially expressed in discovery set (Supplemental Table 4),

significantly and independently associated with the AA diagnosis (Table 3).

Comparison between MDS and HC revealed five miRNAs that exhibited significant

association with MDS: let-7g-5p (P = 0.005), miR-146b-5p (P = 0.0003), miR-26b-5p (P =

0.028), miR-21-5p (P = 0.014), and miR-22-3p (P = 0.003). Subsequent multivariate analysis

of these five miRNAs showed that only miR-22-3p, that were differentially expressed in

discovery set (Supplemental Table 4), significantly associated with MDS (OR = 4.09,

adjusted P = 0.03; Table 3); four other miRNAs were not significantly associated with MDS,

in part due to high correlations of the four miRNAs with each other. When AA was compared

to MDS, four miRNAs displayed significant association with AA: miR-1 (P = 0.024),

miR-22-3p (P = 0.005), miR-29c-3p (P = 0.049), and miR-424-5p (P = 0.004). In

multivariate analysis of these miRNAs, significant, independent association with AA was

observed only for miR-1 (OR = 0.22, adjusted P = 0.01) and miR-22-3p (OR = 0.03, adjusted

P = 0.02; Table 3).

Development of a diagnostic panel for AA composed of three plasma miRNAs

Next, sensitivity and specificity of the miRNAs for the diagnosis of AA were evaluated by

ROC curve analysis using the validation set, indicating strong association of miR-146b-5p

[95% confidence interval (CI), 0.65-0.86, P = 0.0001], miR-150-5p (95% CI, 0.63-0.85, P =

0.0002), and miR-1 (95% CI, 0.62-0.85, P = 0.0004) with AA (AUCs of 0.76, 0.74, and 0.73,

respectively; Figure 3A). Further, logistic regression was employed to determine the best

combination of miRNAs to diagnose AA, demonstrating that a linear combination of

expression levels of miR-150-5p, miR-146b-5p, and miR-1 produced the best model. A

miRNA biomarker panel composed of miR-150-5p, miR-146b-5p, and miR-1 provided

significantly increased AUC of 0.86 (95% CI, 0.78-0.94, P < 0.00001; Figure 3A), compared

with use of each miRNAs alone. Cut-off values of the diagnostic performances of the model

10
were determined based on the maximum of Youden Index of the ROC curve (Cut-off value >

0.44 for AA vs HC). By the 3 miRNA-combined panel, a sensitivity of 82% and a specificity

of 80% were obtained for the diagnosis of AA, compared with HC.

By comparing MDS to HC in the validation set, strong association was observed between

miR-146b-5p and MDS, with AUC of 0.80 (95% CI, 0.68-0.91, P = 0.0001), and miR-22-3p,

with AUC of 0.74 (95% CI, 0.61-0.86) (Figure 3B). In the two miRNA-combined panel

(miR-146b-5p and miR-22-3p), AUC was increased to 0.85 (95% CI, 0.76-0.95, P < 0.00001;

Figure 3B). Comparison between AA and MDS revealed strong association of miR-22-3p,

with AA with AUC of 0.81 (95% CI, 0.70-0.92, P < 0.0001; Figure 3C), miR-424-5p, with

AUC of 0.77 (95% CI, 0.65-0.90), and miR-1 with AUC of 0.71 (95% CI, 0.57-0.85) (Figure

3C). Increased AUC (0.88) was achieved by using a panel composed of these three miRNAs

(95% CI, 0.79-0.96, P < 0.00001; Figure 3C).

Correlations of miRNAs with clinical parameters in AA

In addition to group comparisons, correlation analysis between differentially expressed

miRNAs and clinical parameters [CBCs (ANC, ARC, and platelet count) and age] was

performed using the validation set to assess their correlation with disease severity. At the

onset of AA, miR-150-5p and miR-146b-5p showed modest but significant negative

correlations with platelet counts (r = -0.33, P = 0.025) and ARC (r = -0.34, P = 0.020),

respectively (Supplemental Table 5 and Supplemental Figure 4). MiR-1 similarly positively

correlated with ANC (r = 0.30, P = 0.038; Supplemental Table 5 and Supplemental Figure 4).

These data implicate that expression levels of the three miRNAs (elevated miR-150-5p and

miR-146b-5p and reduced miR-1) were correlated to clinical parameters in AA. There was no

correlation between age and the differentially expressed miRNAs (data not shown).

Effects of IST on miRNA expression in AA patients

11
To address whether IST affected the circulating miRNA profile of AA, 40 AA patients with

plasma samples before IST and 6 months after IST (Supplemental Table 6) were analyzed.

Statistically significant changes after IST were detected in all three miRNA (miR-150-5p,

miR-146b-5p, and miR-1) levels, suggesting restoration of dysregurated miRNA expressions

after therapy (P = 0.0001 for miR-150-5p, P = 0.0263 for miR-146b-5p, and P = 0.0003 for

miR-1; Figure 4A and Table 4). Especially, a sharp miR-150-5p decline was seen after IST

(48%, 95% CI: 34–68%), compared to before IST (Figure 4A). The use of horse-ATG (n =

23) or rabbit-ATG (n = 17) did not significantly affect the miRNA expression changes after

IST: statistically significant changes after IST were detected for all three miRNA levels in

both groups.

Of 40 AA patients, 23 patients (58%) achieved partial responses (PR) or complete

responses (CR) while the rest of 17 patients (42%) were non-responders 6 months after IST.

When we analyzed the effects of IST in responders (either PR or CR) and non-responders, a

reduced miR-150-5p level was observed in responders (P = 0.0005; Figure 4B), but there

were no statistically significant changes of miR-150-5p expression in non-responders (Figure

4C), indicating that the restoration of miR-150-5p levels after IST was associated with

successful treatment. Restoration of miR-1 levels was also observed both in responders and

non-responders after IST (Figure 4B-C). Further, miRNA expression levels were compared

between responders and non-responders before IST to assess whether miRNA signatures

were useful for predicting the response to IST. When the expression levels of 19 miRNAs at

onset were compared between responders (n = 23) and non-responders (n = 17), none of them

was significantly different (Supplemental Figure 5).

12
Discussion

In recent publications, circulating miRNAs have been described as noninvasive biomarkers

for many different diseases.17-21 Challenges in the analysis of circulating miRNAs include

preanalytical decisions (such as sample storage and processing) and postanalytical

assessments (data normalization).17 Detailed analyses of miRNA spectra in serum and plasma

recommend plasma over serum to reduce sample-to-sample variations induced by serum

coagulation,26 and we used EDTA anticoagulated plasma in this study to avoid such

variations. To characterize miRNA signatures in plasma of AA, the Serum/Plasma Focus

microRNA PCR Panel was used, as it is considered as appropriate in terms of reproducibility,

sensitivity, accuracy, and specificity.27 We found that circulating miRNAs were differentially

expressed in AA and MDS vs HC and in AA vs MDS, which was validated in a larger

separate cohort using the different normalization method. By multivariate analysis, we

identified the 5 mRNAs (miR-150-5p, miR-146-5p, miR-1, miR-22-3p, and miR-424-5p) that

were significantly associated with AA or MDS, and these miRNA expression patterns were

similar between the 2 cohorts. Our study showed possible utility of “liquid biopsy" to

distinguish AA and MDS.

Starting with a total of 179 miRNAs, 19 from the discovery set were investigated and

three miRNAs were validated as dysregulated in AA. Two miRNAs (miR-150-5p and

miR-146b-5p) and miR-1 were significantly elevated and decreased in the plasma of AA

patients, respectively. Pathway analysis revealed that these three miRNAs targeted important

immune related functions (Figure 5). One of MiR-150-5p targets is NOTCH3: regulation of

the Notch pathway through miR-150-5p may impact T cell development.28 Another target of

miR-150-5p is the transcription factor C-MYB which plays an essential role in T-cell

differentiation.29 MiR-150-5p is an immuno-miRNA considered to be a crucial regulator of T

cell processes.30 Since AA is a T cell mediated disease, it is interesting that miR-150-5p is

13
selectively expressed in immature, resting T cells with strong upregulation for

maturation/differentiation of the T cell progresses.28 MiR-150-5p is a circulating biomarker in

female MG patients without immunosuppressive drug treatment, as the miR-150-5p level is

reduced with clinical improvement after thymectomy.21 Why miRNA expression changes is

unknown. MiRNAs are released into the circulation as a result of apoptotic and necrotic cell

death.31 Active secretion is also a potential source of cell free miRNAs32 and miR-150-5p is

transferred to plasma with CD8+ T cells-derived exosomes.33 We detected increased

expression of miR-150-5p in plasma, which might be due to the secretion from active T cells

in AA. Of interest, we also observed a negative correlation between miR-150-5p levels and

platelet counts. MiR-150-5p is preferentially expressed in the megakaryocytic lineage cells

and it drives MEP differentiation toward megakaryocytes.34 A mechanistic link between

TPO-induced miR-150-5p up-regulation and megakaryopoiesis has been described.35

Increased plasma levels of TPO are reported in AA.36 Therefore, considering our data

demonstrating high miR-150-5p expression in AA plasma and previous reports, it is plausible

that miR-150-5p promotes megakaryopoiesis in AA. The miR-146b-5p plasma level was able

to differentiate both AA and MDS from HC. Further, pathway analysis identified innate

immune pathways related to Toll-like receptors that might be important in AA and MDS

(Figure 5).5, 37 MiR-146b-5p decreases TNF expression in THP-1 monocytes by targeted

α

repression of IRAK1 and TRAF6, 38 and miR-146b-5p is involved in feedback regulation of

the innate immune response.39 Previous studies have also described that alterations of

miR-146a-5p and/or miR-146b-5p levels were associated with inflammatory diseases.11 A

recent work has reported regulatory roles of miR-146b-5p in erythropoiesis and

megakaryopoiesis.40 MiR-1 has BCL2 as its target 41 and circulating miR-1 is a potential

novel biomarker in acute myocardial infarction.42 MiR-1 exhibits anti-inflammatory roles in

asthma mouse models and in inflammatory myopathies.43, 44 Decreased levels of miR-1 might

14
reflect an active inflammatory status in AA patients. Recovery of the miR-1 levels after IST

might be mediated by immunological effects of ATG+ CsA. Collectively, our data suggest

that the miRNA signature in AA plasma samples may reflect the aberrant immune response

and hematopoiesis in AA.

We included MDS patients in our study to compare AA to another bone marrow failure

syndrome. Others have described an association of miRNA expression with MDS subtypes

and disease outcome.45 For example, reduced expression levels of five miRNAs

(miR-378a-3p, miR-143-3p, miR-143-5p, miR-145-5p, and miR-146a-5p) have been reported

in MDS with del(5q),46, 47 but these reports focused on miRNA expression in HSPCs.

Circulating let-7a and miR-16 from MDS predicted progression-free survival and overall

survival,19 and a 7-miRNA signature (let-7a-5p, miR-144-5p, miR-16-5p, miR-25-3p,

miR-451a, miR-651, and miR-655) was an independent predictor of survival in MDS.25

Based on the literature, we included two miRNAs (let-7a-5p and miR-144-5p) into the

custom PCR panel for validation, but we did not observe significant association of let-7a-5p

and miR-144-5p with MDS, probably due to the heterogeneity of the disease, or variations

between the sample processing and detection protocols, or our particular MDS patient cohort.

In our study, MDS patients were relatively young, compared to the median age of >70 years

for typical MDS. Patients enrolled on the study might be more likely have an

immune-mediated pathology compared to typical MDS, with a higher response rate to

alemtuzumab.24 Thus patient selection might lead to underestimation of our results comparing

AA and MDS. Distinguishing between AA and MDS is often difficult.48 We found that the

miR-146b-5p expression level was significantly elevated both in AA and MDS, and miR-1

and miR-22-3p could distinguish AA and MDS, perhaps reflecting commonalities and

differences between AA and MDS. MiR-22-3p, which could distinguish MDS with AA and

HC in our cohort, is an oncogenic miRNAs and is upregulated in MDS.49 For other

15
autoimmune cytopenias, seven plasma miRNAs (miR-302c-3p, miR-483-5p, miR-410,

miR-544a, miR-302a-3p, miR-223-3p, and miR-597) are differentially expressed in immune

thrombocytopenia (ITP) patients.50 These results also suggest the miRNA signatures in AA

are disease-related and not simply a reflection of blood counts.

Analysis of serial plasma samples in 40 AA patients suggested potential use of three

dysregulated miRNAs (miR-150-5p, miR-146b-5p, and miR-1) as disease biomarkers for

diagnosis, as their expression levels were significantly different from those of healthy donors

and levels tended to normalize after IST. Specifically, miR-150-5p may represent a response

marker in AA, as miR-150-5p expression was significantly decreased in responders but not in

non-responders after IST. Similarly, in cancer patients, not all dysregulated miRNAs at

diagnosis have clinical relevance for monitoring, and only some miRNAs normalize after

successful treatment.18

Our study showed rather modest miRNA expression changes in AA plasma, but miRNAs

may affect biologic functions even with subtle changes in expression levels.51 ROC curve

analysis revealed potential clinical utility of combined miRNA panels for future applications.

Our current study had other limitations, such as the relatively small number of patients. There

may be a limitation in using plasma miRNA expression for predicting response to IST.

Nonetheless, our data strongly suggest that aberrant miRNA expression was predominantly

disease-related, especially as restoration of the miRNA levels was observed after IST. In our

recent study, we identified concurrent downregulation of four miRNAs (miR-126-3p,

miR-145-5p, miR-199a-5p, and miR-223-3p) in both CD4+ and CD8+ T cells from AA

patients,14 but dysregulation of the four miRNAs was not observed in AA plasma samples in

our current work. Previous reports in autoimmune diseases12, 20, 52-59 and cancers,60-63 have

also demonstrated discrepancies of expression profiles between cellular and circulating

miRNAs. SLE patients exhibit distinct miRNA expression profiles in T cells 52-55 and

16
plasma,56 and miRNA profiles in T cells12, 57, 58 and plasma20, 59 are inconsistent in multiple

sclerosis. Although circulating miRNAs originate from cells, not all cellular miRNAs can be

identified as circulating miRNAs in biofluids. Indeed, miRNA expression profiles are

cell-type specific. Of note, regardless of discrepancies between cellular and circulating

miRNA profiles, we could observe some common miRNA signatures in both AA and MG,

such as decreased miR-145-5p in T cells14, 64 and increased miR-150-5p in plasma

specimens.21

In conclusion, we demonstrated that expression levels of three dysregulated miRNAs in

AA plasma associated with clinical parameters and normalized after IST, suggesting them as

potential biomarkers in AA. Importantly, we developed a diagnostic logistic model using

combined miRNA panels for diagnosis. Additional studies in larger patient cohorts are

required to validate miRNAs as disease biomarkers for diagnostic and therapeutic purposes in

bone marrow failure.

17
Authorship and Disclosures

K. Hosokawa, S. Kajigaya, X. Feng, and N.S. Young participated in the design of this

analysis. K. Hosokawa conceptualized, conducted the experiments, analyzed the data,

interpreted the results, and drafted the manuscript. X. Feng, M.J. Desierto, M.D.P. Fernandez

Ibanez, O. Rios, B. Weinstein, P. Scheinberg, D.M. Townsley recruited patients to participate

in the study. S. Kajigaya edited the manuscript. N.S. Young was involved in

conceptualization, interim discussions, interpretation of results, and editing the manuscript.

All authors critically reviewed the manuscript content and agree with the submission of the

final manuscript. The authors declare no conflicts of interest.

18
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23
Table 1. Differentially expressed miRNAs (> 1.5 FC)
in the AA discovery set

FC
miRNA P Elevated Decreased
miR-501-3p .002 2.0
miR-146b-5p <.001 1.9
miR-150-5p .009 1.9
let-7b-5p .014 1.7
miR-200a-3p .021 1.7
miR-1260a .017 1.6
miR-424-5p .033 1.6
miR-1 .018 2.4
miR-29b-3p <.001 2.0
miR-30e-5p <.001 1.6
miR-143-3p .014 1.6
let-7e-5p .048 1.6
let-7c-5p .006 1.5
let-7f-5p .010 1.5

24
Table 2. Association between miRNAs and groups in validation set

AA vs HC MDS vs HC AA vs MDS
miRNA FC P AUC FC P AUC FC P AUC
miR-1 -1.76 .004 0.73 -1.02 .995 0.51 -1.72 .024 0.71
let-7g-5p 1.23 .040 0.64 1.38 .005 0.71 -1.12 .502 0.59
miR-146b-5p 1.80 <.001 0.76 2.12 <.001 0.80 -1.18 .575 0.55
miR-26b-5p 1.34 .049 0.63 1.46 .028 0.66 -1.09 .830 0.58
miR-150-5p 1.72 .015 0.74 1.17 .770 0.54 1.48 .208 0.63
miR-181a-5p 1.48 .029 0.64 1.08 .902 0.53 1.37 .189 0.71
miR-21-5p 1.08 .552 0.55 1.29 .014 0.72 -1.19 .122 0.68
miR-22-3p 1.02 .982 0.53 1.60 .003 0.74 -1.56 .005 0.81
miR-29c-3p -1.12 .680 0.55 1.31 .215 0.62 -1.47 .049 0.67
miR-424-5p -1.18 .429 0.59 1.41 .075 0.65 -1.67 .004 0.77

Bold text represents values that were significant, controlling for a false discovery rate at 0.05.

25
Table 3. Multivariate logistic regression model in the validation set

AA vs HC MDS vs HC AA vs MDS
miRNA OR adjusted P miRNA OR adjusted P miRNA OR adjusted P
miR-1 0.34 .005 let-7g-5p 4.54 .126 miR-1 0.22 .013
let-7g-5p 1.22 .845 miR-146b-5p 2.44 .125 miR-22-3p 0.03 .021
miR-146b-5p 3.82 .029 miR-26b-5p 1.02 .972 miR-29c-3p 1.33 .698
miR-26b-5p 1.62 .477 miR-21-5p 1.52 .604 miR-424-5p 0.37 .259
miR-150-5p 2.19 .041 miR-22-3p 4.09 .029
miR-181a-5p 1.31 .620

All analyses were adjusted for age and sex. OR >1 for AA vs HC and AA vs MDS comparisons demonstrates that
increased expression levels were associated with increased odds of being AA. For the comparisons of MDS vs HC,
OR >1 represents that increased expression levels were associated with increased odds of being MDS. Bold text
indicates values that were significant, controlling for false discovery rate at 0.05.

26
Table 4. Changes in miRNA expression after IST

Lower Upper
Ratio P
miRNA 95% CI 95% CI
miR-1 2.61 1.60 4.30 <.001
miR-146b-5p 0.74 0.56 0.96 .026
miR-150-5p 0.48 0.34 0.68 <.001

CI, confidence interval

27
Figure Legends

Figure 1. Distinct circulating miRNA expression profiles of AA patients compared to

MDS and HC in the discovery set.

(A) A volcano plot of 178 miRNA expression levels in plasma of AA patients (n = 13) and

HC (n = 11) in the discovery set. The x-axis displays the estimated expression difference

measured in log2. Vertical lines refer to a 1.5-fold expression difference between two groups,

showing that MiRNAs highly expressed in AA or HC are on the right or the left, respectively,

in which six miRNAs with higher fold differences are depicted. The y-axis shows

significance of the expression difference measured in −log10 of the P-value. The horizontal

red line represents our cutoff for the significance at P < .05. (B) Heatmap analysis visualizes

hierarchical clustering of 14 miRNAs in plasma from 13 AA patients and 11 HC. A red-blue

color scale indicates normalized miRNA expression levels (red: high, blue: low). (C)

Principal component analysis (PCA) plots of significantly (P < .05) and differentially

expressed miRNAs from the discovery set. Blue circles = AA, red circles = MDS, and green

circles = HC.

Figure 2. Validation of the circulating miRNA expression profiles in the validation set.

(A-B) Volcano plots of 19 miRNA expression levels in plasma of AA (n = 41), MDS (n= 24),

and HC (n = 43) in the validation set. The x-axis is the estimated difference in expression

measured in log2; vertical lines refer to a 1.5-fold difference in expression between the two

groups. MiRNAs highly expressed in AA (MDS) or HC are on the right or the left,

respectively. The y-axis is the significance of the difference measured in −log10 of the

P-value; the horizontal red line represents our cutoff for significance at P < .05. (C) Volcano

plots of 19 miRNA expression levels in plasma of AA (n = 41) and MDS patients (n = 24) in

the validation set. MiRNAs highly expressed in AA or MDS are on the right or the left,

28
respectively. (D) miR-150-5p, miR-146b-5p, miR-1, miR-22-3p, and miR-424-5p expression

in plasma of AA (n = 41), MDS (n =24), and HC (n = 43). *P < .05 (one-way ANOVA)).

Figure 3. ROC curves of dysregulated miRNAs in the validation set.

ROC curves for individual miRNAs in AA vs HC (A), MDS vs HC (B), and AA vs MDS (C).

Logistic regression demonstrated a linear combination of values of miRNAs for the compared

groups: miR-150-5p, miR-146b-5p, and miR-1 produced the model for AA diagnosis (the

equation of the Combined miRNA panel = 4.728 + 0.446 × miR-150-5p + 1.725 ×

miR-146b-5p – 1.022 × miR-1); miR-146b-5p and miR-22-3p produced the model for MDS

diagnosis (the equation of Combined miRNA panel = 9.547 + 1.416 × miR-146b-5p + 1.089

× miR-22-3p), and miR-22-3p, miR-424-5p, and miR-1 produced the model for AA diagnosis

compared to MDS (the equation of Combined miRNA panel = -13.117 – 2.046 × miR-22-3p

– 1.384 × miR-424-5p – 1.221 × miR-1). The ROC curve of miRNA panel was generated

based on the predicted probability (P) for each patient. Predicted probability = Exp

(combined miRNA panel) / [1+ Exp (combined miRNA panel)].

Figure 4. MiRNA expression changes after IST.

(A) MiR-150-5p, miR-146b-5p, and miR-1 expression in plasma of AA patients at onset (n =

40) and after IST at 6 months (n = 40). (B) MiR-150-5p, miR-146-5p, and miR-1 expression

in plasma of AA patients (responders) at onset (n = 23) and after IST at 6 months (n = 23).

(C) MiR-150-5p, miR-146-5p, and miR-1 expression in plasma of AA patients

(non-responders) at onset (n = 17) and after IST at 6 months (n = 17). *P < .05 (paired

two-tailed t-test).

29
Figure 5. Ingenuity Pathway Analysis (IPA) to identify immune targets of the selected

three miRNAs.

Shown are network genes of the dysregulated three miRNAs (miR-1, miR-146b-5p, and

miR-150-5p) in AA plasma, compared to HC. Color intensity indicates upregulation (red) and

downregulation (green). Solid and dotted lines represent direct and indirect relationships

between genes.

30
Supplemental Data:

A plasma microRNA signature as a biomarker for acquired aplastic anemia

Kohei Hosokawa, Sachiko Kajigaya, Xingmin Feng, Marie J. Desierto, Maria del Pilar

Fernandez Ibanez, Olga Rios, Barbara Weinstein, Phillip Scheinberg, Danielle M. Townsley,

Neal S. Young

Address correspondence to:

Kohei Hosokawa, Cell Biology Section, Hematology Branch,

National Heart, Lung, and Blood Institute, National Institutes of Health

Building 10-CRC, Room 3E-5140, 10 Center Drive, Bethesda MD 20892

email: kohei.hosokawa@nih.gov,

phone: 301-496-5093, fax: 301-496-8396

List of Supplementary Information

Supplementary Tables S1-6

Supplementary Figures S1-5
Supplemental Experimental Methods
Supplementary References

1
Supplemental Data

Table S1. Characteristics of patients and healthy controls

Characteristic AA MDS HC
Discovery set (n = 35)
No. 13 11 11
Median age (range) 30 (10-75) 64 (44-80) 34 (23-57)
Sex (M/F) 7/6 4/7 5/6
Disease status (%) SAA 13 (100%) WHO RCUD 5 (45.4%) NA
RCMD 1 (9.1%)
MDS-U 3 (27.3%)
RARS 2 (18.2%)
IPSS Low 1 (9.1%)
Int-1 8 (72.7%)
Int-2 2 (18.2%)
Treatment (%) rATG+CsA 8 (61.5%) Alemtuzumab 4 (36.4%) NA
hATG+CsA 5 (38.5%) TPO-RA 7 (63.6%)
Transfusion yes 13 (100%) yes 8 (72.7%) NA
dependency (%) no 3 (27.3%)

Validation set (n = 108)

No. 41* 24 43
Median age (range) 25 (2-75) 54.5 (26-71) 38 (23-66)
Sex (M/F) 24/17 15/9 22/21
Disease status (%) SAA 41 (100%) WHO RCUD 7 (29.2%) NA
RCMD 7 (29.2%)
MDS-U 5 (20.8%)
RARS 2 (8.3%)
RAEB-1 3 (12.5%)
IPSS Low 2 (8.3%)
Int-1 18 (75.0%)
Int-2 4 (16.7%)
Treatment (%) rATG+CsA 18 (43.9%) Alemtuzumab 24 (100%) NA
hATG+CsA 23 (56.1%)
Transfusion yes 41 (100%) yes 18 (75%) NA
dependency (%) no 6 (25%)
M, male; F, female; NA, not applicable; rATG, rabbit ATG; hATG, horse ATG; CsA,
cyclosporine; IPSS, International Prognostic Scoring System; Int-1, Intermediate 1;
Int-2, Intermediate 2; TPO-RA, thrombopoietin receptor agonist.
*In order to assess the effect of IST, 40 patients of 41 AA who had serial plasma samples
collected both before and after 6 months of IST were analyzed.

2
Table S2. The Serum/Plasma Focus microRNA PCR Panel, 384 well (V4.M) for the
discovery set
Supplemental Table1.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
hsa-miR- hsa-miR- UniSp3 hsa-miR- hsa-miR- hsa-miR- hsa-let-7f- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
A UniSp2
652-3p 502-3p IPC 339-3p 221-3p 409-3p 5p 154-5p 27b-3p 155-5p 374b-5p 140-3p 93-5p 92b-3p 200a-3p 505-3p 23b-3p 484 141-3p 181a-5p 361-5p 106a-5p 27a-3p
hsa-miR- hsa-miR- UniSp3 hsa-miR- hsa-miR- hsa-miR- hsa-let-7f- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
B UniSp2
652-3p 502-3p IPC 339-3p 221-3p 409-3p 5p 154-5p 27b-3p 155-5p 374b-5p 140-3p 93-5p 92b-3p 200a-3p 505-3p 23b-3p 484 141-3p 181a-5p 361-5p 106a-5p 27a-3p
hsa-miR- UniSp3 hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-let-7d- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-let-7e- hsa-miR-
C hsa-miR-1 UniSp4
145-5p IPC 382-5p 486-5p 32-5p 26a-5p 133b 143-3p 5p 30a-5p 133a-3p 222-3p 20b-5p 342-3p 106b-5p 328-3p 324-5p 532-3p 185-5p 15a-5p 5p 532-5p
hsa-miR- UniSp3 hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-let-7d- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-let-7e- hsa-miR-
D hsa-miR-1 UniSp4
145-5p IPC 382-5p 486-5p 32-5p 26a-5p 133b 143-3p 5p 30a-5p 133a-3p 222-3p 20b-5p 342-3p 106b-5p 328-3p 324-5p 532-3p 185-5p 15a-5p 5p 532-5p
hsa-miR- hsa-miR- UniSp3 hsa-miR- hsa-miR- hsa-miR- hsa-let-7a- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-let-7b- hsa-miR- hsa-miR- hsa-miR- hsa-let-7c- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
E UniSp5
139-5p 194-5p IPC 660-5p 451a 574-3p 5p 320c 128-3p 130a-3p 125a-5p 28-5p 485-3p 497-5p 3p 425-3p 132-3p 25-3p 5p 375 18a-5p 33a-5p 29b-3p
hsa-miR- hsa-miR- UniSp3 hsa-miR- hsa-miR- hsa-miR- hsa-let-7a- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-let-7b- hsa-miR- hsa-miR- hsa-miR- hsa-let-7c- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
F UniSp5
139-5p 194-5p IPC 660-5p 451a 574-3p 5p 320c 128-3p 130a-3p 125a-5p 28-5p 485-3p 497-5p 3p 425-3p 132-3p 25-3p 5p 375 18a-5p 33a-5p 29b-3p
hsa-miR- hsa-miR- hsa-let-7b- hsa-miR- hsa-miR- UniSp3 hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- cel-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
G UniSp6
16-5p 136-5p 5p 1260a 152-3p IPC 30c-5p 15b-3p 197-3p 142-5p 99b-5p 100-5p 30e-5p 326 146a-5p 362-3p 39-3p 421 424-5p 223-5p 146b-5p 107 205-5p
hsa-miR- hsa-miR- hsa-let-7b- hsa-miR- hsa-miR- UniSp3 hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- cel-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
H UniSp6
16-5p 136-5p 5p 1260a 152-3p IPC 30c-5p 15b-3p 197-3p 142-5p 99b-5p 100-5p 30e-5p 326 146a-5p 362-3p 39-3p 421 424-5p 223-5p 146b-5p 107 205-5p
hsa-miR- hsa-miR- hsa-miR- Blank hsa-miR- UniSp3 hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-let-7i- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
I
148b-3p 339-5p 20a-5p (H2O) 17-5p IPC 30d-5p 378a-3p 186-5p 425-5p 5p 454-3p 26b-5p 874-3p 34a-5p 193a-5p 320b 885-5p 590-5p 127-3p 191-5p 208a-3p 99a-5p 16-2-3p
hsa-miR- hsa-miR- hsa-miR- Blank hsa-miR- UniSp3 hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-let-7i- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
J
148b-3p 339-5p 20a-5p (H2O) 17-5p IPC 30d-5p 378a-3p 186-5p 425-5p 5p 454-3p 26b-5p 874-3p 34a-5p 193a-5p 320b 885-5p 590-5p 127-3p 191-5p 208a-3p 99a-5p 16-2-3p
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- UniSp3 hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
K
301a-3p 140-5p 151a-5p 130b-3p 122-5p IPC 423-5p 629-5p 101-3p 200c-3p 365a-3p 501-3p 23a-3p 423-3p 215-5p 376a-3p 320a 22-5p 338-3p 2110 223-3p 376c-3p 103a-3p 93-3p
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- UniSp3 hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
L
301a-3p 140-5p 151a-5p 130b-3p 122-5p IPC 423-5p 629-5p 101-3p 200c-3p 365a-3p 501-3p 23a-3p 423-3p 215-5p 376a-3p 320a 22-5p 338-3p 2110 223-3p 376c-3p 103a-3p 93-3p
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-let-7d- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-7- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
M
331-3p 144-5p 142-3p 210-3p 3p 199a-5p 126-5p 766-3p 19a-3p 584-5p 144-3p 92a-3p 126-3p 363-3p 148a-3p 374a-5p 10b-5p 483-5p 195-5p 1-3p 125b-5p 877-5p 192-5p 151a-3p
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-let-7d- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-7- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
N
331-3p 144-5p 142-3p 210-3p 3p 199a-5p 126-5p 766-3p 19a-3p 584-5p 144-3p 92a-3p 126-3p 363-3p 148a-3p 374a-5p 10b-5p 483-5p 195-5p 1-3p 125b-5p 877-5p 192-5p 151a-3p
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-let-7g- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-7- Blank hsa-miR-
O
18b-5p 28-3p 335-5p 324-3p 320d 136-3p 5p 15b-5p 22-3p 106b-3p 199a-3p 29c-3p 19b-3p 335-3p 29a-3p 21-5p 150-5p 30e-3p 30b-5p 543 24-3p 5p (H2O) 495-3p
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-let-7g- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-7- Blank hsa-miR-
P
18b-5p 28-3p 335-5p 324-3p 320d 136-3p 5p 15b-5p 22-3p 106b-3p 199a-3p 29c-3p 19b-3p 335-3p 29a-3p 21-5p 150-5p 30e-3p 30b-5p 543 24-3p 5p (H2O) 495-3p

3
Table S3. The custom microRNA PCR Panel for the validation set
Supplemental Table2.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
A
103a-3p 106a-5p 103a-3p 106a-5p 103a-3p 106a-5p 103a-3p 106a-5p 103a-3p 106a-5p 103a-3p 106a-5p 103a-3p 106a-5p 103a-3p 106a-5p 103a-3p 106a-5p 103a-3p 106a-5p 103a-3p 106a-5p 103a-3p 106a-5p
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
B
23a-3p 148b-3p 23a-3p 148b-3p 23a-3p 148b-3p 23a-3p 148b-3p 23a-3p 148b-3p 23a-3p 148b-3p 23a-3p 148b-3p 23a-3p 148b-3p 23a-3p 148b-3p 23a-3p 148b-3p 23a-3p 148b-3p 23a-3p 148b-3p
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
C
451a 191-5p 451a 191-5p 451a 191-5p 451a 191-5p 451a 191-5p 451a 191-5p 451a 191-5p 451a 191-5p 451a 191-5p 451a 191-5p 451a 191-5p 451a 191-5p
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
D hsa-miR-1 hsa-miR-1 hsa-miR-1 hsa-miR-1 hsa-miR-1 hsa-miR-1 hsa-miR-1 hsa-miR-1 hsa-miR-1 hsa-miR-1 hsa-miR-1 hsa-miR-1
320a 320a 320a 320a 320a 320a 320a 320a 320a 320a 320a 320a
hsa-let-7f- hsa-let-7a- hsa-let-7f- hsa-let-7a- hsa-let-7f- hsa-let-7a- hsa-let-7f- hsa-let-7a- hsa-let-7f- hsa-let-7a- hsa-let-7f- hsa-let-7a- hsa-let-7f- hsa-let-7a- hsa-let-7f- hsa-let-7a- hsa-let-7f- hsa-let-7a- hsa-let-7f- hsa-let-7a- hsa-let-7f- hsa-let-7a- hsa-let-7f- hsa-let-7a-
E
5p 5p 5p 5p 5p 5p 5p 5p 5p 5p 5p 5p 5p 5p 5p 5p 5p 5p 5p 5p 5p 5p 5p 5p
hsa-let-7g- hsa-miR- hsa-let-7g- hsa-miR- hsa-let-7g- hsa-miR- hsa-let-7g- hsa-miR- hsa-let-7g- hsa-miR- hsa-let-7g- hsa-miR- hsa-let-7g- hsa-miR- hsa-let-7g- hsa-miR- hsa-let-7g- hsa-miR- hsa-let-7g- hsa-miR- hsa-let-7g- hsa-miR- hsa-let-7g- hsa-miR-
F
5p 143-3p 5p 143-3p 5p 143-3p 5p 143-3p 5p 143-3p 5p 143-3p 5p 143-3p 5p 143-3p 5p 143-3p 5p 143-3p 5p 143-3p 5p 143-3p
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
G
29b-3p 1260a 29b-3p 1260a 29b-3p 1260a 29b-3p 1260a 29b-3p 1260a 29b-3p 1260a 29b-3p 1260a 29b-3p 1260a 29b-3p 1260a 29b-3p 1260a 29b-3p 1260a 29b-3p 1260a
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
H
30e-5p 146b-5p 30e-5p 146b-5p 30e-5p 146b-5p 30e-5p 146b-5p 30e-5p 146b-5p 30e-5p 146b-5p 30e-5p 146b-5p 30e-5p 146b-5p 30e-5p 146b-5p 30e-5p 146b-5p 30e-5p 146b-5p 30e-5p 146b-5p
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
I
20a-5p 26b-5p 20a-5p 26b-5p 20a-5p 26b-5p 20a-5p 26b-5p 20a-5p 26b-5p 20a-5p 26b-5p 20a-5p 26b-5p 20a-5p 26b-5p 20a-5p 26b-5p 20a-5p 26b-5p 20a-5p 26b-5p 20a-5p 26b-5p
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
J
501-3p 21-5p 501-3p 21-5p 501-3p 21-5p 501-3p 21-5p 501-3p 21-5p 501-3p 21-5p 501-3p 21-5p 501-3p 21-5p 501-3p 21-5p 501-3p 21-5p 501-3p 21-5p 501-3p 21-5p
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
K
150-5p 22-3p 150-5p 22-3p 150-5p 22-3p 150-5p 22-3p 150-5p 22-3p 150-5p 22-3p 150-5p 22-3p 150-5p 22-3p 150-5p 22-3p 150-5p 22-3p 150-5p 22-3p 150-5p 22-3p
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
L
29c-3p 424-5p 29c-3p 424-5p 29c-3p 424-5p 29c-3p 424-5p 29c-3p 424-5p 29c-3p 424-5p 29c-3p 424-5p 29c-3p 424-5p 29c-3p 424-5p 29c-3p 424-5p 29c-3p 424-5p 29c-3p 424-5p
hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR- hsa-miR-
M
181a-5p 144-5p 181a-5p 144-5p 181a-5p 144-5p 181a-5p 144-5p 181a-5p 144-5p 181a-5p 144-5p 181a-5p 144-5p 181a-5p 144-5p 181a-5p 144-5p 181a-5p 144-5p 181a-5p 144-5p 181a-5p 144-5p
Cel-miR- UniSp2 Cel-miR- UniSp2 Cel-miR- UniSp2 Cel-miR- UniSp2 Cel-miR- UniSp2 Cel-miR- UniSp2 Cel-miR- UniSp2 Cel-miR- UniSp2 Cel-miR- UniSp2 Cel-miR- UniSp2 Cel-miR- UniSp2 Cel-miR- UniSp2
N
39-3p CP CP 39-3p CP CP 39-3p CP CP 39-3p CP CP 39-3p CP CP 39-3p CP CP 39-3p CP CP 39-3p CP CP 39-3p CP CP 39-3p CP CP 39-3p CP CP 39-3p CP CP
UniSp4 UniSp5 UniSp4 UniSp5 UniSp4 UniSp5 UniSp4 UniSp5 UniSp4 UniSp5 UniSp4 UniSp5 UniSp4 UniSp5 UniSp4 UniSp5 UniSp4 UniSp5 UniSp4 UniSp5 UniSp4 UniSp5 UniSp4 UniSp5
O
CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP
UniSp6 UniSp3 UniSp6 UniSp3 UniSp6 UniSp3 UniSp6 UniSp3 UniSp6 UniSp3 UniSp6 UniSp3 UniSp6 UniSp3 UniSp6 UniSp3 UniSp6 UniSp3 UniSp6 UniSp3 UniSp6 UniSp3 UniSp6 UniSp3
P
CP IPC CP IPC CP IPC CP IPC CP IPC CP IPC CP IPC CP IPC CP IPC CP IPC CP IPC CP IPC

4
Table S4. Differentially expressed miRNAs in the discovery set
AA vs HC MDS vs HC AA vs MDS
miRNAs P miRNAs P miRNAs P
let-7f-5p .04 miR-339-3p .02 miR-339-3p .03
miR-1 .02 miR-409-3p .009 miR-409-3p .005
miR-143-3p .05 let-7f-5p .001 miR-154-5p .03
miR-29b-3p .005 miR-200a-3p .04 miR-484 .003
miR-1260a .05 miR-484 .006 miR-141-3p .02
miR-30e-5p <.001 miR-181a-5p .001 miR-181a-5p <.001
miR-146b-5p .002 miR-382-5p .01 miR-382-5p <.001
miR-20a-5p .01 miR-32-5p <.001 miR-32-5p .001
miR-26b-5p .02 let-7d-5p .01 miR-143-3p .01
miR-501-3p .02 miR-342-3p .03 let-7d-5p .006
miR-21-5p .02 miR-139-5p .003 miR-342-3p .02
miR-150-5p .05 miR-660-5p .004 miR-139-5p .05
miR-574-3p .02 miR-29b-3p .01
let-7a-5p .05 miR-30e-5p .05
miR-28-5p .03 miR-424-5p .01
miR-1260a .003 miR-146b-5p .04
miR-424-5p <.001 miR-425-5p <.001
miR-30d-5p .01 miR-34a-5p .02
miR-378a-3p .007 miR-191-5p .04
miR-425-5p .04 miR-151a-5p .05
miR-34a-5p .01 miR-101-3p .01
miR-127-3p .03 miR-22-5p .006
miR-423-5p .02 miR-376c-3p .03
miR-629-5p .004 miR-331-3p .03
miR-101-3p .03 miR-766-3p .02
miR-501-3p .02 miR-19a-3p .02
miR-23a-3p .03 miR-148a-3p .05
miR-376a-3p .02 miR-195-5p .002
miR-22-5p .006 let-7g-5p <.001
miR-376c-3p .005 miR-22-3p <.001
miR-331-3p .05 miR-199a-3p .03
miR-126-5p .05 miR-29a-3p .006
miR-766-3p .02 miR-29c-3p <.001
miR-584-5p .01 miR-150-5p .004
miR-483-5p .04 miR-543 .04
miR-195-5p .01 miR-495-3p .04
miR-28-3p .05
let-7g-5p <.001
miR-22-3p <.001
miR-199a-3p .002
miR-335-3p .007
miR-29a-3p .03
miR-29c-3p <.001
miR-21-5p .003
miR-30b-5p .02
miR-543 .02
miR-495-3p .008
Probability values of significantly (P < .05) and differentially expressed miRNAs based on 1-way
ANOVA followed by pair-wise group comparisons in the discovery set.
MiRNAs in bold text were tested using qPCR in the validation set.

5
Table S5. Association
Supplemental Table 5. between miRNAs and clinical and laboratory parameters
Association between miRNAs and Clinical and Laboratory Parameters
ANC Plt ARC
Pearson Pearson Pearson
miRNA correlation P correlation P correlation P
coefficients coefficients coefficients
miR-1 0.296 .038 0.010 .476 0.243 .073
miR-146b-5p -0.130 .222 -0.174 .151 -0.340 .020
miR-150-5p -0.095 .287 -0.325 .025 -0.227 .088
Positive coefficients indicate that an increase in the cycle number is associated with an increase
in ANC, Plt, or ARC.miRNA: microRNA, ANC: Absolute neutrophil count, Plt: Platelet count,
ARC: Absolute reticulocyte count

6
Table S6. Change in miR-150-5p and clinical status after IST in AA patients
% Change in miR- Response at 6
UPN Age Sex IST
150-5p after IST months
1 19 M -23.4 h-ATG PR
2 3 M -74.7 r-ATG NR
3 9 M -26.1 h-ATG PR
4 53 M -74.2 h-ATG CR
5 43 F NA r-ATG PR
6 17 M -79.7 r-ATG NR
7 8 M -72.3 h-ATG PR
8 11 F -86.1 r-ATG NR
9 22 M -93.6 r-ATG NR
10 25 F 88.6 h-ATG PR
11 42 M -66.3 h-ATG CR
12 28 M 109.3 h-ATG NR
13 20 M 109.1 h-ATG NR
14 39 M -78.9 r-ATG PR
15 52 F -74.0 h-ATG PR
16 4 F 13.2 h-ATG NR
17 7 F 133.1 r-ATG NR
18 22 M -60.5 h-ATG PR
19 59 M 4.6 h-ATG NR
20 33 M -81.0 r-ATG PR
21 39 F -74.1 r-ATG PR
22 61 F -53.0 h-ATG PR
23 11 M -37.6 r-ATG NR
24 57 M 29.3 h-ATG NR
25 17 F -75.8 r-ATG NR
26 48 M -55.5 h-ATG PR
27 25 F -97.9 r-ATG CR
28 27 M -15.4 h-ATG PR
29 67 M -22.0 h-ATG PR
30 2 F -85.4 r-ATG NR
31 52 F -62.0 h-ATG PR
32 21 M -57.0 r-ATG NR
33 52 F -44.9 r-ATG NR
34 58 F 105.6 h-ATG PR
35 7 F -13.9 h-ATG CR
36 23 M 2.1 h-ATG PR
37 5 M 135.1 r-ATG NR
38 17 M -45.1 r-ATG PR
39 50 F NA h-ATG NR
40 75 F -62.6 h-ATG PR
IST= immunosuppressive therapy, h= horse, r=rabbit, ATG= Anti-thymocyte globulin,
CR = complete response, PR= partial response, NR = no response, NA = Not available

7
 Supplemental Figure 1, Hosokawa et al.

Figure S1. A summary of all analysis steps of the candidate miRNA.

Shown are flow charts of miRNA analysis in the discovery and validation sets.

8
 Supplemental Figure 2, Hosokawa et al.

Figure S2. Box plots of the 19 miRNA expression levels in the discovery set.

Box plots show 19 miRNAs differentially expressed between AA, MDS, and HC in discovery

set. The Y-axis indicates miRNA expression levels. Bars mean minimum and maximum values

of miRNA expression. Blue bars = AA; red bars = MDS; green bars = HC.

9
 Supplemental Figure 3, Hosokawa et al.

Figure S3. Normofinder represents the stably expressed miRNAs.

Five miRNAs (miR-103a-3p, miR-106a-5p, miR-148-3p, miR-191-5p, and miR-320a, shown in

red color) with superior expression stability among all samples were included in the custom plate

for data normalization in the validation set. Out of five reference genes defined by the discovery

set, miR-106a-5p and miR-320a exhibited to have stable amplification in all of the individual

samples with good expression in the validation cohort, whereby these two miRNAs were used to

calculate ∆CT and further relative expression levels. The y-axis indicates the NormoFinder

stability values.

10
 Supplemental Figure 4, Hosokawa et al.

Figure S4. Correlations between miRNAs and clinical parameters in AA patients at

diagnosis.

Pearson correlation coefficient was calculated between miRNAs and absolute neutrophil count

(ANC), absolute reticulocyte count (ARC), or platelet count, respectively. (A) Negative

correlation between the miR-150-5p expression levels and platelet count. (B) Positive correlation

between the miR-1 expression levels and ANC. (C) Negative correlation between the miR-146b-

5p expression levels and ARC. r, a correlation coefficient value.

11
 Supplemental Figure 5, Hosokawa et al.

Figure S5. Comparison of 19 miRNA expression levels of plasma between responders and

non-responders in AA patients at diagnosis.

Expression levels of 19 plasma miRNA at diagnosis were compared between two patient groups

[responders (n = 23) vs non-responders (n=17)] in AA. None of the miRNAs was significantly

different between the two groups.

12
Supplemental Experimental Methods

MiRNA profiling using the Plasma Focus microRNA PCR Panel

The panel assay includes wells of an interpolate calibrator (UniSp3) to account for run-to-run

differences in amplification signal. Assays were carried out in a final reaction volume of 10

μl/wells, following the manufacturer’s protocol, using 50x diluted cDNA and an equal volume of

2x SYBR Green master mix. qRT-PCR amplification condition was an initial hold at 95°C for 10

min, followed by 40 cycles of amplification (95°C for 10 sec and 60°C for 60 sec) and melting

curve analysis. All qRT-PCR reactions were performed in 384-well plates using the ROX

Reference Dye (Thermo Fisher Scientific, NY) and analyzed with the Applied Biosystems

7900HT Fast Real-Time PCR System (Applied Biosystems, Grand Island, NY).

Raw qRT-PCR amplification data were analyzed using GenEx6 software (Exiqon), according

to the Exiqon’s recommendations, which included the inter-plate calibration, approved quality

controls (RNA-spike-in), and hemolysis test (miR-23a-3p –miR-451a). Plasma was analyzed for

potential cellular miRNA contamination, due to hemolysis during the plasma samples

preparation. A ∆ CT value of established hemolysis markers [∆CT (hemolysis) =CT (miR-23a-

3p)-CT (miR-451a)] is recommended to use as the qualitative measurement of the hemolysis.

Plasma samples with > 7 ∆CT values were not used for further analysis, due to a high risk of

hemolysis. The discovery-set data were normalized using the global mean of all miRNAs with <

34 CT values.1 Candidate reference miRNAs identified by variance analysis of the normalized

dataset were further examined using the established algorithms, Normfinder.2 Five miRNAs

(miR-103a-3p, miR-106a-5p, miR-148-3p, miR-191-5p, and miR-320a) with superior expression

stability among all samples were included in the custom plate for normalization in the

subsequent validation set.

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Validation of the miRNA profiling by custom PCR panel

Based on the results from the discovery set, we designed the custom microRNA PCR Panel for

validation set. This panel covered the analysis of 19 human miRNAs for validation: five

miRNAs (miR-103a-3p, miR-106a-5p, miR-148-3p, miR-191-5p, and miR-320a) as reference

miRNAs, two miRNAs (miR-451a and miR-23a-3p) as hemolysis test, and six miRNAs as

spike-in and other quality controls (Supplemental Table 3). This panel was used for profiling the

validation set (108 of 41 AA patients, 24 MDS patients, and 43 HCs) and 40 AA patients with

plasma samples collected after 6 months of IST.

Amplification conditions of qRT-PCR were an initial hold at 95°C for 10 min, followed by 40

cycles of amplification (95°C for 10 sec and 60°C for 60 sec) and melting curve analysis. All

qRT-PCR reactions were carried out on 384-well plates with ROX Reference Dye (Thermo

Fisher Scientific) and analyzed with the Applied Biosystems 7900HT Fast Real-Time PCR

System (Applied Biosystems). qRT-PCR data from the custom PCR Panels were examined with

GenEx6 software (Exiqon). In this process, the inter-plate calibration, approved quality controls

(RNA-spike-in), and hemolysis test (miR-23a-3p –miR-451a) were included. Reference

miRNAs, miR-106a-5p, and miR-320a were chosen by analyzing the suggested candidate genes

in the applications “NormFinder” available in the GenEx6 software (Supplemental Figure 3).

Normalization of individual miRNA levels was done to the above-mentioned reference genes.

Quantification of relative miRNA expression on the validation set was performed with the

comparative CT method using the formula 2^ ∆CT, where ∆CT = [(CT gene of interest- CT

reference gene) sample A - (CT gene of interest - CT reference gene) sample B] by using miR-

106a-5p and miR-320a as the reference miRNAs.3

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Ingenuity® Pathway Analysis (IPA)

Pathway analysis was carried out to determine differentially regulated biological pathways by

loading differentially expressed miRNAs with statistically significance into the Ingenuity®

Pathway Analysis (IPA) software (www.ingenuity.com).

Statistics

Log conversion of the data in the discovery and validation sets was performed to obtain data

more similar to a normal distribution for statistical testing. Unpaired two-tailed t-test for two-

group comparison and one-way analysis of variance (ANOVA) for three-group comparison were

performed. In the discovery set, candidate miRNAs were selected if significant differences (P

< .05) among individual groups were observed by pair-wise group comparison (AA vs HC, MDS

vs HC, and AA vs MDS). From candidate miRNAs, upregulated or downregulated 19 miRNAs

found in each group were selected for the validation set analysis. A 1.5-fold-change (FC)

threshold was chosen on the basis of its use in the literature.1 Hierarchical clustering analysis was

performed using Cluster 3 and results were displayed as heatmaps generated by TreeView.4 In

the validation set, association between group status and each of the selected miRNAs was

assessed using logistic regression to control for the age and sex. Receiver operating characteristic

(ROC) curves and the area under the ROC curve (AUC) were used to assess sensitivity and

specificity of miRNA biomarkers for the diagnosis. Further, logistic regression was employed to

develop a combined miRNA panel to predict the probability of developing AA, as previously

described.5, 6 Pearson correlation was performed to determine the correlation coefficient between

miRNAs and clinical characteristics [age, absolute neutrophil count (ANC), absolute reticulocyte

count (ARC), platelet count]. Paired two-tailed t-test was carried out based on within-patient

15
change before and after IST. P values from individual analysis were also adjusted for multiple

comparisons using the approach of Benjamini and Hochberg to control the false discovery rate

(FDR) at .05.

Supplemental References
1. Mestdagh P, Van Vlierberghe P, De Weer A, et al. A novel and universal method for
microRNA RT-qPCR data normalization. Genome Biol. 2009;10(6):R64.
2. Andersen CL, Jensen JL, Orntoft TF. Normalization of real-time quantitative reverse
transcription-PCR data: a model-based variance estimation approach to identify genes suited for
normalization, applied to bladder and colon cancer data sets. Cancer research. 2004;64(15):5245-
5250.
3. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method.
Nat Protoc. 2008;3(6):1101-1108.
4. Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide
expression patterns. Proc Natl Acad Sci U S A. 1998;95(25):14863-14868.
5. Xiao B, Wang Y, Li W, et al. Plasma microRNA signature as a noninvasive biomarker for
acute graft-versus-host disease. Blood. 2013;122(19):3365-3375.
6. Jin BX, Zhang YH, Jin WJ, et al. MicroRNA panels as disease biomarkers distinguishing
hepatitis B virus infection caused hepatitis and liver cirrhosis. Scientific reports. 2015;5(15026.

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